U.S. patent application number 09/776696 was filed with the patent office on 2006-12-14 for human fast-1 gene.
Invention is credited to Kenneth W. Kinzler, Bert Vogelstein, Leigh Zawel, Shibin Zhou.
Application Number | 20060281078 09/776696 |
Document ID | / |
Family ID | 22348724 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060281078 |
Kind Code |
A1 |
Zhou; Shibin ; et
al. |
December 14, 2006 |
Human fast-1 gene
Abstract
hFAST-1 is a human forkhead activin signal transducer gene. The
hFAST-1 protein has the ability to bind to human Smad2 and activate
an activin response element (ARE). The hFAST-1-dependent activation
of ARE is dependent on endogenous Smad4 and stimulation of the
TGF-.beta. receptor. The hFAST-1 protein binds to a novel DNA
motif, TGT(G/T)(T/G)ATT, which is present within the ARE. This
motif is important for the activation of genes responsive to
ligands of the TGF-.beta. family. The invention includes tools for
investigating the TGF-.beta. signaling pathway and screening for
compounds which modulate the action of TGF-.beta..
Inventors: |
Zhou; Shibin; (Cockeysville,
MD) ; Zawel; Leigh; (Bowie, MD) ; Vogelstein;
Bert; (Baltimore, MD) ; Kinzler; Kenneth W.;
(BelAir, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
22348724 |
Appl. No.: |
09/776696 |
Filed: |
February 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09521109 |
Mar 7, 2000 |
6225441 |
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09776696 |
Feb 6, 2001 |
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09113309 |
Jul 10, 1998 |
6110738 |
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09521109 |
Mar 7, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/320.1; 435/325; 435/6.14; 435/69.1; 435/7.1; 530/350;
530/388.1; 536/23.2 |
Current CPC
Class: |
C07K 14/4705 20130101;
G01N 33/6872 20130101; G01N 2333/495 20130101; G01N 2500/00
20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/069.1; 435/320.1; 435/325; 530/350; 530/388.1;
536/023.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/475 20060101
C07K014/475; C07K 16/22 20060101 C07K016/22 |
Goverment Interests
[0001] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of USPHS grant CA43460 awarded by the National Institutes of
Health.
Claims
1. An isolated and purified hFAST-1 protein comprising the amino
acid sequence shown in SEQ ID NO:2 and naturally occurring
biologically active variants thereof.
2. A fusion protein which comprises a first protein segment and a
second protein segment fused to each other by means of a peptide
bond, wherein the first protein segment consists of at least
thirteen contiguous amino acids selected from the amino acid
sequence shown in SEQ ID NO:2.
3. An isolated and purified polypeptide which consists of at least
thirteen contiguous amino acids of hFAST-1 as shown in SEQ ID
NO:2.
4. The isolated and purified polypeptide of claim 3 which binds to
a Smad2 protein as shown in SEQ ID NO:3.
5. The isolated polypeptide of claim 3 wherein the at least
thirteen contiguous amino acids of hFAST-1 comprise amino acids
277-364 of SEQ ID NO:2.
6. The isolated polypeptide of claim 3 wherein the at least
thirteen contiguous amino acids of hFAST-1 comprise amino acids
221-365 of SEQ ID NO:2.
7. A preparation of antibodies which specifically bind to an
hFAST-1 protein as shown in SEQ ID NO:2.
8. The preparation of antibodies of claim 7 wherein the antibodies
are monoclonal.
9. The preparation of antibodies of claim 7 wherein the antibodies
are purified from an animal antiserum.
10. The preparation of antibodies of claim 7 wherein the antibodies
are affinity purified.
11. A subgenornic polynucleotide which encodes an hFAST-1 protein
as shown in SEQ ID NO:2.
12. The subgenomic polynucleotide of claim 11 which is
intron-free.
13. The subgenomic polynucleotide of claim 11 which comprises the
sequence shown in SEQ ID NO:1.
14. A vector comprising the polynucleotide of claim 11.
15. A vector comprising the polynucleotide of claim 12.
16. A vector comprising the polynucleotide of claim 13.
17. A recombinant host cell which comprises the polynucleotide of
claim 11.
