U.S. patent application number 10/245175 was filed with the patent office on 2003-03-27 for methods for identifying compounds which modulate circadian rhythm.
This patent application is currently assigned to The General Hospital Corporation, a Massachusetts corporation. Invention is credited to Jin, Xiaowei, Kume, Kazuhiko, Reppert, Steven M., Sathyanarayanan, Sriram, Shearman, Lauren, Weaver, David R., Zylka, Mark.
Application Number | 20030059848 10/245175 |
Document ID | / |
Family ID | 26842892 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059848 |
Kind Code |
A1 |
Reppert, Steven M. ; et
al. |
March 27, 2003 |
Methods for identifying compounds which modulate circadian
rhythm
Abstract
The invention is based, in part, on the discovery that the CRY
proteins and the PER2 protein function as important modulators of
mammalian circadian rhythm. The invention includes methods of
modulating the circadian rhythm and identifying compounds that
modulate the circadian rhythm.
Inventors: |
Reppert, Steven M.; (Newton,
MA) ; Weaver, David R.; (Londonderry, NH) ;
Zylka, Mark; (Pasadena, CA) ; Jin, Xiaowei;
(Boston, MA) ; Kume, Kazuhiko; (Belmont, MA)
; Sathyanarayanan, Sriram; (Somerville, MA) ;
Shearman, Lauren; (Jamaica Plain, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
The General Hospital Corporation, a
Massachusetts corporation
|
Family ID: |
26842892 |
Appl. No.: |
10/245175 |
Filed: |
September 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10245175 |
Sep 17, 2002 |
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09618425 |
Jul 18, 2000 |
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6475744 |
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60203005 |
May 10, 2000 |
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60145363 |
Jul 22, 1999 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 2500/10 20130101;
G01N 33/68 20130101; G01N 2500/20 20130101; G01N 33/6872 20130101;
C12Q 1/6897 20130101; G01N 2500/02 20130101; G01N 2333/4703
20130101; C07K 14/4702 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Claims
What is claimed is:
1. A method for identifying a compound which binds to a mammalian
CRY protein, the method comprising: contacting the CRY protein with
a test compound; and determining whether the CRY protein binds to
the test compound, wherein binding by the test compound to the CRY
protein indicates that the test compound is a CRY protein binding
compound.
2. The method of claim 1, wherein the CRY protein is CRY1 or
CRY2.
3. The method of claim 1, wherein the test compound is
radiolabeled.
4. The method of claim 1, further comprising: contacting the test
compound with the CRY protein in the presence of a PER protein; and
determining whether the test compound disrupts the association of
the CRY protein with the PER protein, wherein a decrease in the
association in the presence of the test compound compared to the
association in the absence of the test compound indicates that the
test compound disrupts the association of the CRY protein and with
PER protein.
5. The method of claim 4, wherein the CRY protein is a mouse CRY 1
or CRY2.
6. The method of claim 4, wherein the PER is a mouse PER1, PER2 or
PER3.
7. The method of claim 1, further comprising: contacting the test
compound with the CRY protein in the presence of a TIM protein; and
determining whether the test compound disrupts the association of
the CRY protein with the TIM protein, wherein a decrease in the
association in the presence of the test compound compared to the
association in the absence of the test compound indicates that the
test compound disrupts the association of the CRY protein with the
TIM protein.
8. The method of claim 1, further comprising: contacting the test
compound with the CRY protein in the presence of a CLOCK:BMAL-1
complex; and determining whether the test compound disrupts the
association of the CRY protein with the CLOCK:BMAL-1 complex,
wherein a decrease in the association in the presence of the test
compound compared to the association in the absence of the test
compound indicates that the test compound disrupts the association
of the CRY protein with the CLOCK:BMAL-1 complex.
9. The method of claim 1, further comprising: contacting the test
compound with the CRY protein in the presence of a BMAL-1 protein;
and determining whether the test compound disrupts the association
of the CRY protein with the BMAL-1 protein, wherein a decrease in
the association in the presence of the test compound compared to
the association in the absence of the test compound indicates that
the test compound disrupts the association of the CRY protein with
the BMAL-1 protein.
10. The method of claim 1, further comprising: contacting the test
compound with the first CRY protein in the presence of a second CRY
protein; and determining whether the test compound disrupts the
association of the first CRY protein with the second CRY protein,
wherein the second CRY protein has an amino acid sequence the same
as or different than the first CRY protein, and wherein a decrease
in the association in the presence of the test compound compared to
the association in the absence of the test compound indicates that
the test compound disrupts the association of the first CRY protein
and the second CRY protein.
11. The method of claim 10, wherein the first CRY protein is CRY1
or CRY2.
12. The method of claim 10, wherein the second CRY protein is CRY1
or CRY2.
13. The method of claim 1, further comprising: providing a cell
comprising a CRY protein, a CLOCK:BMAL-1 complex, and a DNA
comprising an E-box operatively linked to a reporter gene;
introducing the test compound into the cell; and assaying for
transcription of the reporter gene in the cell, wherein an increase
in transcription in the presence of the compound compared to
transcription in the absence of the compound indicates that the
compound blocks CRY-induced inhibition of CLOCK:BMAL-1-mediated
transcription in a cell.
14. The method of claim 13, wherein the cell is a NIH3T3 cell or a
clock neuron.
15. The method of claim 13, wherein the reporter gene encodes
luciferase.
16. A method for identifying a compound which disrupts the
association of a CRY protein and a PER protein, the method
comprising: contacting a test compound with the CRY protein in the
presence of the PER protein; and determining whether the test
compound disrupts the association of the CRY protein and the PER
protein, wherein a decrease in the association in the presence of
the test compound compared to the association in the absence of the
test compound indicates that the test compound disrupts the
association of the CRY protein and the PER protein.
17. The method of claim 16, wherein the CRY protein is a mouse CRY1
or CRY2.
18. The method of claim 16, wherein the PER protein is a mouse
PER1, PER2 or PER3.
19. A method for identifying a compound which disrupts the
association of a CRY protein and a TIM protein, the method
comprising: contacting a test compound with the CRY protein in the
presence of the TIM protein; and determining whether the test
compound disrupts the association of the CRY protein and the TIM
protein, wherein a decrease in the association in the presence of
the test compound compared to the association in the absence of the
test compound indicates that the test compound disrupts the
association of the CRY protein and the TIM protein.
20. The method of claim 19, wherein the CRY protein is a mouse CRY1
or CRY2.
21. The method of claim 19, wherein the TIM protein is a mouse
TIM.
22. A method of identifying a compound that disrupts the
association between a CRY protein and a CLOCK:BMAL-1 complex, the
method comprising: contacting a test compound with the CRY protein
in the presence of a CLOCK protein amd a BMAL-1 protein; and
determining whether the test compound disrupts the association of
the CRY protein with a complex of the CLOCK protein and the BMAL-1
protein, wherein a decrease in the association in the presence of
the test compound compared to the association in the absence of the
test compound indicates that the test compound disrupts the
association of the CRY protein and the CLOCK:BMAL-1 complex.
23. The method of claim 22, wherein the CRY protein is mouse CRY1
or CRY2.
24. The method of claim 22, wherein the CLOCK protein is mouse
CLOCK and the BMAL-1 protein is mouse BMAL-1.
25. A method for identifying a compound which disrupts the
association of a CRY protein and a BMAL-1 protein, the method
comprising: contacting a test compound with the CRY protein in the
presence of the BMAL-1 protein; and determining whether the test
compound disrupts the association of the CRY protein and the BMAL-1
protein, wherein a decrease in the association in the presence of
the test compound compared to the association in the absence of the
test compound indicates that the test compound disrupts the
association of the CRY protein and the BMAL-1 protein.
26. The method of claim 25, wherein the CRY protein is a mouse CRY1
or CRY2.
27. The method of claim 25, wherein the BMAL-1 protein is a mouse
BMAL-1.
28. A method for identifying a compound which disrupts the
association of a first CRY protein and a second CRY protein, the
method comprising: contacting a test compound with the first and
second CRY proteins; and determining whether the test compound
disrupts the association of the first CRY protein with the second
CRY protein, wherein the second CRY protein has an amino acid
sequence the same as or different than the first CRY protein, and
wherein a decrease in the association in the presence of the test
compound compared to the association in the absence of the test
compound indicates that the test compound disrupts the association
of the first CRY protein with the second CRY protein.
29. The method of claim 28, wherein the first CRY protein is a
mouse CRY1 or CRY2.
30. The method of claim 28, wherein the second CRY protein is a
mouse CRY1 or CRY2.
31. A method for identifying a compound that blocks CRY
induced-inhibition of CLOCK:BMAL-1 transcription in a cell, the
method comprising: providing a cell comprising a CRY protein, a
CLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively
linked to a reporter gene; introducing the compound into the cell;
and assaying for transcription of the reporter gene in the cell,
wherein an increase in transcription in the presence of the
compound compared to transcription in the absence of the compound
indicates that the compound blocks CRY-induced inhibition of
CLOCK:BMAL-1-mediated transcription in a cell.
32. The method of claim 31, wherein the cell is a NIH3T3 cell or a
clock neuron.
33. The method of claim 31, wherein the reporter gene encodes a
luciferase.
34. An isolated nucleic acid which encodes a mouse Tim protein.
35. The nucleic acid of claim 34, wherein the nucleic acid encodes
an amino acid sequence which has at least 70% sequence identity to
SEQ ID NO:2.
36. The nucleic acid of claim 34, wherein the nucleic acid encodes
the amino acid sequence of SEQ ID NO:2.
37. A vector comprising the nucleic acid of claim 34.
38. A cell comprising the nucleic acid of claim 34.
39. A substantially pure preparation of a mouse TIM.
40. A substantially pure antibody which specifically binds to mouse
CRY.
41. A substantially pure antibody which specifically binds to mouse
PER.
42. A substantially pure antibody raised against mouse TIM and
which specifically binds to mouse TIM.
43. A purified preparation of a mouse CRY:PER heterodimer.
44. A purified preparation of a CRY:TIM heterodimer.
45. A purified preparation of a mammalian CRY:CRY homodimer.
46. A method for identifying a compound that inhibits the
transcription of Period-2, the method comprising: providing a cell
comprising a Period-2 regulatory sequence operatively linked to a
reporter gene; introducing a test compound into the cell; and
assaying for transcription of the reporter gene in the cell,
wherein a decrease in transcription in the presence of the compound
compared to transcription in the absence of the compound indicates
that the compound inhibits Period-2 transcription in a cell.
47. The method of claim 46, wherein the cell is a NIH3T3 cell, a
Cos-7 cell or a clock neuron.
48. The method of claim 46, wherein the reporter gene encodes a
luciferase, a chloramphenicol acetyl transferase, a
beta-galactosidase, an alkaline phosphate, or a fluorescent
protein.
49. A method for identifying a compound that activates
transcription of a Period-2, the method comprising: providing a
cell comprising a Period-2 regulatory sequence operatively linked
to a reporter gene; introducing a test compound into the cell; and
assaying for transcription of the reporter gene in the cell,
wherein an increase in transcription in the presence of the
compound compared to transcription in the absence of the compound
indicates that the compound activates Period-2 transcription in the
cell.
50. The method of claim 49, wherein the cell is a NIH3T3 cell, a
Cos-7 cell or a clock neuron.
51. The method of claim 49, wherein the reporter gene encodes a
luciferase, a chloramphenicol acetyl transferase, a
beta-galactosidase, an alkaline phosphate, or a fluorescent
protein.
52. A method of modulating circadian-clock controlled rhythms in a
cell comprising introducing into a cell an expression vector
encoding a BMAL-1 protein such that an effective amount of the
BMAL-1 protein is produced in the cell, thereby modulating
circadian-clock controlled rhythms.
53. A method of modulating circadian-clock controlled rhythms in a
cell comprising introducing into the cell an effective amount of an
oligonucleotide antisense to BMAL-1, thereby inhibiting expression
of BMAL-1 in the cell and modulating circadian-clock rhythms.
54. A method of determining if a candidate compound positively
regulates the expression of BMAL-1, the method comprising:
providing a transgenic animal whose somatic and germ cells comprise
a disrupted Period 2 gene, the disruption being sufficient to
inhibit the ability of Period 2 to positively regulate BMAL-1;
administering a test compound to the mouse; and detecting BMAL-1
expression, wherein an increase in the expression of BMAL-1
indicates that the compound positively regulates expression of
BMAL-1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Serial No. 60/203,005 filed May 10, 2000, and U.S.
Provisional Application Serial No. 60/145,363, filed Jul. 22, 1999,
these applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The field of the invention relates to the regulation of
circadian rhythms.
BACKGROUND OF THE INVENTION
[0003] Circadian rhythms in mammals are regulated by a master clock
located in the suprachiasmatic nucleus (SCN) of the brain (Klein et
al., Suprachiasmatic nucleus: The Mind's Clock, Oxford University
Press, New York, 1991; Reppert and Weaver, Cell 89:487-490, 1997).
Environmental light-dark cycles entrain the SCN clock to the 24-hr
day via direct and indirect retinal projections. The timekeeping
capability of the SCN is expressed at the level of single neurons
(Welsh et al., Neuron 14:697-706, 1995).
[0004] The SCN clock mechanism is cell-autonomous, possibly based
on transcriptional and translational negative feedback loops
(Reppert, Neuron 21:1-4, 1998). Precedent for such a mechanism has
been described for circadian clocks in the fly Drosophila
melanogaster.
[0005] In the fly, autoregulatory transcriptional loops occur in
which protein products of clock genes periodically enter the
nucleus to suppress their own transcription. This feedback loop
involves dynamic regulation of the clock genes period (per) and
timeless (Tim). As the levels of PER and TIM rise, they are
phosphorylated, form heterodimers, and are then translocated to the
nucleus where they negatively regulate their own transcription
(Saez and Young, Neuron 17:1-920, 1996; Darlington et al., Science
280:1599-1603, 1998). Negative transcriptional regulation appears
to involve interference with drosophila CLOCK:drosophila dBMAL-1
(dCLOCK:dBMAL-1) and may be mediated by direct interaction of PER
and TIM with dCLOCK. dCLOCK and dBMAL-1 are positive factors which
drive Per and Tim transcriptional activation by binding to CACGTG
E-box enhancers in the promoters of Per and Tim (Allada et al.,
Cell 93:791-804, 1998; Rutila et al., Cell 93:805-814, 1998;
Darlington et al., supra; Hao et al., Mol. Cell Biol. 17:3687-3693,
1997). The temporal phosphorylation of PER provides at least part
of the time delay between transcription and PER-TIM negative
feedback necessary to sustain a 24-hr molecular oscillation in
drosophila (Price et al., Cell 94:83-95, 1998).
SUMMARY OF THE INVENTION
[0006] The invention is based, in part, on the discovery that the
core clockwork in the SCN is comprised of interacting feedback
loops. It was discovered that cryptochrome (CRY) proteins are
critical players in the negative limb of the mammalian clock
feedback loop and Period 2 (PER2) protein is a critical regulator
of the Bmal-1 loop. The CRY proteins and PER2 protein therefore
function as important modulators of mammalian circadian rhythm.
[0007] It was discovered that mammalian CRY proteins can
translocate from the cytoplasm to the nucleus of a cell and inhibit
CLOCK:BMAL-1 induced transcription. It was also discovered that CRY
proteins can homodimerize or heterodimerize with other circadian
proteins. The ability of CRY to heterodimerize with other proteins
provides a mechanism whereby CRY can modulate the activity of other
circadian proteins. For example, mouse CRY proteins can function as
dimeric and potentially trimeric partners for mouse PER proteins;
these interactions lead to the nuclear translocation of PER. Once
in the nucleus, PER can inhibit CLOCK:BMAL-1 induced transcription.
In addition, it was discovered that mouse CRY can form
heterodimeric complexes with mouse TIM. The interaction of TIM with
CRY may have a role in modulating the negative feedback of mouse
PER and/or mouse CRY rhythms. Thus, the compounds which can disrupt
the interaction of CRY with itself and other circadian proteins can
be used to reset the circadian clock.
[0008] In addition, it was discovered that PER2 positively
regulates transcription of the Bmal-1 gene. The ability of PER2 to
positively regulate the transcription of Bmal-1 indicates that PER2
controls the rhythmic regulation of Bmal-1. The availability of
BMAL-1 is critical for restarting the circadian clock loop. When
BMAL-1 is available, it heterodimerizes with CLOCK, thereby driving
the transcription of Per genes (e.g., in the mouse(m), mPER1-3) and
Cryptochrome genes (e.g., mouse mCry1 and mCry2). Compounds which
can disrupt the ability of PER2 to positively activate Bmal-1, or
compounds which can modulate transcription of Bmal-1, can be used
to reset the circadian clock.