18. A recombinant host cell which comprises the polynucleotide of
claim 12.
19. A recombinant host cell which comprises the polynucleotide of
claim 13.
20. A recombinant DNA construct for expressing hFAST-1 antisense
nucleic acids, comprising: a promoter, and a coding sequence for
hFAST-1 consisting of at least 12 contiguous base pairs selected
from SEQ ID NO:1, wherein the coding sequence is in an inverted
orientation with respect to the promoter, such that upon
transcription from said promoter an RNA is produced which is
complementary to native mRNA encoding hFAST-1.
21. The construct of claim 20 further comprising a transcription
terminator, wherein the coding sequence is between the promoter and
the terminator.
22. A method of screening test compounds for those which inhibit
the action of TGF-.beta., comprising the steps of: contacting a
test compound with a first protein which is all or a portion of a
Smad2 protein or a naturally occurring biologically active variant
thereof, wherein the portion of the Smad2 protein is capable of
binding to hFAST-1, and a second protein which is all or a portion
of hFAST-1 or a naturally occurring biologically active variant
thereof, wherein the portion of hFAST-1 is capable of binding to
the portion of the Smad2 protein; and determining an amount
selected from the group consisting of: (a) the first protein bound
to the second protein, (b) the second protein bound to the first
protein, (c) the first protein which is not bound to the second
protein, and (d) the second protein which is not bound to the first
protein, wherein a test compound which decreases the amount of (a)
or (b) or increases the amount of (c) or (d) is a candidate
compound for inhibiting the action of TGF-.beta..
23. The method of claim 22 wherein the step of contacting is
performed in vitro.
24. The method of claim 22 wherein the step of contacting is
performed by contacting a test compound with a cell which expresses
the first protein and the second protein.
25. The method of claim 23 wherein the test compound is contacted
with one of the two proteins prior to contacting with the other
protein.
26. The method of claim 23 wherein one of the two proteins is bound
to a solid support.
27. The method of claim 23 wherein at least one of the two proteins
is radiolabeled.
28. The method of claim 23 wherein at least one of the two proteins
is a fusion protein.
29. The method of claim 23 wherein at least one of the two proteins
is a fusion protein that has a detectable enzyme activity.
30. A method of screening test compounds for the ability to
decrease or augment TGF-.beta. activity, comprising the steps of:
(a) contacting a cell with a test compound, wherein the cell
comprises: i) a first fusion protein comprising (1) a DNA binding
domain or a transcriptional activating domain and (2) all or a
portion of an hFAST-1 protein, wherein the portion consists of a
contiguous sequence of amino acids selected from the amino acid
sequence shown in SEQ ID NO:2, wherein the portion is capable of
binding to Smad2 protein; ii) a second fusion protein comprising
(1) a DNA binding domain or a transcriptional activating domain and
(2) all or a portion of Smad2 protein, or a naturally occurring
biologically active variant thereof wherein the portion is capable
of binding to hFAST-1 protein, wherein when the first fusion
protein comprises a DNA binding domain, the second fusion protein
comprises a transcriptional activating domain, and when the first
fusion protein comprises a transcriptional activating domain, the
second fusion protein comprises a DNA binding domain, wherein the
interaction of the portion of the hFAST-1 protein with the portion
of Smad2 protein reconstitutes a sequence-specific transcriptional
activating factor, ii) a reporter gene comprising a DNA sequence to
which the DNA binding domain of the first or second fusion protein
specifically binds; and iv) hSmad4 protein; and (b) measuring the
expression of the reporter gene, a test compound which increases
the expression of the reporter gene being a potential drug for
increasing TGF-.beta. activity, and a test compound which decreases
the expression of the reporter gene being a potential drug for
decreasing TGF-.beta. activity.
31. A method of screening for drugs with the ability to decrease or
augment TGF-.beta. activity comprising the steps of: (a) contacting
a cell with a test compound and with TGF-.beta., wherein the cell
comprises: (i) all or a portion of Smad2 protein or a naturally
occurring biologically active variant thereof, wherein the portion
of Smad2 protein is capable of binding to hFAST-1; (ii) all or a
portion of hFAST-1 or a naturally occurring biologically active
variant thereof, wherein the portion of hFAST-1 is capable of
binding to Smad2 protein; (iii) a vector which comprises a reporter
gene under the control of an activin response element, wherein the
activin response element comprises an hFAST-1 binding motif
TGT(G/T)(T/G)ATT as shown in SEQ ID NO:4; and (iv) hSmad 4 protein;
and (b) measuring transcription of the reporter gene, a test
compound which increases the amount of reporter gene transcription
being a potential drug for augmenting TGF-.beta. activity, and a
test compound which decreases the amount of reporter gene
transcription being a potential drug for decreasing TGF-.beta.
activity.
32. A recombinant construct which comprises a reporter gene under
the control of an activin response element, wherein the activin
response element comprises an hFAST-1 binding motif
TGT(G/T)(T/G)ATT as shown in SEQ ID NO:4.
33. The recombinant construct of claim 32 wherein the construct
comprises a vector.
34. The recombinant construct of claim 32 wherein the reporter gene
encodes a non-human protein.
35. The recombinant construct of claim 34 wherein the non-human
protein is selected from the group consisting of green fluorescent
protein (GFP), luciferase, chloramphenicol acetyltransferase, and
.beta.-galactosidase.
36. A double-stranded DNA fragment which comprises an activin
response element which comprises an hFAST-1 binding motif
TGT(G/T)(T/G)ATT as shown in SEQ ID NO:4, wherein the fragment is
covalently attached to an insoluble polymeric support.
37. An isolated and purified oligonucleotide which encodes at least
thirteen contiguous amino acids of hFAST-1 protein as shown in SEQ
ID NO:2.
38. An isolated and purified oligonucleotide which comprises at
least 19 contiguous nucleotides of hFAST-1 as shown in SEQ ID
NO:1.
39. The isolated oligonucleotide of claim 38 which is
radiolabeled.
40. The isolated oligonucleotide of claim 38 which is fluorescently
labeled.
41. The isolated oligonucleotide of claim 38 which comprises a
sense strand.
42. The isolated oligonucleotide of claim 38 which comprises an
antisense strand.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] The invention is related to the area of developmental and
cancer genetics. In particular it is related to the field of
transcriptional regulation.
BACKGROUND OF THE INVENTION
[0003] Substantial progress in understanding the responses to
tumor-derived growth factor-.beta. (TGF-.beta.) and related ligands
has been made in the last five years (Derynck and Fang, 1997;
Hoodless and Wrana, 1998; Kretzschmar and Massague, 1998). The
receptors for these ligands have been cloned and shown to be
serine/threonine kinases which are activated by binding to ligand.
The major substrates for these kinases, besides the receptors
themselves, appear to be Smad proteins. The founding member of the
Smad family is the product of the Drosophila gene Mad, identified
by its requirement in signaling by the TGF-.beta. family member Dpp
(Sekelsky et al., 1995). Nine homologs of Mad have since been
identified in vertebrate cells and shown to transduce or inhibit
signals from specific TGF-.beta. like ligands (Heldin et al., 1997;
Derynck and Fang, 1997; Hoodless and Wrana, 1998; Kretzschmar and
Massague, 1998).
[0004] The phosphorylation of Smad1, Smad2, and Smad3 stimulates
their interaction with Smad4 and the transport of the resulting
heteromeric complex to the nucleus (Kretzschmar et al., 1997; Lagna
et al., 1996; Liu, 1997; Macias-Silva et al., 1996; Nakao et al.,
1997; Nakao et al., 1997; Souchelnytskyi et al., 1997). Once in the
nucleus, the Smad complex transcriptionally activates specific
target genes through activation domains present at the carboxyl
termini of these proteins (Liu et al., 1996). Two ways in which
Smad activation could lead to transcriptional activation have been
identified. First, it has been shown that human Smad3 and Smad4,
but not Smad2, can bind to specific DNA sequences and activate
transcription of adjacent reporters (Zawel et al., 1998). A similar
sequence-specific activity is present in Drosophila Mad (Kim et
al., 1997). Second, Smad2 has been shown to bind to the Xenopus
forkhead activin signal transducer protein FAST-1 (xFAST-1) and to
participate in a complex exhibiting sequence specific binding
activity attributable to the xFAST-1 component (Chen et al., 1996;
Chen et al., 1997; Liu, 1997). Although Smad4 does not directly
bind to xFAST-1, Smad4 is recruited to the xFAST-1/Smad2 complex by
Smad2 (Chen et al., 1997; Liu, 1997).