[0009] Accordingly, the invention includes a method for identifying
a compound which binds to a mammalian CRY protein. The method,
which is useful as a quick initial screen for CRY agonists and
antagonists, includes contacting the CRY protein with a test
compound and determining whether the latter binds to the CRY
protein. Binding by the test compound to the CRY protein indicates
that the test compound is a CRY protein binding compound. For ease
of detection, the test compound can be labeled, e.g., radiolabeled.
The CRY protein can any mammalian CRY protein such as a CRY from a
mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep, pig,
non-human, primate, or human. In particular, the CRY protein is a
mouse CRY1 or CRY2 or human CRY1 or CRY2.
[0010] The method may further include contacting the test compound
with: a CRY protein in the presence of a PER protein; a CRY protein
in the presence of a TIM protein; a CRY protein in the presence of
a CLOCK:BMAL-1 complex; or a CRY protein in the presence of a
BMAL-1 protein; and determining whether the test compound disrupts
the association of the CRY protein with the PER, TIM, CLOCK:BMAL-1,
or BMAL-1 protein, as the case may be; wherein a decrease in the
association in the presence of the test compound compared to the
association in the absence of the test compound indicates that the
test compound disrupts the association of the CRY protein with the
indicated binding partner. The PER protein can any mammalian PER
protein such as mouse, rat, rabbit, goat, horse, cow, pig, dog, cat
or human. For example, the PER protein may be mouse or human PER1,
PER2 or PER3.
[0011] The method can further include contacting the test compound
with the first CRY protein in the presence of a second CRY protein
and determining whether the test compound disrupts the association
of the first CRY protein with the second CRY protein, wherein the
second CRY protein has an amino acid sequence the same as or
different than the first CRY protein, and wherein a decrease in the
association in the presence of the test compound compared to the
association in the absence of the test compound indicates that the
test compound disrupts the association of the first CRY protein and
the second CRY protein. The first and second CRY proteins can be
any mammalian CRY protein such as a CRY from a mouse, rat, rabbit,
goat, horse, cow, pig, dog, cat, sheep, non-human, primate or
human. For example, each CRY protein can be a mouse or human CRY1
or CRY2 and the second CRY protein is a mouse CRY1 or CRY2.
[0012] The method can further include providing a cell or cell-free
system which includes a CRY protein, a CLOCK:BMAL-1 complex, and a
DNA comprising an E-box operatively linked to a reporter gene. The
method includes introducing the test compound into the cell or
cell-free system and assaying for transcription of the reporter
gene, wherein an increase in transcription in the presence of the
compound compared to transcription in the absence of the compound
indicates that the compound blocks CRY-induced inhibition of
CLOCK:BMAL-1-mediated transcription in a cell. The cell can be any
cell type, such as a cultured mammalian cell, e.g., a NIH3T3 cell,
a COS7 cell, or a clock neuron. The reporter gene can be a gene
that encodes a detectable marker, e.g., luciferase.
[0013] The invention further includes a method for identifying a
compound which disrupts the association of a CRY protein and a
second protein or protein complex, which can be any of the
following: a PER protein, a TIM protein, a BMAL-1 protein, a second
CRY protein, or a CLOCK:BMAL-1 complex. The method includes
contacting a test compound with the CRY protein in the presence of
the second protein (or protein complex) and determining whether the
test compound disrupts the association of the CRY protein and the
second protein (or protein complex), wherein a decrease in the
association in the presence of the test compound compared to the
association in the absence of the test compound indicates that the
test compound disrupts the association of the CRY protein and the
second protein. The first and second CRY proteins can be any
mammalian CRY protein such as a CRY protein from a mouse, rat,
rabbit, goat, horse, cow, sheep, pig, dog, cat, non-human primate
or human, e.g., a mouse or human CRY1 or CRY2. The PER protein can
be any mammalian PER protein as described above, e.g., a mouse
PER1, PER2 or PER3. The TIM protein can be any mammalian TIM
protein as described above, e.g., a mouse or human TIM protein. The
CLOCK and the BMAL-1 proteins can be any mammalian CLOCK and BMAL-1
proteins as described above, particularly mouse or human.
[0014] Also within the invention is a method for identifying a
compound that blocks CRY-induced inhibition of CLOCK:BMAL-1
transcription in a cell. The method includes providing a cell
comprising a CRY protein, a CLOCK:BMAL-1 complex, and a DNA
comprising an E-box operatively linked to a reporter gene;
introducing the compound into the cell or a cell-free transcription
system; and assaying for transcription of the reporter gene,
wherein an increase in transcription in the presence of the
compound compared to transcription in the absence of the compound
indicates that the compound blocks CRY-induced inhibition of
CLOCK:BMAL-1-mediated transcription. The cell can be any cell type,
such as a cultured mammalian cell, e.g., a NIH3T3 cell, a COS7 cell
or a clock neuron. The reporter gene can be gene that encodes a
detectable marker, e.g., luciferase.
[0015] The invention further includes a method for identifying a
compound that activates or inhibits the transcription of Per2. The
method includes providing a cell including a mammalian Per2
regulatory sequence operatively linked to a reporter gene,
introducing a test compound into the cell, and assaying for
transcription of the reporter gene in the cell. A decrease in
transcription in the presence of the compound compared to
transcription in the absence of the compound indicates that the
compound inhibits Per2 transcription in a cell. Likewise, an
increase of transcription in the presence of the compound compared
to transcription in the absence of the compound indicates that the
compound inhibits Per2 transcription in a cell. The cell can be any
cell that can generate circadian rhythms, such as a NIH3T3 cell, a
Cos-7 cell or a clock neuron. The reporter gene can be any
detectable marker, e.g., a luciferase, a chloramphenicol acetyl
transferase, a betagalactosidase, an alkaline phosphate, or a
fluorescent protein such as green fluorescent protein. The Per2
regulatory sequence can be any mammalian Per2 regulatory sequence,
e.g., from a mouse, a rat, a rabbit, a goat, a horse, a cow, a pig,
a dog, a cat, a sheep, a non-human primate, or a human. In
particular, the Per2 regulatory sequence can be a mouse Per2
regulatory sequence (SEQ ID NO:3).
[0016] Also within the invention is a method of determining if a
candidate compound positively regulates the expression of Bmal-1.
The method includes providing a transgenic animal whose somatic and
germ cells comprise a disrupted Per2 gene, the disruption being
sufficient to inhibit the ability of Per2 to positively regulate
Bmal-1, administering a test compound to the mouse, and detecting
Bmal-1 expression, wherein an increase in the expression of Bmal-1
indicates that the compound can positively regulate expression of
Bmal-1.
[0017] The invention also features a method of modulating
circadian-clock controlled rhythms in a cell including comprising
introducing into a cell an expression vector encoding a BMAL-1
protein such that an effective amount of the BMAL-1 protein is
produced in the cell, thereby modulating circadian-clock controlled
rhythms. The BMAL-1 can be any mammalian BMAL-1, e.g., that of a
mouse, a rat, a rabbit, a goat, a horse, a cow, a dog, a cat, a
sheep, a non-human primate, or a human BMAL-1.
[0018] Also within the invention is a method of modulating
circadian-clock controlled rhythms in a cell comprising introducing
into the cell an effective amount of an oligonucleotide antisense
to a part, or all, of a mammalian Bmal-1, thereby inhibiting
expression of Bmal-1 in the cell and modulating circadian-clock
rhythms. Oligonucleotides can be antisense to any mammalian Bmal-1,
e.g., Bmal-1 from a mouse, a rat, a rabbit, a goat, a horse, a cow,
a sheep, a non-human primate, or a human.
[0019] The invention further includes isolated nucleic acid
molecules which are at least about 60% (or 65%, 75%, 85%, 95%, or
98%) identical to the nucleotide sequence of mouse TIMELESS (TIM)
(SEQ ID NO:1). The invention also features isolated nucleic acid
molecules which include a fragment of at least 100 (e.g., at least
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,
3000, 3500, or 3745) nucleotides of the nucleotide sequence of SEQ
ID NO:1, or a complement thereof. The invention also features
nucleic acid molecules which include a nucleotide sequence encoding
a protein having an amino acid sequence that is at least about 60%
(or 70%, 75%, 85%, 95%, or 98%) identical to the amino acid
sequence of SEQ ID NO:2. In a preferred embodiment, the isolated
nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1,
or a complement thereof.
[0020] Also within the invention is an isolated polypeptide having
an amino acid sequence that is at least about 60%, preferably 70%,
75%, 85%, 95%, or 98%, identical to the amino acid sequence of SEQ
ID NO:2. Also within the invention are isolated polypeptides
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under stringent hybridization conditions to the
complement of SEQ ID NO: 1.
[0021] The invention also features isolated nucleic acid molecules
which are at least about 60% (or 65%, 75%, 85%, 95%, or 98%)
identical to the mouse Per2 upstream sequence (SEQ ID NO:3)
containing a sequence controlling expression of mouse Per2. The
invention also features isolated nucleic acid molecules which
include a fragment of at least 100 (e.g., at least 200, 300, 400,
500, 600, 700, 800, 900, or 950) nucleotides of the nucleotide
sequence of SEQ ID NO:3, or a complement thereof.
[0022] The invention also includes nucleic acid molecules that
hybridize under stringent conditions to a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. The
nucleic acid molecules can be, for example, at least 20 (e.g. at
least about 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700,
800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 3745) nucleotides
in length.
[0023] Another aspect of the invention provides vectors, e.g.,
recombinant expression vectors, comprising a nucleic acid molecule
described herein. The vector or nucleic acid molecule can be
provided in a host cell. Such cells may be utilized for producing a
polypeptide of the invention by culturing the cells in a suitable
medium.
[0024] Also within the invention are a substantially pure
preparation of a mouse or human TIM, a mouse or human CRY:PER
heterodimer, a CRY:TIM heterodimer, and a mammalian CRY:CRY
homodimer.
[0025] Isolated antibodies, which specifically bind to mouse CRY,
mouse PER, mouse TIM, mouse BMAL-1 are also within the
invention.
[0026] As used herein, "isolated DNA" means either DNA with a
non-naturally occurring sequence or DNA free of the genes that
flank the DNA in the genome of the organism in which the DNA
naturally occurs. The term therefore includes a recombinant DNA
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote. It also includes a separate molecule such as a cDNA, a
genomic fragment, a fragment produced by polymerase chain reaction
(PCR), or a restriction fragment.
[0027] As used herein, an regulatory sequence which is "operably
linked" to a second sequence (or vise versa) means that both are
incorporated into a genetic construct so that the regulatory
sequence effectively controls expression of a second sequence.
[0028] As used herein, a "substantially pure" protein refers to a
protein which either (Klein et al., (1991). Suprachiasmatic
nucleus: The Mind's Clock, Oxford University Press, New York. has a
non-naturally occurring sequence (e.g., mutated, truncated,
chimeric, or completely artificial), or (D. R. Weaver, J. Biol.
Rhythms 13, 100 (1998) has a naturally occurring sequence but is
not accompanied by or at least partially separated from, components
that naturally accompany it. Typically, the protein is
substantially pure when it is at least 60% (by weight) free from
the proteins and other naturally-occurring organic molecules with
which it is naturally associated. Preferably, the purity of the
preparation is at least 75%, more preferably at least 90%, and most
preferably at least 99%, by weight. A substantially pure protein
can be obtained, for example, by extraction from a natural source,
by expression of a recombinant nucleic acid encoding the protein or
by chemical synthesis. Purity can be measured by any appropriate
method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis. A chemically synthesized protein
or a recombinant protein produced in a cell type other than the
cell type in which it naturally occurs is, by definition,
substantially free from components that naturally accompany it.
Accordingly, substantially pure proteins include those having
sequences derived from eukaryotic organisms but synthesized in E.
coli or other prokaryotes.
[0029] As used herein, the term "vector" refers to a replicable
nucleic acid construct. Examples of vectors include plasmids and
viral nucleic acids.
[0030] As used herein, a "circadian protein" refers to a protein
that participates in the circadian timing system and controls
circadian rhythm. Examples of circadian proteins include PER, TIM,
CLOCK, and BMAL-1.
[0031] As used herein, an antibody that "specifically binds" a
mouse or human CRY, PER or TIM, respectively, is an antibody that
binds only to mouse or human CRY, PER or TIM and does not bind to
(i) other molecules in a biological sample or (ii) CRY, PER or TIM
of another organism.
[0032] As used herein, a "therapeutically effective amount" is an
amount of the nucleic acid of the invention which is capable of
producing a medically desirable result in a treated animal.
[0033] As used herein, a "reporter gene" means a gene whose
expression can be assayed.
[0034] As used herein, the terms "heterologous DNA" or
"heterologous nucleic acid" is meant to include DNA that does not
occur naturally as part of the genome in which it is present, or
DNA which is found in a location or locations in the genome that
differs from that in which it occurs in nature, or occurs
extra-chromasomally, e.g., as part of a plasmid.
[0035] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. The
preferred methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention. All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. In case of conflict, the present document,
including definitions, will control. Unless otherwise indicated,
materials, methods, and examples described herein are illustrative
only and not intended to be limiting.
[0036] Various features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a histogram showing dose-response studies on
inhibition of CLOCK:BMAL-1-induced transcription by the mPER and
mTIM proteins.
[0038] FIGS. 2A-2B are line graphs showing mouse Cry1 and Cry2 mRNA
levels in SCN (FIG. 2A) and mouse Cry1 and Cry2 RNA levels in
skeletal muscle (FIG. 2B).
[0039] FIGS. 3A-D is a histogram showing inhibition of
CLOCK:BMAL1-mediated transcription from the vasopressin (AVP)
promoter (FIGS. 3A, 3C-D) or mPer1 promoter (FIG. 3B) by mPER1,
mCRY1 and mCRY2 (250 ng each).
[0040] FIG. 4 is a schematic representation of epitope-tagged mouse
CRY1 and CRY2 proteins evaluated for cellular location and
inhibition of Clock:Bmal-1 mediated transcription.
[0041] FIGS. 5A-D are histograms depicting the specificity of mouse
PER and mouse CRY in inhibiting transcription of Mop4:Bmal-1
mediated transcription.
[0042] FIG. 6 is a representation of the nucleotide sequence of
mouse TIM (SEQ. ID NO:1).
[0043] FIG. 7 is a representation of the amino acid sequence of
mouse TIM (SEQ. ID NO:2).
[0044] FIG. 8 is a representation of the nucleotide sequence of the
regulatory sequence of mouse Per2 (SEQ. ID NO:3).
[0045] FIG. 9 is a line graph depicting temporal profiles of Bmal1
RNA levels in the SCN of wild type (solid) and Clock/Clock (dashed)
mice. Each value is the mean.+-.SEM of 5-9 animals. Data at CT 2,
3, 22, and 24 are double-plotted. Gray bar, subjective day; black
bar, subjective night.
[0046] FIG. 10 is a line graph depicting CLOCK mRNA levels in the
SCN of wild-type (solid line) or Clock/Clock (dashed line) mice.
Each value is the mean.+-.SEM of 5-9 animals. Data at CT 2, 3, 22,
and 24 are double-plotted. Gray bar, subjective day; black bar,
subjective night.
[0047] FIG. 11 is a line graph depicting temporal profiles of Bmal1
RNA levels in the SCN of wild-type (solid line) and mPER2.sup.Brdm1
mutant (dashed line) mice. Each value is the mean.+-.SEM of 4
animals.
[0048] FIG. 12 is a line graph depicting temporal profiles of mCry1
RNA levels in the SCN of wildtype (solid line) and mPER2.sup.Brdm1
mutant (dashed line) mice are shown. Each value is the mean.+-.SEM
of 4 animals.
[0049] FIG. 13 is a schematic representation of different mPER2
constructs with a V5 epitope tagged at the carboxyl terminus of
mPER2. Also shown is the cellular location of immunofluorescence of
V5-tagged mPER2 constructs expressed in COS-7 cells either with (+)
or without (-) mCRY1. The cellular location of immunofluorscence
was scored as one of three categories: cytoplasm only (C), both
cytoplasm and nucleus (B), or nucleus only (N). Values shown are
the mean percentages from two experiments; all values were within
17% of the mean. Gray bars are PAS domain.
[0050] FIG. 14 is a histogram depicting attenuated peak levels of
Bmal1 RNA in mCry-deficient mice. Quantitation of Bmal1 RNA levels
in the SCN of wild-type (solid bars) and mCry-deficient (open bars)
mice. Values are the mean.+-.SEM of 5 animals. Mice were studied on
the first day in DD. * is the significance difference in Bmal1 RNA
levels between CT 6 and CT 18 in wild-type mice; P<0.0001.
[0051] FIG. 15 is a histogram depicting quantitation of Clock RNA
levels in the SCN of wildtype (solid bars) and mCry-deficient (open
bars) mice. Values are the mean.+-.SEM of 5 animals.
[0052] FIG. 16 is a histogram depicting the effects of mCRY
proteins on transcriptional activation in Drosophila S2 cells.