[0005] TGF-.beta.-like responses are remarkably widespread in
eukaryotes, and are important not only in development but also in
cancer (Fynan and Reiss, 1993; Hartsough and Mulder, 1997). Further
progress in understanding the varied developmental and oncogenic
ramifications of these pathways in mammalian cells depends on
knowledge of the relevant mammalian genes. Thus, there is a need in
the art for the identification, isolation, purification, and
analysis of mammalian and human genes which mediate physiological
and pathological responses to TGF-.beta. and related ligands.
SUMMARY OF THE INVENTION
[0006] It is an object of the invention to provide reagents and
methods for altering TGF-.beta. activity. These and other objects
of the invention are provided by one or more of the embodiments
described below.
[0007] One embodiment of the invention provides an isolated and
purified hFAST-1 protein comprising the amino acid sequence shown
in SEQ ID NO:2 and naturally occurring biologically active variants
thereof.
[0008] Another embodiment of the invention provides a fusion
protein which comprises a first protein segment and a second
protein segment fused to each other by means of a peptide bond. The
first protein segment consists of at least thirteen contiguous
amino acids selected from the amino acid sequence shown in SEQ ID
NO:2.
[0009] Still another embodiment of the invention provides an
isolated and purified polypeptide which consists of at least
thirteen contiguous amino acids of hFAST-1 as shown in SEQ ID
NO:2.
[0010] Even another embodiment of the invention provides a
preparation of antibodies which specifically bind to an hFAST-1
protein as shown in SEQ ID NO:2.
[0011] Yet another embodiment of the invention provides a
subgenomic polynucleotide which encodes an hFAST-1 protein as shown
in SEQ ID NO:2.
[0012] Still another embodiment of the invention provides a vector
comprising a subgenomic polynucleotide which encodes an hFAST-1
protein as shown in SEQ ID NO:2.
[0013] Another embodiment of the invention provides a vector
comprising a subgenomic polynucleotide which encodes an hFAST-1
protein as shown in SEQ ID NO:2 and which is intron-free.
[0014] Yet another embodiment of the invention provides a vector
comprising a subgenomic polynucleotide which comprises the sequence
shown in SEQ ID NO:1.
[0015] Even another embodiment of the invention provides a
recombinant host cell which comprises a polynucleotide. The
polynucleotide encodes an hFAST-1 protein as shown in SEQ ID
NO:2.
[0016] Still another embodiment of the invention provides a
recombinant host cell which comprises a polynucleotide. The
polynucleotide encodes an hFAST-1 protein as shown in SEQ ID NO:2
and which is intron-free.
[0017] Yet another embodiment of the invention provides a
recombinant host cell which comprises a polynucleotide. The
polynucleotide comprises the sequence shown in SEQ ID NO: 1.
[0018] A further embodiment of the invention provides a recombinant
DNA construct for expressing hFAST-1 antisense nucleic acids. The
recombinant DNA construct comprises a promoter and a coding
sequence for hFAST-1. The coding sequence consists of at least 12
contiguous base pairs selected from SEQ ID NO:1. The coding
sequence is in an inverted orientation with respect to the
promoter. Upon transcription from the promoter an RNA is produced
which is complementary to native mRNA encoding hFAST-1.
[0019] Another embodiment of the invention provides a method of
screening test compounds for those which inhibit the action of
TGF-.beta.. A test compound is contacted with a first protein and a
second protein. The first protein is all or a portion of a Smad2
protein or a naturally occurring biologically active variant
thereof. The portion of the Smad2 protein is capable of binding to
hFAST-1. The second protein is all or a portion of hFAST-1 or a
naturally occurring biologically active variant thereof. The
portion of hFAST-1 is capable of binding to the portion of the
Smad2 protein. An amount selected from the group consisting of (a)
the first protein bound to the second protein, (b) the second
protein bound to the first protein, (c) the first protein which is
not bound to the second protein, and (d) the second protein which
is not bound to the first protein is determined. A test compound
which decreases the amount of (a) or (b) or increases the amount of
(c) or (d) is a candidate compound for inhibiting the action of
TGF-.beta..