Values are luciferase activity expressed as relative to the
response in presence of activators (100%). Each value is the
mean.+-.SEM of three replicates from a single assay.
[0053] FIG. 17 is a histogram depicting the effects of mCRY
proteins on transcriptional activation in COS-7 cells. Presence (+)
or absence (-) of luciferase reporter (pGL3-Basic) (10 ng) and
expression plasmids (0.25 ug mClock, shBmal1, hMop4, dclock; 0.1 ug
mCry1, mCry2) is denoted. Values are luciferase activity expressed
as relative to the response in presence of activators (100%). Each
value is the mean.+-.SEM of three replicates from a single assay.
The results shown are representative of three independent
experiments.
[0054] FIG. 18 is a schematic drawing depicting a model of
circadian clockwork within an individual SCN neuron.
DETAILED DESCRIPTION
[0055] It has been discovered that members of the mouse PER family
(PER1, PER2, and PER3), the mouse CRY family (CRY1, and CRY2) and
mouse TIM can interact directly with each other. The ability of
these proteins to interact is critically involved in regulating
circadian rhythm. More specifically, PER, CRY and TIM control
circadian rhythm by inhibiting the transcriptional feedback loop
which is at the heart of the mammalian circadian clock.
[0056] It was also discovered that PER2 positively regulates the
transcription of Bmal-1, thereby controlling the rhythmic
regulation of Bmal-1. BMAL1-1 functions as a positive regulator in
the circadian loop. More specifically, BMAL-1 forms a heterodimeric
protein with CLOCK, which heterodimer in turn positively regulates
the expression of the circadian genes such as PER or CRY.
[0057] Based on the discovery made herein, the SCN clockwork is
predicted to include three types of interacting molecular loops
(FIG. 18). The Cry genes comprise one loop that has true
autoregulatory, negative feedback features, with the protein
products feeding back to turn off their transcription. The second
loop is that manifested by each of the Per genes and some
clock-controlled genes (CCGs) (for example, vasopressin
prepropressophysin). This loop type is driven by the same positive
elements (CLOCK(C):BMAL1(B)) as the CRY loop, but is not turned off
by the respective gene products. Instead, these loops use the CRY
proteins as negative regulators, leaving the generated protein
products free to transduce other actions. For example, PER2 is used
for the positive transcriptional regulation of the Bmal-1 gene. The
rhythmic regulation of Bmal-1 comprises the third loop, whose
rhythmicity is controlled by the cycling presence and absence of a
positive element dependent upon mPER2. This positive feedback loop
functions to augment the positive regulation of the first two
loops.
[0058] This model of interacting loops proposes that at the start
of the circadian day PER and CRY transcription are driven by
accumulating CLOCK:BMAL1 heterodimers acting through E box
enhancers. After a delay, the PER and CRY proteins are
synchronously expressed in the nucleus where the CRY proteins shut
off Clock:Bmal1-mediated transcription by directly interacting with
these transcription factors. At the same time that the CRY proteins
are inhibiting Clock:Bmal-1-mediated transcription, PER2 either
shuttles a transcriptional activator into the nucleus or
coactivates a transcriptional complex to enhance Bmal-1
transcription. The importance of the Bmal-1 RNA rhythm is to drive
a Bmal-1 rhythm after a 4 to 6 hour delay. This delay in the
protein rhythm would provide increasingly available CLOCK:BMAL1
heterodimers at the appropriate circadian time to drive Per and Cry
transcription, thereby restarting the cycle. It is thus predicted
that BMAL-1 availability is rate limiting for heterodimer formation
and critical for restarting the loops.
[0059] TIM Nucleic Acid Molecules
[0060] The invention pertains to isolated nucleic acid molecules
that encode mouse TIM proteins or biologically active portions
thereof, as well as nucleic acid molecules which can serve as
hybridization probes to identify TIM-encoding nucleic acids (e.g.,
TIM mRNA), or as PCR primers for the amplification or mutation of
TIM nucleic acid molecules. The nucleic acid encoding mouse TIM
(SEQ ID NO:1) (and/or the complement of that nucleic acid) can be
used as a probe to identify nucleic acids related to the mouse TIM
gene, e.g., other naturally occurring mammalian TIM DNA's.
[0061] Fragments of SEQ ID NO:1 and its complement can be used as
probes or primers, so long as they are at least 10, and preferably
at least 15 (e.g., at least 18, 20, 25, 50, 100, 150, or 200)
nucleotides in length. TIM probes and primers can be produced using
any of several standard methods (see, e.g., Ausubel et al., 1989,
Current Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., NY). For
example, the probe can be generated using PCR amplification methods
in which oligonucleotide primers are used to amplify a portion of
SEQ ID NO:1 that can be used as a specific probe. Such probes and
primers are part of the invention.
[0062] Hybridization under stringent conditions can be used to
identify nucleic acid sequences which encode mouse TIM or other
related TIMs, e.g., other mammalian TIM proteins. A related nucleic
acid sequence has at least 50% sequence identity to mouse TIM cDNA
(SEQ ID NO:1). Standard hybridization conditions (e.g., moderate or
highly stringent conditions) are known to those skilled in the art
and can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, hereby incorporated by
reference. Moderate hybridization conditions are defined as
equivalent to hybridization in 2.times.sodium chloride/sodium
citrate (SSC) at 30.degree. C., followed by one or more washes in
1.times.SSC, 0.1% SDS at 60.degree. C. Highly stringent conditions
are defined as equivalent to hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at 45.degree. C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0063] Nucleic acids which hybridize to the above-described probes
under stringent conditions can be used as probes themselves to
analyze the expression of mouse TIM mRNA in the SCN. These nucleic
acids can also be used to express mouse TIM polypeptides or
immunogenic fragments thereof for raising mouse TIM antibodies.
[0064] Genomic fragments of the TIM locus that are hybridizable to
the above-described probes are also included in the invention. Such
fragments are useful starting materials for generating, e.g.,
knockout constructs that are used to create non-human transgenic
mammals containing null mutations at the TIM locus.
[0065] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence of SEQ ID NO:1 due to
degeneracy of the genetic code, and thus encode the same TIM
protein as that encoded by the nucleotide sequence shown in SEQ ID
NO:1.
[0066] Mutations which change the nucleotide sequence of SEQ ID
NO:1 without altering the functional activity of the TIM protein
are also within the scope of the invention. For example, one can
make nucleotide substitutions leading to amino acid substitutions
at "non-essential" amino acid residues. A "non-essential" amino
acid residue is a residue that can be altered from the wild-type
sequence of mouse TIM (e.g., the sequence of SEQ ID NO:2) without
altering the biological activity, whereas an "essential" amino acid
residue is required for biological activity. For example, amino
acid residues that are conserved among the TIM proteins of various
species are predicted to be particularly unamenable to alteration.
These can be identified by sequence comparison among the known TIM
proteins (yeast, Drosphylia and now, mouse) Thus, the invention
encompasses nucleic acid molecules encoding mouse TIM proteins that
contain changes in amino acid residues that are not essential for
activity. Such TIM proteins differ in amino acid sequence from SEQ
ID NO:2, yet retain biological activity.
[0067] An isolated nucleic acid molecule encoding a TIM protein
having a sequence which differs from that of SEQ ID NO:2 can be
created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID NO:1
such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Generally, additions or
deletions of nucleotides will be done in multiples of three, so as
to avoid a frame shift.
[0068] TIM Polypeptides
[0069] A mouse TIM polypeptide can be isolated and purified from a
natural source. Alternatively, it can be produced recombinantly or
chemically synthesized by conventional methods. A TIM polypeptide,
full-length or truncated, can also be part of a fusion protein, for
example, by linking it to an antigenic determinant to facilitate
purification. The TIM polypeptides can be prepared for a variety of
uses, e.g., generation of antibodies which can be used to detect
TIM, and in screening assays which identify compounds that disrupt
the association of TIM with CRY.
[0070] Techniques for generating substantially pure polypeptide
preparations are well known in the art. A typical method involves
transfecting host cells (e.g., bacterial cells such as E. coli, or
mammalian cells such as COS7) with an expression vector carrying a
nucleic acid that encodes a mouse TIM protein. The recombinant
polypeptide so produced can be purified from the culture medium or
from lysates of the cells.
[0071] Conventional site-directed mutagenesis techniques can be
applied to a TIM coding sequence, e.g., SEQ ID NO:1, to generate
TIM sequence variants optimized for expression in a given type of
host cell.
[0072] Furthermore, one skilled in the art can prepare not only a
natural mouse TIM protein with a naturally occurring sequence (SEQ
ID NO:2), but also proteins with substantially the same function as
that of the natural protein, by replacing amino acids in the
protein. Methods for amino acid alteration include, for example, a
site-directed mutagenesis system using PCR (GIBCO-BRL,
Gaithersburg, Md.); the oligonucleotide-mediated site-directed
mutagenesis method (Kramer, Methods in Enzymol. 154:350-367 1997);
and the Kunkel method (Methods Enzymol. 85:2763-2766, 1988).
Usually ten or fewer, preferably six or fewer, and more preferably
three or fewer amino acids (e.g., one or two) are substituted.
Proteins functionally equivalent to the TIM protein can be produced
by conservative amino acid substitutions at one or more amino acid
residues. A "conservative amino acid substitution" is one in which
the amino acid residue is replaced with an amino acid residue
having a chemically similar side chain. Families of amino acid
residues having similar side chains have been defined in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0073] Biologically active portions of a mouse TIM protein include
peptides comprising amino acid sequences identical to or derived
from the amino acid sequence of the mouse TIM protein (e.g., the
amino acid sequence shown in SEQ ID NO:2).
[0074] A TIM protein that has a high sequence identity to SEQ ID
NO:2 is also included in the invention. A useful TIM protein has an
amino acid sequence at least 60% identical, preferably at least
70%, more preferably at least 80%, and even more preferably at
least 90, 95, 96, 97, 98 or 99% identical to the amino acid
sequence of SEQ ID NO:2, and retains the functional activity of the
TIM protein of SEQ ID NO:2.
[0075] To determine the percent sequence identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
homology between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length.
[0076] To determine percent homology between two sequences, the
algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA
87:2264-2268, 1990), modified as in Karlin and Altschul (Proc.
Natl. Acad. Sci. USA 90:5873-5877, 1993), is used. Such an
algorithm is incorporated into the NBLAST and XBLAST programs of
Altschul et al. (J. Mol. Biol. 215:403-410, 1990. BLAST nucleotide
searches are performed with the NBLAST program, score=100,
wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid molecules of the invention. BLAST protein searches are
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to mouse TIM protein. To obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402,
1997). When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) are
used. See http://www.ncbi.nlm.nih.gov.
[0077] Per2 Regulatory Sequence
[0078] The invention pertains to an isolated genomic nucleic acid
molecule that includes the mouse Per2 regulatory sequence
(promoter/enhancer sequence), as well as nucleic acid molecules
which can serve as hybridization probes to identify a Per2
regulatory sequence, or as PCR primers for the amplification or
mutation of a Per2 regulatory sequence.
[0079] Fragments of mouse Per2 regulatory sequence (SEQ ID NO:3)
and its complement can be used as probes or primers, so long as
they are at least 10, and preferably at least 15 (e.g., at least
18, 20, 25, 50, 100, 150, or 200) nucleotides in length. PER2
regulatory sequence probes and primers can be produced using any of
several standard methods described above. For example, the probe
can be generated using PCR amplification methods in which
oligonucleotide primers are used to amplify a portion of SEQ ID
NO:3 that can be used as a specific probe.
[0080] Other uses for the Per2 regulatory sequence include use as a
starting material for generating, e.g., knockout constructs that
are used to create non-human transgenic marnmals that contain a
disruption in the Per2 regulatory sequence and that are unable to
express Per2. Alternatively, the Per2 regulatory sequence may be
operably linked to a DNA sequence encoding a polypeptide that is
not PER2 (i.e., a heterologous polypeptide).
[0081] Hybridization under stringent conditions can be used to
identify nucleic acid sequences that contain a regulatory sequence
of mouse PER2, or other related PER2 regulatory seqeuences. A
related nucleic acid sequence has at least 50% sequence identity to
mouse PER2 regulatory sequence (SEQ ID NO:3). Standard
hybridization conditions are described above.
[0082] Circadian Proteins
[0083] The invention includes screening methods which are used to
identify compounds which can disrupt the association of mammalian
circadian proteins, e.g., the association of TIM with CRY, CRY with
CRY, CRY with PER, CRY with BMAL-1, and CRY with CLOCK:BMAL-1. The
invention also features antibodies generated against CRY, PER, and
TIM proteins. These various uses require a source of CRY, TIM, PER,
CLOCK, BMAL-1, and CLOCK:BMAL-1.
[0084] Circadian proteins can be isolated and purified from a
natural source. Alternatively, the proteins can be produced
recombinantly or chemically synthesized by conventional methods.
Typically the proteins will be produced recombinantly. The
nucleotide and amino acid sequences of the circadian proteins are
publicly available to one skilled in the art, e.g., mouse CRY1
(Genbank accession # AB000777), mouse CRY2 (Genbank Accession #
AB003433), mouse TIM (Genbank accession # AF071506), mouse PER3
(Genbank accession # AF050182), CLOCK (Genbank accesssion #
AF000998) and BMAL-1 (Genbank accession # AB015203).
[0085] Methods of generating a recombinant circadian protein or a
recombinant circadian fusion protein, e.g., CLOCK:GST, are well
known in the art. For example, the circadian proteins can be
generated by cloning the nucleic acid sequence encoding a circadian
protein into an expression vector, where it is operably linked to
one or more regulatory sequences. The need for, and identity of,
regulatory sequences will vary according to the type of cell in
which the circadian protein sequence is to be expressed. Examples
of regulatory sequences include transcriptional promoters,
enhancers, suitable mRNA ribosomal binding sites, and sequences
that terminate transcription and translation. Suitable regulatory
sequences can be selected by one of ordinary skill in the art.
Standard methods can be used by the skilled person to construct
expression vectors. See, generally, Sambrook et al., 1989,
Cloning--A Laboratory Manual (2nd Edition), Cold Spring Harbor
Press.
[0086] Vectors useful in this invention include plasmid vectors and
viral vectors. Viral vectors can be those derived from, for
example, retroviruses, adenovirus, adeno-associated virus, SV40
virus, pox viruses, or herpes viruses. Once introduced into a host
cell (e.g., bacterial cell, yeast cell, insect cell, or mammalian
cell), the vector can remain episomal, or be incorporated into the
genome of the host cell.
[0087] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
gene product being expressed. For example, when a large quantity of
such a protein is to be produced, e.g., for studying the
interaction of a CRY protein with other proteins or for raising
antibodies to the protein, a vector capable of directing the
expression of high levels of a fusion protein (e.g., a GST fusion
protein) that is readily purified may be desirable. Alternatively,
in mammalian host cells, a number of viral-based expression systems
can be utilized.
[0088] Construction of GST Fusion Proteins
[0089] In certain screening assays (see below) it may be desirable
to immobilize the circadian protein. One method of immobilizing a
circadian protein is to express the protein as a fusion protein
with GST. To do this a chimeric gene encoding a GST fusion protein
can be constructed by fusing DNA encoding a circadian protein to
the DNA encoding the carboxyl terminus of GST (see e.g., Smith et
al., Gene 67:31, 1988). The fusion construct can be transformed
into a suitable expression system, e.g., E. coli XA90 in which
expression of the GST fusion protein can be induced with
isopropyl-.beta.-D-thiogalactopyranoside (IPTG).
[0090] Purification of GST Fusion Proteins
[0091] After transformation of the construct into a suitable
expression system, induction with IPTG should yield the fusion
protein as a major constituent of soluble, cellular proteins. The
fusion proteins can be purified by methods known to those skilled
in the art, including purification by glutathione affinity
chromatography. The purity of the product can be assayed by methods
known to those skilled in the art, e.g., gel electrophoresis.
[0092] Binding of Circadian Proteins to Immobilized GST
[0093] GST fusion proteins can be complexed to glutathione which is
attached to a matrix material, e.g., glutathione Sepharose, by
methods known to those skilled in the art.
[0094] Antibodies
[0095] Antibodies which specifically bind to mouse or human CRY,
mouse or human TIM, or mouse or human PER, or mouse or human BMAL-1
are also included in the invention. An antibody that specifically
binds a mouse or human CRY, PER, TIM, or BMAL-1 is an antibody that
binds only to mouse or human CRY, PER, TIM or BMAL-1 and does not
bind to (i) other molecules in a biological sample or (ii) CRY,
PER, TIM or BMAL-1 of another organism (e.g., Drosophila or
yeast).
[0096] Antibodies against mouse or human CRY, PER, TIM or BMAL-1
can be used, for example, to inhibit the interaction between these
circadian proteins. Anti-CRY, -TIM or PER antibodies (e.g.,
monoclonal antibodies) can also be used to isolate a CRY, TIM or
-PER protein using techniques well known in the art, such as
affinity chromatography or immunoprecipitation. The antibodies are
also useful in the screening assays described below. Compounds
bound to the immunopreceipitated protein can then be
identified.
[0097] Antibodies specific for mouse CRY, TIM, PER or BMAL-1 can be
raised by immunizing a suitable subject (e.g., rabbit, goat, mouse
or other mammal) with an immunogenic preparation which contains the
mouse or human CRY, TIM, PER or BMAL-1 protein. An appropriate
immunogenic preparation can contain, for example, a recombinantly
expressed or chemically synthesized CRY, or an immunogenic fragment
thereof. The preparation can further include an adjuvant, such as
Freund's complete or incomplete adjuvant, or similar
immunostimulatory agent. Immunization of a suitable subject with an
immunogenic CRY, TIM, PER or BMAL-1 preparation induces a
polyclonal anti-CRY, TIM, PER or BMAL-1 antibody response.
[0098] The term antibody refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules.
Examples of immunologically active portions of immunoglobulin
molecules include F(ab) and F(ab').sub.2 fragments, which can be
generated by treating the antibody with an enzyme such as pepsin.
The term monoclonal antibody or monoclonal antibody composition
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of the polypeptide. A monoclonal antibody
composition thus typically displays a single binding affinity for
the CRY, TIM or PER with which it immunoreacts.
[0099] Polyclonal anti-CRY, -TIM or -PER antibodies can be prepared
by immunizing a suitable subject with a mouse CRY, TIM or PER
immunogen. The anti-CRY, -TIM or -PER antibody titer in the
immunized subject can be monitored over time by well known
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized polypeptide. If desired, the antibody
molecules directed against CRY, TIM, PER or BMAL-1 can be isolated
from the mammal (e.g., from the blood) and further purified by
well-known techniques, such as protein A chromatography, to obtain
the IgG fraction.
[0100] Monoclonal antibodies can be generated by immunizing a
subject with an immunogenic preparation containing a CRY, TIM, PER
or BMAL-1. At an appropriate time after immunization, e.g., when
the anti-CRY, -TIM, -PER or BMAL-1 antibody titers are highest,
antibody-producing cells are obtained from the subject and used to
prepare monoclonal antibodies by techniques well known in the art,
such as the hybridoma technique originally described by Kohler et
al., Nature 256:495-497, 1975, the human B cell hybridoma technique
(Kozbor et al., Immunol Today 4:72, 1983), the EBV-hybridoma
technique (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology
for producing monoclonal antibody hybridomas is well known (see
generally Current Protocols in Immunology (1994) Coligan et al.
(eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, an
immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with a CRY, TIM,
PER or BMAL-1, immunogen as described above, and the culture
supernatant of the resulting hybridoma cells that screened to
identify a hybridoma producing a monoclonal antibody that binds the
CRY, TIM, PER or BMAL-1.
[0101] The anti-CRY, -TIM, -PER or BMAL-1 antibody may be coupled
to a detectable substance. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, and
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a luminescent material is luminol;
examples of bioluminescent materials include luciferase, luciferin,
and aequorin; and examples of suitable radioactive materials
include .sup.125I, .sup.131I, .sup.35S and .sup.3H.
[0102] Screening Assays
[0103] The invention encompasses methods for identifying compounds
that bind to CRY; disrupt the association of TIM:CRY, CRY:CRY,
CRY:PER, CRY:BMAL-1, and CRY:CLOCK:BMAL-1; inhibit or activate the
transcription of Per2; or positively regulate the transcription of
Bmal-1. Candidate compounds that can be screened in accordance with
the invention include polypeptides, oligopeptides, antibodies, and
monomeric organic compounds, i.e., "small molecules."
[0104] Identification of a Compound that Binds to CRY
[0105] A useful first step to identifying a compound which disrupts
the association between different circadian proteins (e.g.,
TIM:CRY, CRY:CRY, CRY:PER, CRY:BMAL-1,and CRY:CLOCK:BMAL-1) is to
identify a compound that binds to CRY or another circadian protein.
Once a circadian binding compound is identified, the ability of the
compound to disrupt the association of different circadian proteins
can be assayed. Below are a number of assays which can be used to
identify a compound which binds to a CRY protein, e.g., CRY1 or
CRY2. The examples are not meant to be limiting and the assays can
be performed with other circadian proteins, e.g., TIM, PER, CLOCK
and BMAL-1.
[0106] Methods of identifying a compound which binds a protein of
interest are well known in the art. In one screening method, test
compounds are evaluated for their ability to bind CRY, e.g., CRY1
or CRY2. Control reactions which do not contain the compound can be
performed in parallel. The method includes immobilizing CRY using
methods known in the art such as binding a GST-CRY to a polymeric
bead containing glutathione or binding a CRY protein to an anti-CRY
antibody which is attached to a solid support. The immobilized CRY
is incubated with a test compound for a period of time that permits
binding of the test compound to CRY. Following the incubation
period, unbound test compound is removed and bound test compound
detected. For example, a detectable moiety such as a radionuclide
or a fluorescent label can be attached to the compound for ease of
detection. Examples of radionuclide and fluorescent labels include
.sup.125I, .sup.131I, .sup.35S, .sup.3H, umbelliferone,
fluorescein, fluorescein isothiocyanate, and rhodamine.
[0107] Alternatively, the screening method can involve incubating a
labeled test compound, with an epitope-tagged CRY protein.
Following incubation, the ability of the test compound to bind to
the CRY protein is determined using immunoprecipitation with an
antibody directed against the epitope tag (e.g., Flag or myc). The
recovery of a labeled test compound, e.g., a radioactive compound,
following immunoprecipitation indicates that the test compound
binds to the CRY protein.
[0108] Display libraries can also be used to identify compounds
which bind to a CRY protein. In this approach, the test peptides
are displayed on the surface of a cell or viral particle, and the
ability of particular cells or viral particles to bind an
appropriate CRY protein, e.g., CRY1 or CRY2, via the displayed
product can be detected in a "panning assay" (Ladner et al., WO
88/06630).
[0109] Identifying Compounds which Disrupt the Interaction of
CRY:TIM, CRY:CRY CRY:PER, CRY:BMAL-1, and CRY:CLOCK:BMAL-1
[0110] The two-hybrid expression system can be used to screen for
compounds capable of disrupting CRY:TIM, CRY:CRY, CRY:PER,
CRY:BMAL-1, or CRY:CLOCK:BMAL-1 associations in vivo. In this
system, a GAL4 binding site, linked to a reporter gene such as
lacZ, is contacted in the presence and absence of a test compound
with a GAL4 binding domain linked to a circadian protein, e.g.,
CRY, TIM, PER, CLOCK or BMAL-1 and a GAL4 transactivation domain
linked to a circadian protein, e.g., CRY, TIM, PER, CLOCK, or
BMAL-1. Expression of the reporter gene is monitored and a decrease
in said expression is an indication that the test compound inhibits
the interaction of CRY with TIM, CRY with CRY, CRY with PER, CRY
with BMAL-1, or CRY with CLOCK:BMAL-1.
[0111] Another method of identifying compounds which disrupt an
association between circadian proteins involves the determination
of whether the test compounds can disrupt the ability of, e.g.,
CRY:PER, to block CLOCK:BMAL-1-mediated transcriptional activation.
In this system, an E-box sequence linked to a reporter gene such as
a luciferase gene is contacted with a CLOCK:BMAL-1 heterodimer.
Binding of the CLOCK:BAML-1 heterodimer to the E-box results in
expression of the reporter gene. The system is then contacted with
a test compound and a circadian protein (e.g., a CRY protein or a
circadian protein complex, e.g., CRY:PER), and expression of the
reporter gene is monitored. Since CRY and PER block
CLOCK:BMAL-1-mediated transcription, an increase in expression of
the reporter gene in the presence of the test compound as compared
to the expression in the absence of the compound indicates that the
compound disrupts the ability of CRY and PER to block
CLOCK:BMAL-1-mediated transcription. The transcription assay can be
preformed in any cell that expresses the necessary proteins, either
naturally or recombinantly, e.g., NIH 3T3 cells, COS-7 cells, or
clock neuron cells.
[0112] In yet another screening method, one of the components of
the CRY:TIM, CRY:CRY, CRY:PER, CRY:BMAL-1, or CRY:CLOCK:BMAL-1
binding complex is immobilized. The circadian protein can be
immobilized using methods known in the art, such as adsorption onto
a plastic microtiter plate or specific binding of a GST-fusion
protein to a polymeric bead containing glutathione. For example, to
determine a compound which binds CRY:PER, a GST-CRY can be bound to
glutathione-Sepharose beads. The immobilized CRY is then contacted
with a labeled circadian protein to which it binds (PER in this
case) in the presence and absence of a test compound. Unbound PER
can then be removed and the complex solubilized and analyzed to
determine the amount of bound labeled PER. A decrease in binding is
an indication that the test compound inhibits the interaction of
CRY with PER.
[0113] A variation of the above-described screening method involves
screening for test compounds which are capable of disrupting a
previously-formed CRY:TIM, CRY:CRY, CRY:PER, CRY:BMAL-1, or
CRY:CLOCK:BMAL-1 interaction. For example, a complex comprising
CRY:PER is immobilized as described above and contacted with a test
compound. The disassociation of the complex by the test compound
correlates with the ability of the test compound to disrupt or
inhibit the interaction of CRY with PER.
[0114] Identifying Compounds that Activate Transcription of
PER2
[0115] A screening method used to identify a compound that
activates or inhibits the transcription of Per2 includes providing
a cell that includes a Per2 regulatory sequence operatively linked
to a reporter gene. The Per2 regulatory sequence is preferably
mammalian, e.g., mouse PER2 (SEQ ID NO:3; see FIG. 8). In one
example, the mouse Per2 regulatory sequence is operably linked to a
reporter gene such as a luciferase, a chloramphenicol acetyl
transferase, a beta-galactosidase, an alkaline phosphate, or a
fluorescent protein gene. A test compound is then contacted with
the cell and expression of the reporter gene monitored. An increase
in expression of the reporter gene in the presence of the test
compound as compared to the expression in the absence of the
compound indicates that the compound activates Per2 transcription.
Alternatively, a decrease in expression of the reporter gene in the
presence of the test compound as compared to expression in the
absence of the test compound indicates that the compound inhibits
Per2 transcription. The transcription assay can be preformed in any
cell which undergoes a circadian rhythm, e.g., NIH 3T3 cells, COS-7
cells, or clock neuron cells.
[0116] Identifying Compounds that Positively Regulate Expression of
BMAL-1
[0117] A screening method that uses a non-human transgenic animal
whose somatic and germ cells comprise a disrupted Per2 gene can be
used to identify a compound that regulates expression of Bmal-1.
The method includes administering a test compound to the transgenic
mouse and detecting Bmal-1 expression. An increase in expression of
Bmal-1, compared to a control non-human transgenic animal,
indicates that the compound positively regulates expression of
Bmal-1. Expression of Bmal-1 can be detected using any appropriate
method, e.g., detecting Bmal-1 mRNA levels using Northern blot
analysis or BMAL-1 protein levels using a BMAL-1 specific antibody
or an activity assay.
[0118] The transgenic non-human animal used in the method described
above includes a non-human animal that contains a disruption in the
Per2 gene that is sufficient to inhibit the ability of PER2 to
positively regulate Bmal-1. A transgenic non-human animal is
preferably a mammal such as a rat or mouse, in which one or more of
the cells of the animal include a disruption in the Per2 gene.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, amphibians, and the like. The
transgenic non-human animal is one in which the Per2 gene has been
altered, e.g., by homologous recombination between the endogenous
gene and an exogenous DNA molecule introduced into a cell of the
animal, e.g., an embryonic cell of the animal, prior to development
of the animal. Appropriate PER2 transgenic animals which can be
used in the method described above are known in the art, e.g., the
homozygous mPer2.sup.brdm1 described by Zheng et al. (Nature,
400:1667 (1999)) the contents of which are incorporated herein by
reference.
[0119] Modulating the Circadian Clock
[0120] Based on the discoveries described herein, it is apparent
that expression of Bmal-1 is critical for restarting the circadian
loop. The importance of Bmal-1 mRNA rhythm is to drive a Bmal-1
rhythm after a four to six hour delay in the circadian loop. The
expression of Bmal-1 makes BMAL-1 available to heterodimerize with
CLOCK to drive transcription of circadian proteins, such as Per or
Cry. The transcription of Per or Cry restarts the cycle. Therefore,
a method of modulating a circadian-clock controlled rhythm
includes, for example, altering the endogenous expression of
Bmal-1. In one example, an effective amount of a ribozyme, or an
oligouncleotide antisense to Bmal-1, can be introduced into a SCN
in vivo, thereby inhibiting expression of Bmal-1 in the cell and
modulating circadian-clock rhythms.
[0121] Antisense Bmal-1 nucleic acid molecules include molecules
which are complementary to a sense nucleic acid encoding a BMAL-1
protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to a mRNA sequence.
Accordingly, an antisense nucleic acid can hydrogen bond to a sense
nucleic acid. Antisense Bmal-1 nucleic acids can be designed
according to the rules of Watson and Crick base pairing. The
antisense nucleic acid molecule can be complementary to full length
Bmal-1 mRNA, but more preferably is an oligonucleotide that is
antisense to only a portion of the Bmal-1 mRNA, e.g., part or all
of the transcription start site, and/or part or all of the coding
region. An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
[0122] The Bmal-1 antisense nucleic acid molecules are typically
administered to a subject such that they hybridize with or bind to
cellular mRNA and/or genomic DNA encoding a protein to thereby
inhibit Bmal-1 expression of the protein. An example of a route of
administration of antisense nucleic acid molecules of the invention
includes direct injection at a tissue site. Alternatively,
antisense nucleic acid molecules can be modified to target selected
cells and then administered systemically. For example, for systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies which bind to clock neuron cell surface
receptors or antigens. In another example, the antisense nucleic
acid molecule is linked to TAT, a HIV leader sequence, that can
target the antisense to the SCN (Lisziewicz et al., Hum Gene Ther
11:807-15, 2000).
[0123] Alternatively, an expression vector encoding BMAL-1 protein
can be introduced into a clock neuron using gene therapy methods.
For example, methods of targeting a vector containing a Bmal-1
sequence into an SCN include using a gene therapy vector which
includes a tat sequence operably lined to a Bmal-1 nucleic acid
sequence. Expression of TAT targets the vector to the SCN.
[0124] The gene therapy expression vector can be in the form of a
recombinant plasmid, phagemid or attenuated virus in which a
mammalian BMAL-1 is operably linked to an appropriate regulatory
sequence. Examples of suitable viral vectors include recombinant
retroviral vectors (Valerio et al., 1989, Gene, 84:419; Scharfman
et al., 1991, Proc. Natl. Acad. Sci., USA, 88:462; Miller, D. G.
& Buttimore, C., 1986, Mol. Cell. Biol., 6:2895), recombinant
adenoviral vectors (Freidman et al., 1986, Mol. Cell. Biol.,
6:3791; Levrero et al., 1991, Gene, 101:195), and recombinant
Herpes simplex viral vectors. The regulatory sequence can be the
same as the endogenous regulatory sequence, or different. It can be
inducible or constitutive. Suitable constitutive regulatory
sequences include the regulatory sequence of a housekeeping gene
such as the .alpha.-actin regulatory sequence, or may be of viral
origin such as regulatory sequences derived from mouse mammary
tumor virus (MMTV) or cytomegalovirus (CMV).
[0125] Utility of the Compounds
[0126] Compounds found to disrupt the interaction of CRY:TIM,
CRY:CRY, CRY:PER, CRY:BMAL-1, or CRY:CLOCK:BMAL-1 or bind to CRY
can be used to manipulate the circadian clock. For example, the
association of PER with CRY in the cytoplasm of a clock neuron is
necessary for the translocation of PER into the nucleus of the
cell. Once PER is in the nucleus, PER has a negative feedback
effect on the circadian loop, i.e., inhibits CLOCK:BMAL-1-mediated
transcription. A compound which disrupts the ability of CRY and PER
to associate in the cytoplasm would prevent the translocation of
PER to the nucleus and would therefore be useful for blocking PER's
negative feedback effect on the circadian loop. Similarly, a
compound that binds to CRY is potentially useful for blocking CRY's
negative feedback effect on the circadian loop.
[0127] Compounds that can modulate the transcription of the Per2
gene can be used to advance or delay restarting the circadian loop.
For example, a compound that inhibits transcription of Per2 will
inhibit the transcription of Bmal-1. Since BMAL-1 is needed to
restart the circadian loop, a compound that inhibits transcription
of Per2 will inhibit the restarting of the circadian loop.