[0020] Even another embodiment of the invention provides a method
of screening test compounds for the ability to decrease or augment
TGF-.beta. activity. A cell is contacted with a test compound. The
cell comprises a first fusion protein, a second fusion protein, a
reporter gene, and hSmad4 protein. The first fusion protein
comprises (1) a DNA binding domain or a transcriptional activating
domain and (2) all or a portion of an hFAST-1 protein. The portion
of hFAST-1 consists of a contiguous sequence of amino acids
selected from the amino acid sequence shown in SEQ ID NO:2. The
portion of hFAST-1 is capable of binding to Smad2 protein. The
second fusion protein comprises (1) a DNA binding domain or a
transcriptional activating domain and (2) all or a portion of Smad2
protein, or a naturally occurring biologically active variant
thereof. The portion of Smad2 is capable of binding to hFAST-1
protein. When the first fusion protein comprises a DNA binding
domain, the second fusion protein comprises a transcriptional
activating domain. When the first fusion protein comprises a
transcriptional activating domain, the second fusion protein
comprises a DNA binding domain. The interaction of the portion of
the hFAST-1 protein with the portion of Smad2 protein reconstitutes
a sequence-specific transcriptional activating factor. The reporter
gene comprises a DNA sequence to which the DNA binding domain of
the first or second fusion protein specifically binds. The
expression of the reporter gene is measured. A test compound which
increases the expression of the reporter gene is a potential drug
for increasing TGF-.beta. activity. A test compound which decreases
the expression of the reporter gene is a potential drug for
decreasing TGF-.beta. activity.
[0021] Still another embodiment of the invention provides a method
of screening for drugs with the ability to decrease or augment
TGF-.beta. activity. A cell is contacted with a test compound and
with TGF-.beta.. The cell comprises all or a portion of Smad2
protein or a naturally occurring biologically active variant
thereof and all or a portion of hFAST-1 or a naturally occurring
biologically active variant thereof. The portion of Smad2 protein
is capable of binding to hFAST-1. The portion of hFAST-1 is capable
of binding to Smad2 protein. The cell also comprises a vector and
hSmad4 protein. The vector comprises a reporter gene under the
control of an activin response element. The activin response
element comprises a DNA motif TGT(G/T)(T/G)ATT as shown in SEQ ID
NO:4. Transcription of the reporter gene is measured. A test
compound which increases the amount of reporter gene transcription
is a potential drug for augmenting TGF-.beta. activity. A test
compound which decreases the amount of reporter gene transcription
is a potential drug for decreasing TGF-.beta. activity.
[0022] Another embodiment of the invention provides a recombinant
construct which comprises a reporter gene under the control of an
activin response element. The activin response element comprises an
hFAST-1 binding motif TGT(G/T)(T/G)ATT as shown in SEQ ID NO:4.
[0023] A further embodiment of the invention provides a
double-stranded DNA fragment which comprises an activin response
element. The activin response element comprises an hFAST-1 binding
motif TGT(G/T)(T/G)ATT as shown in SEQ ID NO:4. The fragment is
covalently attached to an insoluble polymeric support.
[0024] Even another embodiment of the invention provides an
isolated and purified oligonucleotide which encodes at least
thirteen contiguous amino acids of hFAST-1 protein as shown in SEQ
ID NO:2.
[0025] Yet another embodiment of the invention provides an isolated
and purified oligonucleotide which comprises at least 19 contiguous
nucleotides of hFAST-1 as shown in SEQ ID NO: 1.
[0026] The invention thus provides the art with novel tools and
systems with which to probe and modify the molecular events of the
TGF-.beta. signal transduction pathway which result in
transcriptional activation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 displays the sequence of hFAST-1 and compares it to
the Xenopus homolog. Conserved amino acids are shaded. The forkhead
domain encompasses xFAST-1 residues 108 to 219 (Chen et al., 1996).
The C-terminal Smad-interacting domain (SID) encompasses xFAST-1
residues 380 to 506 (Chen et al., 1997).
[0028] FIG. 2 illustrates the expression of hFAST-1 and its
interaction with Smad2. FIG. 2A demonstrates that hFAST-1 was
expressed in all tissues tested. RNA samples prepared from the
indicated tissues were used as templates for RT-PCR analysis. The
PCR primers used span a 100-bp intron and discriminate the spliced
(423 bp) and unspliced (523 bp) RT-PCR products. The unspliced
products arose from either genomic DNA or from unprocessed
transcripts. FIG. 2B shows that both full-length hFAST-1 (FAST-FL)
and its carboxyl-terminus (FAST-SID) could interact with the
carboxyl-terminal (MH2) domain of Smad2 in vitro. Polypeptides
encoding full-length hFAST1 or its SID domain were generated by in
vitro translation in the presence of .sup.35S-labeled methionine
and incubated with a GST-fusion protein containing the carboxyl
terminus of Smad2 (GST-Smad2/MH2) immobilized on agarose beads. An
irrelevant protein (PIG3, Polyak et al., 1997) was also translated
and incubated with GST-Smad2MH2 as a control. After extensive
washing, the bound proteins were eluted and separated in a 4-20%
SDS-Tris-glycine gel which was dried and autoradiographed. Ten
percent of the in vitro translated proteins used for binding to
Smad2 were applied to the lanes labeled "Total."
[0029] FIG. 3 demonstrates hFAST-1 mediated transcriptional
activation. FIG. 3A shows that hFAST-1 mediated transcriptional
activation requires TGF-.beta.. MvLu1 cells were transfected with
pAR3-lux with or without pCMV-hFAST-1 (FAST-1). The transfected
cells were cultured in the presence or absence of TGF-.beta.1 (1
ng/ml). Twenty hours following transfection, cells were harvested
and luciferase activity measured. The results were normalized to
the control in which cells were neither transfected with
pCMV-hFAST-1 nor treated with TGF-.beta.. Bars and brackets
represents the means and standard deviations calculated from
triplicate transfections. FIG. 3B shows that activin signaling
leads to hFAST-1 mediated transcriptional activation. HCT116 cells
were cotransfected with pAR3-lux, pCMV-hFAST-1 (FAST-1), and the
constitutively active activin receptor ActRIB* as indicated.
Luciferase activity was analyzed 20 hours later and the results
normalized to controls transfected with reporter but without
pCMV-hFAST-1 or ActR1B*. FIG. 3C demonstrates that hFAST-1-mediated
transcriptional activation requires Smad4 and a functional hFAST-1
forkhead domain. HCT116 cells or their Smad4-deficient derivatives
(5-18) were transfected with pAR3-lux plus pCMV-hFAST-1 (wt
[FAST-1] or mutant H83R [FAST-1]) plus the RII receptor for
TGF-.beta. as indicated. All cells were treated with TGF-.beta.1
for 20 hours prior to harvest. Luciferase activity was normalized
to the control in which cells were not transfected with
pCMV-hFAST-1 or RII.
[0030] FIG. 4 demonstrates the sequence-specific DNA binding of
hFAST-1. FIG. 4A shows examples of an electrophoretic mobility
shift analysis (EMSA) of mock-selected or hFAST-1-selected clones.
.sup.32P-labeled PCR products generated from individual clones were
incubated with a GST-fusion protein containing full length hFAST-1
sequences. Derivation of clones and EMSA were performed as
described in Example 7. Mock selected clones were used for
comparison ("C" lanes). The positions of free probe and hFAST-1
bound probe ("shift") are indicated. FIG. 4B provides a sequence
summary of clones that bound to hFAST-1. The sequences of the
relevant segment of 17 hFAST-1-binding clones were determined and
the fractions of clones containing the nucleotides at the indicated
positions relative to the consensus are shown. FIG. 4C demonstrates
the binding of FBE to hFAST-1. Wild-type (FBE) or mutant (FBE*)
oligonucleotides were incubated with 0.5-2.0 .mu.g of GST fusion
proteins containing full-length hFAST-1 (FAST-FL) or only its
forkhead domain (FAST-FH). GST fusion proteins containing the MH1
or MH2 domains of Smad2 (S-N and S-C, respectively; Zawel et al.,
1998) were used as controls. The FBE* oligonucleotide contained the
sequence TCTGTATC in place of the consensus TGTGTATT but was
otherwise identical to FBE. FIG. 4D demonstrates the binding of ARE
oligonucleotides to hFAST-1. EMSA was performed with (+) or without
(-) 1 .mu.g of GST fusion protein containing full length FAST-1
("FAST-FL"). The sequence of the 50 bp ARE oligonucleotide differed
by only one bp from ARE*.