Moreover, delivery of an expression vector encoding a mammalian
Bmal-1 protein to a clock neuron can also be used to manipulate the
circadian rhythm and advance restarting of the circadian loop.
[0128] A compound identified as described above is therefore useful
as an agent that can reset the circadian clock. The compound can be
used to prevent jet lag or facilitate resetting the clock in shift
workers. In addition, the compound can be used to improve
rhythmicity, i.e., the co-ordinated regulation of outputs from
cells within the SCN. Disruption of rhythmicity is common in the
elderly and affects the ability to sleep. The compound described
herein can be used to improve the interactions between neurons to
allow them to arrive at a common phase or directly reset individual
neurons to a common phase. Compounds can also be used to alleviate
circadian rhythm disorders such as winter depression or seasonal
affective disorder.
[0129] Administration
[0130] The compounds described herein can be administered to a
subject, e.g., a mammal such as a human, to treat a circadian
rhythm disorder, e.g., jet lag, winter depression and shift work
disturbance. The compounds can be used to specifically advance or
delay the phase of certain circadian rhythms. The ability of a
compound to reset the clock to a specified phase will depend on the
nature of the agent and its biological half-life.
[0131] The compound can be administered alone, or in a mixture, in
the presence of a pharmaceutically acceptable excipient or carrier
(e.g., physiological saline). Given that the different CRY and PER
proteins are redundant, it is preferable that the compound
administered have the specificity to affect all members of a given
family, e.g., CRY1 and CRY2 (CRY protein family members) or PER1,
PER2 or PER3 (PER protein family members). Alternatively, a
combination of compounds specific for each member of a family can
be administered.
[0132] Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection or local administration (see U.S.
Pat. No. 5,328,470). The pharmaceutical preparation of the gene
therapy vector will typically include the gene therapy vector in an
acceptable carrier.
[0133] The excipient or carrier is selected on the basis of the
mode and route of administration. Suitable pharmaceutical carriers,
as well as pharmaceutical necessities for use in pharmaceutical
formulations, are described in Remington's Pharmaceutical Sciences
(E. W. Martin), a well known reference text in this field, and in
the USP/NF (United States Pharmacopeia and the National
Formularly). A pharmaceutical composition is formulated to be
compatible with its intended route of administration. Examples of
routes of administration include oral, rectal, and parenteral,
e.g., intravenous, intradermal, and subcutaneous, transdermal
(topical), and transmucosal, administration. Compounds which are
unable to cross the blood-brain barrier are administered locally to
the SCN.
[0134] As is well known in the medical arts, dosage for any given
patient depends upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and other
drugs being administered concurrently. Dosages for the compounds of
the invention will vary, but determination of optimal dosage is
well within the abilities of a pharmacologist of ordinary
skill.
[0135] Transgenic Animals
[0136] Based on the discovery made herein, Tim is predicted to be
essential for embryonic development in animals. In order to
delineate the region(s) of Tim essential for development, the
invention includes non-human transgenic animals that have a
selected region of Tim disrupted. The role of this region in
embryonic development can be determined by analyzing homozygous
embryos for developmental defects, e.g., determining cellular
organization in whole embryos that are fixed and embedded in
paraffin around embryonic day 7.5.
[0137] Transgenic non-human animals that have a Tim disruption are
also useful for screening for compounds that ameliorate the
developmental defects caused by the disruption of Tim, e.g., a test
compound can be administered to a female Tim.sup.+/Tim.sup.-
heterozygote non-human animal during and/or subsequent to mating
with a male Tim.sup.+/Tim.sup.-0 heterozygote of the same species.
The ability of the test compound to ameliorate Tim-associated
defects occurring during embryonic development can be determined by
analyzing Tim.sup.- homozygous embryos for developmental defects,
e.g., determining cellular organization in whole embryos that are
fixed and embedded in paraffin around embryonic day 7.5.
[0138] Transgenic Tim animals which overexpress TIM are also be
useful for studying the function and/or activity of a TIM protein
in circadian rhythm. For example, transgenic non-human animals are
generated where an endogenous Tim regulatory element, e.g., a
promoter, is replaced with an exogenous regulatory element such
that the exogenous regulatory element drives a higher level of
expression of TIM in a cell of the transgenic animal as compared to
a non-transgenic animal. The cell is preferably a neuron. The role
of TIM in circadian rhythm in the transgenic animal can be
determined by analyzing circadian rhythms in locomoter activity,
e.g., rhythmic wheel turning.
[0139] As used herein, a "transgenic animal" is a non-human animal,
the nucleated cells of which include a transgene. The animal is
preferably a mammal, e.g., a rodent such as a rat or mouse. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, rabbits, amphibians, and the like. A
transgene is exogenous DNA or a rearrangment, e.g., a deletion of
endogenous chromosomal DNA, which is integrated into or occurs in
the genome of the animal's cells. A transgene can direct the
expression of an encoded gene product in one or more cell types or
tissues of the transgenic animal. Other transgenes, e.g., a
knockout, reduce expression. Thus, a transgenic animal can be one
in which an endogenous Tim gene has been altered, e.g., by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal. The animal can be heterozygous or homozygous for the
transgene.
[0140] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a TIM protein in particular cells. A transgenic
founder animal can be identified based upon the presence of a TIM
transgene in its genome and/or expression of TIM mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a TIM protein can
further be bred to other transgenic animals carrying other
transgenes.
[0141] TIM proteins or polypeptides can be expressed in transgenic
animals or plants, e.g., a nucleic acid encoding the protein or
polypeptide can be introduced into the genome of an animal. In
preferred embodiments, the nucleic acid is placed under the control
of a tissue specific promoter, e.g., a milk or egg specific
promoter, and recovered from the milk or eggs produced by the
animal. Suitable animals are mice, pigs, cows, goats, sheep, and
chickens.
[0142] The invention also includes a population of cells from a
transgenic animal, as discussed herein.
[0143] Any technique known in the art may be used to generate the
transgene non-human animals discussed herein. For a review, see
Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229 and
Hogan et al. "Manipulating the Mouse Embryo" (Cold Spring Harbor
Press, Cold Spring Harbor, N.Y., 1986.
[0144] Experimental Information
EXAMPLE 1
[0145] mPER Proteins Interact in Mammalian Cells
[0146] The importance of mPER:mPER interactions in the negative
limb of the clock feedback loop was examined. Previous studies
using the yeast two-hybrid assay showed that all of the mPERs
interact with one another and that mPER1 and mPER2 can homodimerize
(Zylka et al., Neuron 21:1103-1115, 1998). No interactions were
detectable between mTIM and any of the mPER proteins in the yeast
system. Co-immunoprecipitation experiments were performed in
mammalian cells using epitope-tagged proteins expressed in COS7
cells.
[0147] Expression plasmids were constructed that contain
full-length coding regions for each mPER protein and mTIM with
either a hemaglutinin (HA) or a V5 epitope tag at the carboxyl
terminus. For cloning, the coding regions of mPER2 (AF035830),
mPER3 (AF050182), and mTIM (AF071506) were ligated into pcDNA 3.1
containing either an N terminal or C terminal HA tag. Full-length
coding regions were amplified with Pfu TurboJ (Stratagene, La
Jolla, Calif.) from plasmid DNA (mPER1). Correct orientation of
each construct was verified by sequence analysis. Clones were also
transcribed and translated in vitro using TnT T7 QuickJ (Promega,
Madison, Wis.) to confirm that a protein of the correct size was
produced. Moreover, clones were transiently transfected into NIH3T3
cells and into COS7 cells. Crude cell extracts were prepared,
western blotted and probed with anti-V5 or anti-HA antibodies to
detect full-length, epitope-tagged proteins.
[0148] Once the constructs were generated, COS7 cells were
transiently cotransfected with expression plasmids encoding
mPER3-HA and either mPER1-V5, mPER2-VS, mPER3-V5, or mTIM-VS. Cell
lysates were immunoprecipitated with anti-HA antibody, and the
immunoprecipitated material was blotted and probed with anti-V5
antibodies to assess interactions. Briefly, co-immunoprecipitations
were performed as described by Lee and colleagues (Neuron
21:857-867, 1998) with the following modifications. COS7 cells
(5.times.10.sup.6) were seeded in 10 cm dishes and transfected the
following day with the expression plasmids described above.
Forty-eight hours post transfection, the cells were washed twice
with PBS, homogenized in binding buffer (20 mM HEPES, pH 7.5, 100
mM KCl, 2.5 mM EDTA, 5 mM DTT, 2.5 mM PMSF, 0.05% Triton X-100, 10%
glycerol, 10 .mu.g/ml leupeptin, 10 .mu.g/ml aprotonin) and
clarified by centrifugation. Protein concentrations were determined
by the Bradford method according to the manufacturer's instructions
(Pierce, Iselm, N.J.). Total protein (30 .mu.g) from the clarified
supernatant was combined with 15 .mu.l of protein A/G agarose beads
(Santa Cruz Biotechnology, Santa Cruz, Calif.) and incubated for 1
hr at 4EC to remove non-specific interactions. The samples were
centrifuged and the supernatant was incubated for 3 hrs at 4EC with
anti-HA mouse monoclonal antibodies (Babco, 1:50 dilution) and 15
.mu.l of protein A/G agarose beads. Subsequently, the beads were
washed four times (400 .mu.l binding buffer for 10 min. per wash),
mixed with 5 .mu.l of 4.times.sodium dodecyl sulfate (SDS) gel
loading buffer, boiled, and centrifuged. The supernatant was
analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and
western blotted as follows. Total protein (5 .mu.g) from COS7 cells
was extracted as described above, separated by SDS-PAGE, and
transferred to a nitrocellulose membrane using a semi-dry blotting
apparatus. Membranes were blocked with 5% non-fat milk. Blots were
incubated with either the mouse anti-HA antibody (1:10,000) or the
mouse anti-V5 antibody (1:5,000) overnight at 4EC. A goat
anti-mouse horseradish peroxidase secondary antibody (1:10,000) was
used in combination with enhanced chemiluminescence (NEN) to detect
proteins.
[0149] Following detection of epitope-tagged proteins with one
antibody, the blots were stripped in stripping buffer (62.5 mM
Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol, 2% SDS) at 50EC for 30
minutes. The membrane was washed extensively (20 mM Tris, pH 7.6,
137 mM NaCl, 0.05% Tween-20) then blocked again and processed for
detection of the second epitope-tagged protein.
[0150] Western blotting of cell lysates prior to
immunoprecipitation showed that all four proteins tagged with the
V5 epitope were expressed at detectable levels. The
co-immunoprecipitation data showed that mPER3 homodimerized and
heterodimerized with mPER1 and mPER2, but did not interact at
detectable levels with mTIM. When the blot was stripped and
re-probed with the anti-HA antibody, similar amounts of mPER3-HA
were precipitated in each sample. Thus, the lack of detection of an
mPER3:mTIM interaction was not due to a transfection or expression
artifact. A similar pattern of interactions was obtained when the
coimmunoprecipitation experiments were performed using mPER1-HA in
place of mPER3-HA; that is, co-immunoprecipitation of the mPER
proteins but not mTIM. These results in mammalian cells confirm the
findings in yeast: each mPER can homodimerize with itself or
heterodimerize with another mPER but does not detectably interact
with mTIM. Our results do not rule out the possibility of
biologically relevant mPER:mTIM interactions in the mammalian
clockwork. But the data do suggest that such mPER:mTIM interactions
must be much weaker than the strong mPER:mPER interactions found in
both yeast and mammalian cells.
EXAMPLE 2
[0151] Subcellular Location of mPER3 Changes in the Presence of
mPER1 or mPER2
[0152] To determine whether mPER:mPER interactions may be important
for the nuclear translocation of the mPERs and their subsequent
negative feedback on transcription, mPER:mPER interactions were
examined by first evaluating the subcellular location of the HA-and
V5-epitope tagged constructs when transfected into NIH3T3 and COS7
cells.
[0153] Immunofluorescence of epitope-tagged proteins was used to
observe protein location within cells. Briefly, cells
(3.times.10.sup.5) were seeded on glass coverslips in 6-well dishes
and transfected the following day as described above with 1 .mu.g
of total DNA per well. Forty-eight hours after transfection, cells
adherent to the coverslip were washed twice with phosphate buffered
saline (PBS), fixed with -20EC methanol (10 min), washed, and
blocked in 5% normal goat serum/0.1%Triton X-100 in PBS (1 hr).
Mouse anti-V5 IgG (1:500; Invitrogen, Calsbad, Calif.) or rabbit
anti-HA IgG (1:200; Santa Cruz Biotechnology, Santa Cruz, Calif.)
was applied for 1.5 hrs. Cells were washed and then incubated in
the dark (1 hr) with secondary antibodies. These consisted of
either goat anti-rabbit IgG conjugated to Cy2 (1:200) or goat
anti-mouse IgG conjugated to Cy3 (1:200; Jackson ImmunoResearch).
Cells were washed, and the nuclei were stained with bisBenzimide
and then mounted for fluorescence microscopy. A random population
of 30-60 cells from each coverslip was examined by epifluorescence
microscopy and the subcellular distributions of the transfected
proteins were recorded without knowledge of the treatment. At least
three independently transfected coverslips were analysed. The
cellular location was scored as one of three categories: both
cytoplasm and nucleus, cytoplasm alone, or nucleus alone.
[0154] When expressed singly in NIH3T3 cells, mPER1 and mPER2 were
each found predominantly in both cytoplasm and nucleus of
individual cells (78% and 61% of transfected cells, respectively;
n=3 experiments), but were also detected in the nucleus alone (15%
and 29%, respectively). In contrast, mPER3 was mostly in cytoplasm
alone (95% of transfected cells), and mTIM was mostly nucleus alone
(89%).
[0155] To determine whether co-expression promotes nuclear entry of
the proteins, all possible pairwise combinations of the mPER and
mTIM plasmids were co-transfected. mTIM co-expressed with any of
the mPER proteins did not affect subcellular location of mTIM or
the mPER proteins (p>0.05). The most obvious example of this was
observed when mPER3 and mTIM were coexpressed: mPER3 remained
cytoplasmic, and mTIM remained nuclear. The inability of mTIM to
influence subcellular location of the mPER proteins provides
further evidence that mTIM does not interact functionally with the
mPER proteins in a manner analogous to the interactions of PER and
TIM in Drosophila.
[0156] When mPER3 was co-expressed with either mPER1 or mPER2,
mPER3 was dramatically redistributed from cytoplasm only to both
cytoplasm and nucleus (p<0.01, n=3 experiments). mPER1 was more
effective than mPER2 in promoting nuclear entry of mPER3; that is,
nucleus-only location was found in 3 times more cells with mPER1
co-transfections, compared with mPER2. The same redistribution
profile was observed when the amounts of the mPER1 and mPER3
plasmids transfected were decreased by 75% (from 500 ng to 125 ng).
All of the subcellular localization experiments described above in
NIH3T3 cells were also performed in COS7 cells with similar
results. Despite trying all possible combinations of mPER proteins
with mTIM, including adding all four proteins at once, we were
unable to induce a "nucleus-only" location of mPER1 or mPER2 in
>30% of NIH3T3 cells. Thus, it would appear that the tested
combinations do not completely reconstitute mPER function in NIH3T3
cells. This suggested that there are other clockrelevant factors
important for the nuclear translocation of the mPER proteins.
EXAMPLE 3
[0157] mPER:mPER Interactions do not Augment Inhibition of
CLOCK:BMAL-1-Induced Transcription
[0158] The ability of mPER1/2:mPER3 interactions to promote the
nuclear entry of mPER3 and augment the inhibition of
CLOCK:BMAL1-induced transcription was examined. For these studies,
a luciferase reporter gene assay in NIH3T3 cells was used. The
reporter construct utilizes a 200 bp fragment of the promoter
region of the mouse arginine vasopressin (prepropressophysin) gene
containing a CACGTG E box, as previously described (Jin et al.,
Cell 96:57-68, 1999). This reporter gene construct is activated by
CLOCK and BMAL1 acting together on the E box enhancer (Jin et al.,
supra). Briefly, luciferase reporter gene assays were performed in
NIH3T3 cells as previously described (Gekakis et al., Science
280:1564-1569, 1998; Jin et al., supra). Cells (3.times.10.sup.5)
were seeded in six-well plates and transfected the following day.
Each construct contained the vasopressin promoter (10 ng) or 1.8 kb
of the 5' flanking region of the mPerl, gene each cloned into pGL3
BasicJ (Promega, Madison, Wis.) (10 ng of each reporter) and CMV
.beta.galactosidase (25 ng). cDNAs encoding Mouse CLOCK, hamster
BMAL-1 and human MOP4, each subcloned into pcDNA3.1-V5, were each
used at 250 ng per transfection. Amounts of the mPER and mTIM
constructs transfected varied depending on the experiment. The
total amount of DNA per well was adjusted to 1 .mu.g by adding
pcDNA 3.1 vector as carrier. Forty-eight hours after transfection,
cells were harvested to determine .beta.-galactosidase activity and
luciferase activity by luminometry.
[0159] Dose-response studies of inhibition of CLOCK:BMAL-1-induced
transcription by the mPER proteins and mTIM are shown in FIG. 1.
Data from 16 transcription assays were combined by normalizing the
relative luciferase activity values in each experiment to the
activity from CLOCK:BMAL-1 alone (set at 100%). The amounts of the
mPER or mTIM expression constructs transfected are listed (in ng)
at the extremes of the triangles. Individual experiments were done
in duplicate or triplicate. Values are plotted as the mean %+SEM
when three or more experiments were performed with a given amount
of expression construct. All other values represent averages from
two experiments.
[0160] Results showed that CLOCK:BMAL-1-induced transcription was
maximally inhibited transfection of 250 ng of each of the mPer and
mTim constructs. Maximal inhibition reached 55-70% for each
construct and was not substantially augmented by any pairwise
transfection of the mPer and mTim constructs (at 250 ng each). As
the amount of each expression plasmid transfected was decreased,
there was decreasing inhibition of CLOCK:BMAL-1 transcription (FIG.
1). From the dose-response curves, the amount of each expression
construct that was at the threshold of causing transcriptional
inhibition was identified.
[0161] Using threshold amounts of each expression construct, all
possible pairwise mPER:mPER and mPER:mTIM combinations were next
examined to look for synergistic or additive interactions. In no
instance, however, was there observed a consistent augmentation of
transcriptional inhibition with low-dose, pairwise combinations of
mPER expression constructs or mPER plus mTIM expression constructs
(n=4 experiments). Co-expression experiments with low doses of
mPER1 and mPER3 did show a consistent trend toward inhibition of
CLOCK:BMAL-1-induced transcription, but the effects were
significant (p<0.05) in only one of three experiments.
[0162] The data hint that mPER1:mPER3 heterodimers may be
functionally relevant for transcriptional inhibition. The
endogenous expression of the mPer1, mPER2, mPer3, and mTim genes in
NIH3T3 cells may obscure finding a more robust inhibitory effect on
transcription. Based on the modest effects of mPER:mPER
interactions on nuclear localization and transcriptional
inhibition, however, it seemed more likely that there were other
factors necessary for nuclear translocation and/or retention of the
mPER proteins and for their subsequent inhibition of
CLOCK:BMAL-1-induced transcription.
EXAMPLE 4
[0163] mCry1 and mCry2 RNA Levels in the SCN and in Peripheral
Clocks are Regulated by CLOCK
[0164] It was next determined if cryptochromes were involved in the
CLOCK:BMAL1-driven mPer feedback loop. mCry1 and mCry2 gene
expression in wild-type and homozygous Clock mutant mice was
examined, because a decrease in gene expression in Clock/Clock mice
(i.e., mice homozygous for the mutation) would place the
cryptochrome genes within the CLOCK-driven feedback loop.
[0165] Northern analysis was used to examine gene expression of
CRY1 and CRY2. Briefly, total RNA was extracted from tissues using
the Ultraspec RNA isolation reagent. Polyadenylated (polyA+) RNA
was prepared using oligotex poly dT spin columns (Qiagen, Valencia,
Calfi.). PolyA+ RNA was separated by electrophoresis through a 1%
agarose-formaldehyde gel, blotted onto GenScreenJ (New England
Nuclear), and hybridized with random prime-labeled probe
(S.A.=2.times.10.sup.6 cpm/ml). The blots were hybridized with
Express HybridizationJ Solution (Clontech, Palo Alto, Calif.) and
washed following the manufacturer's protocol. Probes used were
mCry1 (nt 1081-1793 of Act. No. AB000777) and mCry2 (nt 1060-1664
of Act. No. AB003433). Probe for actin was from human B-actin,
purchased from Clontech (Palo Alto, Calif.). Blots were exposed at
-80EC to film with 2 intensifying screens.
[0166] Four blots were prepared from the RNA samples, with each
blot consisting of the eight time-points from one genotype and a
standard lane. One microgram of polyA +RNA was loaded per lane for
each genotype. Each blot was probed, stripped, then reprobed to
detect mCry1, mCry2, and actin. To calculate relative RNA
abundance, optical densities of mCry1 and mCry2 hybridization were
divided by densities from actin hybridization to the same blot.
Normalized values were then averaged for the two replicate blots
prepared from a single set of RNA samples. Comparison across blots
probed and exposed under similar conditions suggested that the
absolute level of expression of the mCry genes was lower in
Clock/Clock mice than in wild-type mice. This difference in
absolute expression level was confirmed using two additional blots
that included selected (peak-trough) RNA samples from the two
genotypes side-by-side, and were probed for both mCry1, mCry2, and
actin.
[0167] mCry RNA levels in SCN are depicted in FIG. 2A. Panels
depict the temporal profiles of mCry1 RNA levels (left) and mCry2
RNA levels (right) in the SCN of wild-type mice (solid lines) and
Clock/Clock mice (dashed lines). Each values is the mean+SEM of 4
animals. The horizontal bar at the bottom of the panels represents
lighting cycle prior to placement in DD; the stippled areas
represent subjective day; and the filled areas represent subjective
night. Photomicrographs showed representative autoradiographs of
mCry1 and mCry2 gene expression from coronal brain sections (15
.mu.m) at the level of the SCN from wild-type (+/+) and Clock/Clock
(CZk/CZk) mice at CT 9. The brain sections were examined by in situ
hybridization using cRNA probes as follows. A breeding colony of
mice carrying the Clock mutation was established on a BALB/c
background. For studies, both males and female mice 5-15 weeks of
24 age were used. Mice were housed in LD, except as noted. Animals
were killed by decapitation. Genotypes were determined using a PCR
mutagenesis method, as previously described (Jin et al.,
supra).
[0168] Antisense and sense cRNA probes were generated from each
plasmid by in vitro transcription in the presence of .sup.35S-UTP
(1200 Ci/mmol). Probe for mCry1 (AB000777) was nucleotides
1081-1793 and for mCry2 (AB003433) was nucleotides 1060-1664. Probe
quality and size was confirmed by determining .sup.35S
incorporation into TCA-precipitable material, and by gel
electrophoresis and subsequent autoradiography of the gel.
[0169] Prehybridization, hybridization, and wash procedures were
performed as described by Weaver. Probe (50 .mu.l at 107 cpm/ml)
was applied to each slide. Coverslipped slides were then incubated
in humidified chambers overnight at 55E C. Following completion of
the wash steps, slides were air dried and exposed to Kodak BioMax
MR film for 8 days.
[0170] Densitometric analysis of hybridization intensity was
accomplished using NIH Image software on a Macintosh computer; data
are expressed as absolute optical density values as determined by
calibration with Kodak photographic step tablet #3. .sup.14C
standards included in each cassette were used to verify that the
optical density values measured were within the linear response
range of the film.
[0171] The results showed that mCry1 RNA levels exhibited a
prominent circadian rhythm in the SCN of wild-type animals (ANOVA,
p<0.05; FIG. 2A). The phase of the mCry1 RNA rhythm was most
similar to the phase of the mPER2 RNA oscillation in the SCN. In
sharp contrast to wild-type mice, no mCry1 RNA rhythm was apparent
in the SCN of Clock/Clock mice (ANOVA, p>0.05; FIG. 2A). Thus,
the mCry1 RNA rhythm is dependent on a functional CLOCK protein.
These results are similar to the finding that the amplitude of RNA
rhythms for each of the three mPer genes is markedly reduced in
Clock/Clock mice (Jin et al., supra).
[0172] mCry2 RNA levels in the SCN of wild-type animals did not
show a circadian rhythm (FIG. 2A; p>0.05). Interestingly, mean
steady-state mCry2 RNA levels were nonetheless significantly lower
in Clock/Clock mice, compared to those in wildtype controls (ANOVA,
p<0.005). This finding suggests that mCry2 transcription is also
at least partially dependent on a functional CLOCK protein. It is
worth noting that of 5 genes studied whose RNA levels do not
manifest a circadian rhythm in the SCN, mCry2 is the only one in
which mRNA levels in Clock/Clock animals were observed (see Jin et
al., supra). Since circadian clocks also appear to exist in
peripheral tissues (Balsalobre et al., Cell 93:929-937, 1998; Zylka
et al., Neuron 20:1110, 1998b; Sakamoto et al., J. Biol. Chem.
273:27039-27042, 1998), the temporal profiles of mCry1 and mCry2
RNA levels in skeletal muscle were examined. This tissue was chosen
because the three mPer genes manifest robust RNA rhythms there
(Zylka et al., 1998b, supra). mCry RNA levels in skeletal muscle
are shown in FIG. 2B. Autoradiograms (upper panels) illustrate
Northern blots of mCry1 (3.0 kb transcript, left) and mCry2 (4.4 kb
transcript, right) RNA levels at each of 8 time points in 12L:12D,
with lights on from Zeitgeber Times (ZT) 0-12. The lower panels
depict quantitative assessment of mCry1 and mCry2 RNA levels in
skeletal muscle of wildtype (solid lines) and Clock/Clock mice
(dashed lines). The values are the average relative intensity of
two replicate blots with each probe. Data were normalized and
expressed relative to hybridization intensity of actin control
probe. Data at ZT 21, ZT0/24, and ZT3 are double plotted. In
contrast to the situation in the SCN, both mCry1 and mCry2 RNA
levels in muscle exhibited a daily rhythm under 12 hrs light:12 hrs
dark (LD) (FIG. 2A) and a circadian rhythm under constant darkness.
The peak of the mCry2 rhythm preceded that of mCry1 by 6 to 9 hrs,
and the mCry1 RNA rhythm was delayed by several hrs relative to the
phase of its RNA rhythm in the SCN. A phase delay between the SCN
and peripheral oscillations is also observed in the RNA rhythms of
the three mPer genes (Zylka et al., 1998b, supra). In skeletal
muscle of Clock/Clock animals, the mCry1 RNA rhythm was dampened
and phase advanced, while the mCry2 RNA rhythm was abolished (FIG.
2B). For both genes, RNA levels were lower in Clock/Clock animals
at all times, compared to wild-type controls.
[0173] Taken together, these data indicate that the transcriptional
regulation of mCry 1 and mCry2 is under CLOCK control in both the
SCN and in peripheral clocks. These findings provide strong
evidence that the mouse cryptochromes are components of the CLOCK:
BMAL-1-driven feedback loop. Moreover, the occurrence of a CACGTG E
box 300 bp upstream of the mCry1 transcription start site suggests
that CLOCK directly participates in rhythmic mCry1 transcription
through an E box enhancer in its promoter.
EXAMPLE 5
[0174] mCRY1 and mCRY2 Block CLOCK:BMAL-1-induced Transcription in
NIH3T3 Cells
[0175] The involvment of mammalian cryptochrome within the negative
limb of the feedback loop was analyzed by determining whether mCRY1
and/or mCRY2 can inhibit CLOCK: BMAL-1-induced transcription. For
this phase of study, 14 luciferase reporter gene studies were
performed in NIH3T3 cells using either the vasopressin promoter
(Jin et al., supra) or 1.8 kb of the 5' flanking region of the
mPer1 gene subcloned into a promoterless luciferase reporter
vector.
[0176] Inhibition of CLOCK:BMAL-1-mediated transcription from the
vasopressin (AVP) promoter (FIG. 3A) or mPer1 promoter (FIG. 4B) by
mPER1, mCRY1 and mCRY2 (250 ng each) was determined. Each value is
the mean+SEM of three replicates from a single assay. The results
are representative of three independent experiments. Dose-response
curves for mCRY1 (Fig. cD) or mCRY2 (FIG. 3D) inhibition of
CLOCK:BMAL-1-mediated transcription from the vasopressin (AVP)
promoter. Each value is the mean+SEM of three replicates from a
single assay. Similar results were found in replicate
experiments.
[0177] Results show that when vasopressin and mPer1 promoters were
used in the reporter vectors, mPER1 caused a maximal inhibition of
61% and 30%, respectively. mCRY1 and mCRY2, on the other hand,
inhibited CLOCK:BMAL-1-induced transcription by >90% from either
reporter. This dramatic effect on transcriptional inhibition was
dose dependent for each of the two mCRY proteins. These results
indicate that mCRY1 and mCRY2 are each potent inhibitors of
CLOCK:BMAL-1-mediated transcription. The mCRY-induced
transcriptional inhibition must occur through direct or indirect
interaction with the CLOCK:BMAL-1:E box complex because this is the
only complex common to both the vasopressin and mPer1 promoters
EXAMPLE 6
[0178] Both mCRY1 and mCRY2 are Nuclear Proteins
[0179] For the mCRY proteins to interact with the CLOCK:BMAL-1:E
box complex, they must be present in the nucleus. Previous studies
have shown that mCRY2 is indeed a nuclear antigen (Kobayashi et
al., Nucleic Acids Res. 26:5086-5092, 1998; Thresher et al.,
Science 282:1490-1494, 1998). The situation with mCRY1 is ambiguous
because previous studies of the endogenous protein and green
fluorescent protein (GFP)-tagged mCRY1 fragments indicate
localization mainly in mitochondria (Kobayashi et al., supra). To
determine the localization of CRY1 or CRY2, the CRY proteins were
tagged at the ends of the protein with a number of different
epitopes. For example, the coding regions of mCRY1 (AB000777) were
ligated into the pcDNA 3.1 VS-His expression vector containing
either an N terminal or C terminal HA tag. For mCRY2, the
nucleotide sequence encoding the amino terminal portion of the
coding region was not available in GenBank (partial clone accession
no. AB003433). The 5'end of the mCRY2 coding region was thus cloned
by 5'rapid amplification of cDNA ends. The full-length coding
region was then amplified as described above, sequenced, and
deposited in GenBank as Accession Number AF156987. The constructs
(FIG. 4) were transfected into NIH3T3 cells and both their cellular
localization (by immunofluorescence) and ability to inhibit
CLOCK:BMAL-1-induced transcription were assessed.
[0180] The results clearly showed that mCRY1 translocates to the
nucleus when tagged with either the V5 or HA epitope. This was true
when HA was placed at either the N-terminal or C-terminal ends, as
well as when epitope tags were placed on both ends of the protein.
In each instance, the protein was nuclear and inhibited
CLOCK:BMAL1-induced transcription by >90%. Interestingly, when
enhanced (E)GFP was fused to either end of mCRY1,
immunofluorescence was found diffusely throughout the cell and
there was no transcriptional inhibition. The same diffuse staining
and lack of transcriptional inhibition was found with EGFP alone.
When EGFP was fused to an N-terminal fragment of mCRY1 containing a
putative signal sequence for transport into mitochondria, the
cellular location was mainly cytoplasmic, punctate and appeared to
be in mitochondria. Using a specific anti-mCRY1 antibody,
endogenous mCRY1 protein was shown to be nuclear in non-transfected
NIH3T3 cells and in SCN. Thus, mCRY1 is normally a nuclear protein
and that GFP fused to CRY alters the location of the native protein
by changing its conformation. mCRY2-V5 was found in the nucleus,
consistent with previous findings (Kobyashi et al., supra; Tresher
et al., supra), and the tagged protein inhibited
CLOCK:BMAL-1-induced transcription by >90%.
EXAMPLE 7
[0181] mCRY1 and mCRY2 Directly Interact with the mPER Proteins and
Translocate them into the Nucleus
[0182] To evaluate the potential for protein: protein interactions
between the mCRY and mPER families, co-immunoprecipitation using
epitope-tagged proteins was utilized.
[0183] COS7 cells co-transfected with expression plasmids encoding
mCRY 1-HA and either mPER1-V5, mPER2-VS, mPER3-V5, or mTIM-V5
expressed each V5-tagged protein prior to immunoprecipitation.
Immunoprecipitation with the HA antibody and analysis of the
immunoprecipitated material with anti-V5 antibodies indicated the
presence of heterodimeric interactions between mCRY1 and each of
the mPER and mTIM proteins. There was no interaction between mCRY1
and .beta.galactosidase which served as a specificity control.
Co-immunoprecipitation experiments using mCRY2-HA instead of mCRY
1-HA similarily showed the presence of heterodimeric interactions
between mCRY2 and each of the mPER and mTIM proteins.
[0184] Having shown that mCRY:mPER heterodimers could exist, the
ability of such interactions to translocate the mPER proteins to
the nucleus was determined. In marked contrast to the lack of
effect of any pairwise combination of mPER:mPER or mPER:mTIM
interactions to translocate mPER1 and mPER2 to the nucleus, each
mCRY protein profoundly changed the location all three mPER
proteins in NIH3T3 and COS7 cells. This was most apparent for mPER1
and mPER2 which were almost entirely nuclear after co-transfection
with either mCRY1 or mCRY2. Curiously, each mCRY protein changed
mPER3 from mainly cytoplasm only (>80%) to both cytoplasm and
nucleus (>80%) to a degree similar to that induced by
co-transfection of mPER3 with mPER1. When mPER3 was co-transfected
with mPER1 and either mCRY1 or mCRY2, however, each of the three
protein combinations changed mPER3's location from 13-20% nucleus
only to predominantly nucleus only (54-68% of transfected cells).
Co-transfection of either mCRY1 or mCRY2 with mTIM did not change
the predominantly nucleus only location (>90% of transfected
cells) of any of the three proteins.
[0185] These data indicate that the mCRY proteins can
heterodimerize with the mPER proteins and mTIM. The mCRY:mPER
interactions mimic the in vivo situation where the interaction of
mCRY and mPER results in the almost complete translocation of mPER1
and mPER2 to the nucleus. Moreover, trimeric interactions among the
mPER and mCRY proteins appear necessary for complete nuclear
translocation of mPER3. The data also suggest that the nuclear
translocation of the mPER proteins is dependent on mCRY 1 and
mCRY2. The mCRY proteins, however, appear to be able to translocate
to the nucleus independent of the mPERs. Even with massive
overexpression of mCRY proteins in cell culture they are always
>90% nuclear.
EXAMPLE 8
[0186] mCRY1 and mCRY2 Levels Express Synchronous Circadian Rhythms
in the SCN
[0187] If nuclear entry of mPER1 and mPER2 is dependent on the mCRY
proteins as suggested by the cell culture experiments, then
similarily synchronous circadian oscillations of endogenous mCRY1
and mCRY2 levels in the nuclei of SCN neurons might be expected. To
determine this the oscillations of endogenous CRY in neurons was
determined. Briefly, mice entrained to a schedule of 12L:12D were
transferred to constant dim red light. Circadian Time (CT) was
initially defined relative to predicted lights-off (CT12), and on
the day of sampling was confirmed by the coincident onset of group
activity, as monitored by passive infra-red movement detectors.
After 20 (CT8) to 42 (CT6) hours in constant dim red light, mice
were killed with an anesthetic overdose, and perfused (4%
paraformaldehyde). Brains were removed, post-fixed, transferred to
cryoprotectant buffered sucrose solution (20%) and then sectioned
on a freezing microtome. Alternate freefloating sections (40 .mu.m)
were incubated with affinity purified anti-mCRY1 or anti-mCRY2
(both at 0.5 .mu.g/ml) primary sera (Alpha Diagnostic
International). The sera were raised against synthetic peptides
corresponding to specific sequences close to the C-terminals of the
mCRY1 (26 amino acids) and mCRY2 (22 amino acids)proteins. To test
for specificity of the sera, some SCN sections were incubated with
affinity purified sera to which synthetic peptide (10 .mu.g/ml) had
been added. Immunoreaction was visualised by
avidin-biotin/peroxidase in conjunction with diaminobenzidine
chromogen (Vector Labs, Peterborough, U.K.). Counts of the number
of immunoreactive nuclear profiles in the SCN were made using an
image analysis system as described previously.
[0188] Immunocytochemical analysis of mCRY1 and mCRY2 in the brains
of mice sampled at Zeitgeber Time (ZT) 15 (3 h after lights off)
identified them both as nuclear antigens in the SCN and elsewhere,
including piriform cortex (mCRY2) and hippocampus (mCRY1, mCRY2).
The majority of SCN neurons appeared to be immunoreactive for the
antigen tested, and the immunoreactivities were specific, being
blocked by pre-incubation with the peptide (10 .mu.g/ml) used to
raise the respective serum. In contrast, the SCN from animals
sampled at ZT3 contained very few mCry1-or mCRY2-immunoreactive
nuclei, and those which were evident were located in a
dorso-lateral position comparable to that reported for mPER1
immunoreactive nuclei at this phase. Rhythmic expression of mCRY1
and mCRY2 was sustained under free-running conditions, with low
levels at Circadian Time (CT)2 and high expression throughout the
SCN at CT14 was observed. Quantitative analysis of the number of
immunoreactive nuclei in the SCN sampled at 2 h intervals over 24 h
in DD showed a clear circadian variation. The abundance of both
proteins was low in the early subjective day, rising in later
subjective day to peak at CT12-CT16. There was a progressive
decline during subjective night to basal counts at CT24. This
temporal profile of mCRY1 and mCRY2-immunoreactivity in the SCN is
directly comparable with that observed for mPER1 and mPER2,
indicative of a synchronous nuclear accumulation of these proteins
in the SCN.
[0189] In contrast, expression of mCry1- and mCRY2-immunoreactivity
in other areas did not exhibit appreciable circadian variation,
consistent with the constitutive expression of mPER proteins in
brain sites outside the SCN.
[0190] These in vivo data, in conjunction with our cell culture
data, strongly suggest that the mCRY proteins are the dominant
movers of the mPER1 and mPER2 proteins from cytoplasm to nucleus.
We do not yet know the temporal pattern of mPER3 immunoreactivity
in the SCN, but we have no reason to believe it will be any
different from that found for mPER1 and mPER2.
EXAMPLE 9
[0191] Dissociation Between the Inhibitory Effects of the mPER
Proteins and the mCRY Proteins on Transcription
[0192] By varying the amounts of mPER and mCRY plasmids in
co-transfection experiments, we have observed at best additive
effects of pairwise combinations of mPER with mCRY proteins on the
inhibition of CLOCK:BMAL-1-mediated transcription. Although these
studies in cell culture are confounded by the endogenous expression
of the mPer1, mPER2, mPer3, mTim, mCry1 and mCry2 genes in the cell
lines used, the lack of synergism of pairwise combinations on
transcriptional inhibition suggested that the mPER and mCRY
proteins have independent effects on the transcriptional machinery.
To examine this in more detail, the fact that
MOP4:BMAL-1-heterodimers also activate transcription via a CACGTG E
box was exploited (Hogenesch et al., Proc. Natl. Acad. Sci. USA
95:5474-5479, 1998).
[0193] CLOCK, MOP4, and BMAL-1 alone or in pairwise combinations
were tested for transcriptional activation (FIG. 8A). Significant
transcriptional activation was seen only when CLOCK and BMAL-1
(10-fold increase) or MOP4 and BMAL 1 (37-fold increase) were
co-expressed. Transcriptional activation was dependent on the
E-box, because no transcriptional activation was detected when the
vasopressin promoter with a mutated E-box was used. The greater
levels of transcriptional activation with MOP4:BMAL-1 than with
CLOCK:BMAL-1 appeared due to much higher levels of MOP4 protein
expression compared with CLOCK based on western blot analysis of
epitope tagged proteins.
[0194] Each mPER alone, mTIM, or each mCRY alone was tested for its
ability to inhibit MOP4:BMAL-1-induced transcription. Even though
each mPER protein can inhibit CLOCK:BMAL-1-induced transcription,
the mPER proteins (500 ng of each plasmid) did not affect
MOP4:BMAL-1-induced transcription (FIG. 5B). When the amount of
MOP4 was reduced so that the relative luciferase values were equal
to those seen with CLOCK and BMAL-1 activation, the mPER expression
plasmids were still unable to inhibit transcription. In contrast to
the lack of inhibition of the mPER proteins, mTIM (at 500 ng) was
able to inhibit MOP4:BMAL-1-induced transcription by about 40%
(FIG. 5; p>0.01). Combinations of each mPER and the mTIM
expression plasmids, or pairwise combinations of mPER expression
plasmids did not inhibit more effectively than when the mTIM
plasmid was transfected alone. Remarkably, each mCRY protein (250
ng each) abrogated MOP4:BMAL-1-mediated transcription (FIGS. 5C and
5D).
[0195] These data suggest that the mPER proteins have their action
on CLOCK, perhaps as mPER:mCRY heterodimers, while the mCRY
proteins appear capable of interacting directly with either BMAL-1
or the CACGTG E box. It is worth noting that MOP4 does not appear
to play a major role in circadian function, as its RNA is not
detectably expressed in the SCN of either wild-type or Clock-mutant
mice.
EXAMPLE 10
[0196] Bmal1 RNA Rhythm in Clock/Clock Mutant Mice
[0197] BMAL-1 RNA rhythm was first documented in mouse SCN using
quantitative in situ hybridization (Jin et al., Cell 96:57 (1999))
with an antisense riboprobe that recognizes the two major Bmal1
transcripts in the SCN (Yu et al., Biochem. Biophys. Res. Commun.
260:760 (1999)). Wild-type mice exhibited a robust circadian rhythm
in Bmal1 RNA levels, with low levels from circadian time (CT) 6-9
and peak levels from CT 15-18.
[0198] The phase of the Bmal1 rhythm is opposite that of the mouse
Per1-3(mPer1-3) RNA rhythms (Zylka et al., Neuron 20:1103 (1998);
Oishi et al., Biochem. Biophys. Res. Commun. 268:164 (2000); Honma
et al., Biochem. Biophys. Res. Commun. 250:83 (1998)). In addition
to driving rhythmic transcription of the mPer and mCry genes (Jin
et al., Cell 96: 57 (1999); Kume et al., Cell 98:193 (1999)), it
seemed possible that CLOCK:BMAL1 heterodimers might simultaneously
negatively regulate Bmal1 gene expression, similar to a proposed
model of clock gene regulation in Drosophila. If CLOCK:BMAL1
heterodimers are negatively regulating Bmal1 gene expression and if
the mutant CLOCK protein is ineffective in this negative
transcriptional activity, then Bmal1 RNA levels should be elevated
and less rhythmic in homozygous Clock mutant mice. Compared to
wild-types, however, Clock/Clock animals expressed a severely
dampened circadian rhythm of Bmal1 RNA levels in the SCN
(significant difference between genotypes; ANOVA, P<0.001) (FIG.
9). Trough Bmal1 RNA levels did not differ between Clock/Clock mice
and wild-types. The peak level of the RNA rhythm in homozygous
Clock mutant mice was only .apprxeq. 30% of the peak value in
wild-types. A similar blunting of the Bmal1 RNA rhythm in the SCN
of Clock/Clock mice has been reported by others (Oishi et al.,
Biochem. Biophys. Res. Commun. 268:164 (2000)).
[0199] The temporal profile of Clock RNA levels was examined in the
SCN of Clock/Clock mutant animals, since it has been reported that
Clock RNA levels (assessed by Northern blot analysis) are decreased
in the eye and hypothalamus of Clock/Clock mutant mice (King et
al., Cell 89:641 (1991)). Consistent with previous reports (Tei et
al., Nature 389:512 (1997); Shearman et al., Neuroscience 89:387
(1999) Clock RNA levels did not manifest a circadian oscillation in
mouse SCN. Surprisingly, Clock RNA levels in the SCN of Clock/Clock
mutant mice were not significantly different from those in the SCN
of wild-type animals (FIG. 10; ANOVA, P>0.05). Thus, the Clock
mutation appears to alter regulation of Bmal1 gene expression in
SCN, but not the regulation of the Clock gene itself. Clock
expression may be decreased in other hypothalamic regions.
[0200] The low levels of Bmal1 RNA in the SCN of homozygous Clock
mutant animals show that CLOCK is not required for the negative
regulation of Bmal1. Instead, these data indicate that CLOCK is
actually necessary for the positive regulation of Bmal1. The
positive effect of CLOCK on Bmal1 levels is probably indirect and
may occur via the mPER and/or mCRY proteins, which are expressed in
the nucleus of SCN neurons at the appropriate circadian time to
enhance Bmal1 gene expression (Kume et al., Cell 98:193 (1999);
Field et al., Neuron 25:437 (2000)). In addition, the mPer1-3 and
mCry1-2 RNA oscillations are all down-regulated in Clock/Clock
mutant mice (Jin et al., Cell 96:57 (1999); Kume et al., Cell
98:193 (1999)). Reduced levels of the protein products of one or
more of these genes may lead to the reduced levels of Bmal1 in the
mutant mice, through loss of a positive drive on Bmal1
transcription.
EXAMPLE 11
[0201] Bmal1 and mCry1 RNA Rhythms in mPER2.sup.Brdm1 Mutant
Mice
[0202] Homozygous mPER2.sup.Brdm1 mutant animals have depressed
mPer1 and mPER2 RNA rhythms (Zheng et al., Nature 400:167 (1999)).
The Bmal1 rhythm in homozygous mPER2.sup.Brdm1 mutants was examined
to determine whether the positive drive on the Bmal1 feedback loop
might come from the mPER2 protein. The effects of this mutation on
the mCry1 RNA rhythm were also examined.
[0203] The temporal profiles of gene expression were analyzed at
six time points over the first day in DD in homozygous
mPER2.sup.Brdm1 mutant mice and wild-type littermates. The Bmal1
RNA rhythm expressed in the SCN of wild-type animals was
substantially altered in the SCN of mutant mice (ANOVA,
P<0.05)(FIG. 11). Trough RNA levels did not differ between
wild-type and mutant animals, but the increase in Bmal1 RNA levels
was advanced and truncated in the mutants, compared to the
wild-type rhythm.
[0204] The mCry1 RNA rhythm was also significantly altered. In the
SCN of mPER2.sup.Brdm1 mutant mice (ANOVA, P<0.0001)(FIG. 12),
the peak levels of the mCry1 RNA rhythm were suppressed by
.apprxeq.50%, as reported for mPer1 and mPER2 RNA rhythms in this
mouse line (Zheng et al., Nature 400:167 (1999)).
[0205] These data suggest that maintenance of a normal Bmal1 RNA
rhythm is important for the positive transcriptional regulation of
the mPer and mCry feedback loops. Thus, rhythmic Bmal1 RNA levels
may drive rhythmic BMAL 1 levels which, in turn, regulate
CLOCK:BMAL1-mediated transcriptional enhancement in the master
clock. Indeed, mPer1, mPER2, and mCry1 RNA rhythms are all blunted
in the SCN of mPER2.sup.Brdm1 mutant mice, in which the Bmal1
rhythm is also blunted. In addition, the homozygous mPER2.sup.Brdm1
mutation is associated with a shortened circadian period and
ensuing arrhythmicity in constant darkness (DD).
[0206] These data, along with the fact that Clock RNA levels are
unaltered in the SCN of homozygous mPER2.sup.Brdm1 mutants (Zheng
et al., Nature 400:167 (1999), also provide evidence that mPER2 is
a positive regulator of the Bmal1 RNA rhythm. This effect may be
unique to mPER2. For example, the diurnal oscillation in mPer2 RNA
is not altered in the SCN of mPer1-deficient mice, and mPer1,
mPer2, and Bmal1 RNA circadian rhythms are not altered in the SCN
of mPer3-deficient mice. Moreover, circadian rhythms in behavior
are sustained in mice deficient in either mPer1 or mPer3.
EXAMPLE 12
[0207] mCRY-Mediated Nuclear Translocation of mPER2 is
PAS-Independent
[0208] There are at least two ways that the mPER2.sup.Brdm1
mutation could alter the positive drive of the clock feedback
loops. The mutation could disrupt mPER:mCRY interactions important
for the synchronous oscillations of their nuclear localization
and/or alter the protein's ability to interact with other proteins
(e.g., transcription factors). We examined whether the PAS domain
is necessary for functionally relevant mPER2:mCRY interactions,
using immunofluorescence of epitope-tagged proteins in COS-7 cells.
Briefly, COS-7 cells (3.times.10.sup.5) were seeded on glass
coverslips in 6-well dishes and transfected with Lipofectamine
Plus.TM. (Gibco BRL) with 0.5 ug of total DNA per well. Forty-eight
hrs after transfection, cells were processed as described (Sangoram
et al., Neuron 21:1101 (1998)). A random population of 30-60 cells
from each covership was examined by epiflourescence microscopy and
the subcellular distribution of expressed proteins was recorded
without knowledge of treatment. At least three independently
transfected coverslips were analyzed.
[0209] Coexpression of mPER1 or mPER2 with either mCRY1 or mCRY2 in
COS-7 cells translocates >90% of mPER1 and mPER2 into the
nucleus (Kume et al., Cell 98:193 (1999)). To determine whether the
PAS domain of mPER2 is required for this translocation an mPER2
fragment containing residues 1-337 of PER2 (mPER2.sup.1-337), which
includes the PAS domain, was examined in COS-7 cells.
mPER2.sup.1-337 was localized to both cytoplasm and nucleus (89% of
transfected cells)(FIG. 13) and the localization was not changed by
co-expression with mCRY1. Co-expression of mPER2.sup.338-1257 with
mCRY1, however, dramatically changed the cellular location of the
mPER2.sup.338-1257 fragment from cytoplasm only (12%) to nucleus
only (85%). Co-expression of mPER2.sup.Brdm1 (missing residues
348-434) with mCRY1 also moved mutant mPER2.sup.Brdm1 into the
nucleus, from cytoplasm only (100% when expressed alone) to
predominantly both cytoplasm and nucleus (81%) when co-expressed
with mCRY1 (FIG. 13). The same patterns of cellular localization
were found when mCRY2 was co-expressed with these mPER2 constructs
instead of mCRY1. Thus, functional mPER2:mCRY interactions are not
mediated through the PAS domain. Similarly, the PAS domain was not
important for the mCRY-mediated nuclear translocation of mPER1 in
COS-7 cells.
[0210] The data show mPER:mCRY interactions necessary for nuclear
transport of the mPER1 and mPER2 proteins occur through domains
outside the PAS region. Thus, the PAS domain of an mPER2:mCRY
heterodimer might be free to bind to an activator (e.g.,
transcription factor) and shuttle it into the nucleus to activate
Bmal1 transcription. Alternatively, once in the nucleus, mPER2:mCRY
heterodimers or mPER2 monomers could coactivate Bmal1 transcription
through a PAS-mediated interaction with a transcription factor
(Glossop et al., Science 286:766 (1999)). mPER2 itself does not
possess a DNA binding motif (Shearman et al., Neuron 19:1261
(1997)).
EXAMPLE 13
[0211] Bmal1 RNA Levels in Mice Lacking mCry1 and mCry2
[0212] The tonic mid-to-high mPer1 and mPer2 RNA levels in
mCRY-deficient mice (van der Horst et al., Nature 398:627 (1999)
suggest that CLOCK:BMAL1 heterodimers might be constantly driving
mPer1 and mPer2 gene expression in the absence of transcriptional
inhibition by the mCRY proteins. To examine whether Bmal1 RNA
levels would also be modestly elevated, Bmal1 RNA levels in the SCN
of mCRY-deficient mice were compared to those in the SCN of
wild-type mice of the same genetic background at CT 6 and at CT 18
on the first day in DD. The mCRY-deficient (double mutant) colony
of mice had a C57BL/6 X 129 hybrid background, and wild-type
controls were of the same genetic background (van der Horst et al.,
Neuroreport 10:3165 (1999)). Sex ratios of male and female mice
were balanced across time points. We also examined Clock RNA levels
in these animals.
[0213] In wild-type animals, the typical circadian variation in
Bmal1 RNA levels was apparent with high levels at CT 18 and low
levels at CT 6 (P<0.001)(FIG. 14). In mCry-deficient mice, on
the other hand, Bmal1 RNA levels were low at both circadian times
(P>0.05)(FIG. 15). Clock RNA levels did not differ as a function
of circadian time or genotype (P>0.05)(FIG. 15).
[0214] The unexpectedly low Bmal1 gene expression in the SCN of
mCry-deficient mice suggests that the Bmal1 feedback loop is
disrupted in the mutant animals, with a resultant non-functional
circadian clock. Nevertheless, enough Bmal1 gene expression and
protein synthesis occurs for heterodimerization with CLOCK so that,
without the strong negative feedback normally exerted by the mCRY
proteins, mPer1 and mPer2 gene expression is driven sufficiently by
the heterodimer to give intermediate to high RNA values (depending
on RNA stability).
EXAMPLE 14
[0215] mPER1 and mPER2 Localization in mCry-Deficient Mice
[0216] The mid to high mPer1 and mPer2 RNA levels in the SCN of
mCry-deficient mice, and simultaneous low Bmal1 levels, suggests
that mPER1 and mPER2 proteins may not be exerting much positive or
negative influence on the core feedback loops. To test this,
immunocytochemistry was used to determine whether mPER1 and mPER2
were tonically expressed in the nuclei of SCN cells in
mCry-deficient mice, since nuclear location is necessary for action
on transcription (Kume et al., Cell 98:193 (1999); Field et al.,
Neuron 25:437 (2000)).
[0217] mPER1 immunoreactivity exhibited a robust rhythm of nuclear
staining in the SCN of wild-type mice, with high values at CT 12
(328.+-.3.5, mean.+-.SEM of positive nuclei per 30 .mu.m section,
n=3) and significantly lower values at CT 24 (54.+-.5, n=3;
P<0.01). These values are very similar to those previously
reported in other strains of mice (Field et al., Neuron 25:437
(2000)).
[0218] The pattern of mPER1 immunoreactivity in the SCN of
mCry-deficient mice was quite different, however. mPER1
immunoreactivity was detected in the nucleus of a similar number of
SCN neurons at each of the two circadian times (CT 12, 140.+-.9,
n=3; CT 24, 152.+-.21, n=3), and the counts at each time were at
.apprxeq.40% of those seen at peak (CT 12) in wild-type
animals.
[0219] The double mCry mutation also altered the sub-cellular
distribution of mPER1 staining in the SCN. In wild-type mice, mPER1
staining viewed under contrast interference was clearly nuclear
with a very condensed immunoreaction and a clear nucleolus. The
neuropil of the SCN in wild-types was devoid of mPER1
immunoreactivity. In the SCN of mCry-deficient animals, mPER1
staining was clearly nuclear, but the nuclear profiles were less
well defined and less intensely stained, and perinuclear,
cytoplasmic immunoreaction could be observed. In addition, the
neuropil staining for mPER1 was higher in mCry deficient mice,
although dendritic profiles were not discernible. In the same
brains, the constitutive nuclear staining for mPER1 normally seen
in the piriform cortex was not altered in mCry-deficient
animals.
[0220] mPER2-immunoreactivity also exhibited a robust rhythm of
nuclear staining in the SCN of wild-type mice, with high counts at
CT 12 (371.+-.11, n=3) and significantly lower counts at CT 24
(31.+-.3, n=3; P<0.01), similar to that previously reported in
another strain (Field et al., Neuron 25:437 (2000)). In striking
contrast, the pattern of mPER2-immunoreactivity in the SCN of
mCry-deficient mice was dramatically altered. There were extremely
few mPER2-immunoreactive cells in the SCN of mCry-deficient animals
at either circadian time (CT 12, 12.+-.1, n=3; CT 24, 8.+-.2,
n=3).
[0221] In the wild-type mice, the mPER2 staining profiles were
clearly nuclear, with well-defined outlines and nucleoli devoid of
reaction product. In the few mPER2-immunoreactive cells in the SCN
of mCry-deficient mice, low level mPER2 staining was observed in
the nucleus, but the profiles were poorly defined and low intensity
perinuclear staining could also be observed. As for mPER1, genotype
had no discernible effect on nuclear mPER2 immunoreactivity in the
piriform cortex, although there was evidence of a low level of
perinuclear immunoreactivity for mPER2 in piriform cortex of
mCRY-deficient mice.
[0222] The marked reduction of mPER2 staining in the SCN of
mCry-deficient animals suggests that the mCRY proteins are either
directly or indirectly important for mPER2 stability, as mPER2 RNA
levels are at tonic intermediate to high levels in mCry-deficient
mice, similar to those found for mPer1 RNA levels (Okamura et al.,
Science 286:2531 (1999)). It seems unlikely that our assay is
incapable of detecting mPER2 in the cytoplasm of mCry-mutants,
since the PER2 antibody can detect cytoplasmically localized
antigen in SCN cells (Field et al., Neuron 25:437 (2000)).
[0223] The low levels of mPER2 immunoreactivity in the SCN of
mCry-deficient mice, in conjunction with tonically low Bmal1 RNA
levels, is consistent with an important role of mPER2 in the
positive regulation of the Bmal1 loop. Since mPER1 is present in
SCN nuclei in mCry-deficient mice, yet Bmal1 RNA is low, it appears
likely that mPER1 likely has little effect on the positive
regulation of the Bmal1 feedback loop or negative regulation of the
mPer1-3 cycles.
[0224] The immunohistochemical data also indicate that mPER1 and
mPER2 can each enter the nucleus even in the absence of mCRY:mPER
interactions. mPER1 is expressed in the nucleus of SCN neurons from
mCry-deficient mice, and both mPER1 and mPER2 are constitutively
expressed in the nucleus of cells in the piriform cortex of
mCry-deficient animals. The phosphorylation state of mPER1 dictates
its cellular location in the absence of mPER1:mCRY interactions,
since its phosphorylation by casein kinase I epsilon leads to
cytoplasmic retention in vitro. Thus, the nuclear location of both
mPER1 and mPER2 in vivo may depend on several factors, including
interactions with mCRY and other proteins and their
phosphorylation.
EXAMPLE 15
[0225] mCRY-Induced Inhibition of Transcription
[0226] The intermediate to high levels of mPer1 and mPER2 gene
expression throughout the circadian day in mCry-deficient mice
(Okamura et al., Science 286:2531 (1999); Vitaterna et al., Proc.
Natl. Acad. Sci. USA 96:12114 (1999)) is consistent with a
prominent role of the mCRY proteins in negatively regulating
CLOCK:BMAL1-mediated transcription, as in vitro data have suggested
(Kume et al., Cell 98:193 (1999)). The endogenous expression of the
mCry1, mCry2, and mPer1-3 genes in mammalian cell lines, however,
has obscured rigorous in vitro analysis of the mechanism.
Therefore, an insect cell line, Schneider (S2) cells, a Drosophila
cell line that expresses cycle (the Drosophila Bmal1) but not per,
Tim, and clock (Saez et al., Neuron 17:911 (1996); Darlington et
al., Science 280:1599 (1998)), was used to study the negative
regulation of mCRY1 and mCRY2 on E box-mediated transcription with
a luciferase reporter that consists of a tandem repeat of the
Drosophila per E box (CACGTG) and flanking nucleotides fused to
hsp70 driving luciferase (Darlington et al., Science 280: 1599
(1998)). Briefly, S2 cells were transfected with Cellfectin.TM.
(Gibco BRL). Each transfection consisted of 10 to 100 ng of
expression plasmid with indicated inserts in pAC5.1-V5, 10 ng
luciferase reporter, and 25 ng of .beta.-gal internal control
plasmid (driven by baculovirus immediate-early gene, ie-1
promoter). Total DNA for each transfection was normalized using
pAC5.1-V5. Cells were harvested 48 hrs after tranfection.
Luciferase activity was normalized by determining
luciferase:.beta.-gal activity ratios and averaging the values from
triplicate wells.
[0227] Since S2 cells express endogenous cyc, transfection with
dclock alone caused a large increase in transcriptional activity
(265-fold), as described (Darlington et al., Science 280: 1599
(1998)). As for dCRY (Ceriani et al., Science 285:553 (1999)), this
activation was not inhibited by either mCRY1 or mCRY2. When
co-transfected, mCLOCK and syrian hamster (sh)BMAL1 heterodimers
induced a large increase in transcriptional activity (1744-fold)
that was reduced by >90% by mCRY1 or mCRY2 (FIG. 16). Moreover,
cotransfection of shBmal1 and human (h)Mop4, but not transfection
of hMop4 alone, similarly caused a large increase in
transcriptional activity in S2 cells (539-fold), like that
previously found for hMOP4:shBMAL1 heterodimers in mammalian cells
(Hogenesch et al., Proc. Natl. Acad. Sci. USA. 95:5474 (1998); Kume
et al., Cell 98:193 (1999)). hMOP4:shBMAL1-mediated transcription
was also blocked by either mCRY1 or mCRY2 (FIG. 16). The
mCLOCK:shBMAL1- and hMOP4:shBMAL1-induced transcription in S2 cells
was dependent on an intact CACGTG E box, because neither
heterodimer caused an increase in transcription when a mutated E
box reporter was used in the transcriptional assay.
Immunofluorescence of epitope-tagged mCRY1 or mCRY2 expressed in S2
cells showed that each was >90% nuclear in location, as in
mammalian cells (Kume et al., Cell 98:193 (1999)).
[0228] These data indicate that mCRY1 and mCRY2 are nuclear
proteins that can each inhibit mCLOCK:shBMAL1-induced transcription
independent of the mPER and mTIM proteins and of each other. The
results also show that the inhibitory effect is not mediated by the
interaction of either mCRY1 or mCRY2 with the E box itself, since E
box-mediated transcription was not blocked by the mCRY proteins
when transcription was activated by dCLOCK:CYC heterodimers. It
thus appears that the mCRY proteins inhibit mCLOCK:shBMAL1-mediated
transcription by interacting with either or both of the
transcription factors, since a similar inhibition was found with
hMOP4:shBMAL1-induced transcription. The system was performed as
described in Gekakis et al. (Science 270:811 (1995)). Yeast
two-hybrid assays revealed strong interactions of each mCRY protein
with mCLOCK and shBMAL1. Weaker interactions were detected between
each mCRY protein and hMOP4. This is further evidence of
functionally relevant associations of each mCRY protein with each
of the three transcription factors (Griffin et al., Science 286:768
(1999)).
[0229] Next it was determined whether the mCRY-induced inhibition
of transcription was through interaction with CLOCK and/or BMAL1.
Since neither mCRY1 or mCRY2 inhibited dCLOCK:CYC mediated
transcription, the ability of each to inhibit
dCLOCK:shBMAL1-mediated transcription was examined. This aspect of
study could not be examined in S2 cells, because of the strong
activation induced by transfecting dclock alone in S2 cells where
there is strong endogenous cyc expression. Briefly, luciferase
reporter gene assays were performed in COS-7 cells as described
(Jin et al., Cell 96:57 (1999)). mCRY1 and mCRY2 completely
inhibited mCLOCK:shBMAL1- and hMOP4:shBMAL1-induced transcription
in COS-7 cells (FIG. 17, Left and Middle, respectively), while the
cryptochromes did not inhibit dCLOCK:shBMAL1-mediated transcription
by more than 20% (FIG. 17). Thus, mCRY inhibits
mCLOCK:shBMAL1-induced transcription through interaction with
either mCLOCK alone or through an association with both mCLOCK and
BMAL1 in a multiprotein complex. Unfortunately, the examination of
the inhibition of mCLOCK:CYC heterodimers was not possible, because
co-transfection of mClock and cyc did not activate transcription in
either insect cells or mammalian cells, even though strong
interactions between mCLOCK and CYC were detected in yeast.
EXAMPLE 16
[0230] Identifying a Role for Mouse Tim
[0231] To delineate potential functions for mTim, the gene was
disrupted by targeted mutagenesis. A targeting vector was designed
from a 15 kb genomic clone in which a portion of the gene was
replaced with a PGK-Neo cassette; this deletion-insertion disrupts
mTIM after codon 178 (of 1197). Homologous recombination of the
targeted allele was obtained in 129/Sv J1 embryonic stem cells, and
two clones were microinjected into C57BL/6 mouse blastocysts.
Chimeric offspring were mated and germline transmission was
obtained.
[0232] When heterozygous animals were crossed, the resulting
litters contained a 1:2 ratio of wild-type to heterozygous
offspring, but no homozygous mutants. Of the offspring analyzed by
Southern blotting, 29 contained only the wild-type allele and 58
were heterozygous for the mTim mutation. These results are
consistent with mTim being essential for mouse survival.
[0233] Heterozygous mTim mutant embryos had reduced mTIM protein
levels, confirming the targeting event; wild-type
levels=8.04.+-.2.07 (mean.+-.SEM; n=4) versus heterozygote
levels=2.98.+-.0.61 (n=5; p<0.05, unpaired t test). Heterozygous
mTim mutants had no obvious developmental or behavioral
abnormalities.
[0234] Heterozygous mTim mutant animals displayed circadian rhythms
in locomotor activity indistinguishable from wild-type mice of
isogenic background. Rhythmic wheel-running activity of both groups
persisted in constant conditions (>25 days). Furthermore, the
period of locomotor activity was unchanged; wild-type mice
displayed a period of 23.52.+-.0.22 hrs (n=4) vs. 23.73.+-.0.13 hrs
(n=8) for heterozygotes (p>0.05, Student's t-test). The lack of
period change in heterozygotes does not rule out a clock-relevant
function for mTim, because the null Tim mutation in Drosophila is
recessive.
[0235] The mortality rate of homozygous mTim mutant embryos at
different gestational ages was next determined. Histological
analysis of embryos from 13 litters from heterozygous mTim mutant
crosses spanning embryonic day (ED) 6.5 to 11.5 showed a mortality
rate of 41%. When corrected for naturally occurring prenatal
attrition (14%, determined from heterozygous female X wild-type
male matings), the lethality rate was 25.5%, consistent with the
predicted Mendelian rate for a mutation that is lethal when
homozygous.
[0236] Developmental defects due to the mTim mutation were striking
at ED 7.5. At this stage, presumptive homozygous embryos lack any
cellular organization, with necrotic cell debris filling the
amniotic cavity, and resorption by surrounding maternal tissues has
already begun. Developmental abnormalities were observed in embryos
as early as ED 5.5 (data not shown), indicating that mTim is
essential for development around the time of implantation. The
mechanism behind the essential role of mTIM for mouse development
is currently not known. At ED 7.5, in situ hybridization showed
that mTim RNA is expressed throughout the embryo, particularly in
the embryonic germ cell layers and in the ectoplacental cone.
[0237] The results show that mTim is essential for embryonic
development.
* * * * *
References