DETAILED DESCRIPTION OF THE INVENTION
[0031] We have isolated and characterized a human homolog of
Xenopus FAST-1, termed hFAST-1. hFAST-1 mediates transcriptional
responses to TGF-.beta. and activin in a ligand-, receptor-, and
Smad-dependent fashion.
[0032] hFAST-1 protein consists of 365 amino acid residues which
are shown in SEQ ID NO:2 and FIG. 1. A nuclear localization domain
is found at residues 22-30, and the adjacent downstream region
(approximately residues 33-154) is presumed to contain the forkhead
DNA-binding domain. The Smad2 binding domain is found near the
carboxy terminus.
[0033] The invention also includes naturally occurring biologically
active variants of hFAST-1. Naturally occurring biologically active
variants of hFAST-1 include proteins which have, for example,
conservative amino acid substitutions of amino acids of SEQ ID
NO:2. Such variants can result, for example, from polymorphisms in
an hFAST-1 coding sequence. Biologically active variants of hFAST-1
possess similar biological activity to that of the hFAST1 protein
shown in SEQ ID NO:2, such as the ability to bind to Smad 2, to
bind to the ARE binding motif of SEQ ID NO:4, and to activate
transcription.
[0034] hFAST-1 polypeptides consist of at least 13, 14, 15, 17, 20,
25, 30, 35, 40, 45, 50, 60, 70, 80, 87, 88, 100, 120, 140, 144,
145, or 150 contiguous amino acids of hFAST-1 as shown in SEQ ID
NO:2. Polypeptides can also comprise regions of the hFAST-1 amino
acid sequence which are involved in the binding of hFAST-1 to
Smad2. Such regions are located near the carboxy terminus of
hFAST-1, e.g., in the region from positions 277-364 or 221-365 of
SEQ ID NO:2. Polypeptides can also comprise the nuclear
localization region of hFAST-1, amino acids 22-30. An hFAST-1
protein or polypeptide can be isolated by physical separation from
the cells in which it is produced and separation from most of the
other proteins produced by the cells. Standard purification
techniques such as affinity or ion exchange chromatography, as well
as any other technique known in the art, can be used to purify the
protein or polypeptide. A protein or polypeptide preparation is
purified when it exists as a nearly homogeneous mixture consisting
of at least about 70, 75, 80, 85, 90, 95, 98, or 99% of the desired
molecular species.
[0035] hFAST-1 protein or polypeptides can also be produced by
recombinant DNA methods or by synthetic chemical methods. For
production of recombinant hFAST-1 protein or polypeptides, for
example, the coding sequence shown in SEQ ID NO:1 can be expressed
in known prokaryotic or eukaryotic expression systems. Bacterial,
yeast, insect, or mammalian expression systems can be used, as is
known in the art. Alternatively, synthetic chemical methods, such
as solid phase peptide synthesis, can be used to synthesize an
hFAST-1 protein or polypeptide.
[0036] The invention also provides non-naturally occurring fusion
proteins which comprise all or a portion of hFAST-1. In such a
fusion protein, a first protein segment is fused to a second
protein segment by means of a peptide bond. The first protein
segment consists of at least 13, 14, 15, 17, 20, 25, 30, 35, 40,
45, 50, 60, 70, 80, 87, 88, 100, 120, 140, 144, 145, or 150 amino
acids of hFAST-1 as shown in SEQ ID NO:2. The second protein
segment can be all or a portion of any protein whose structure or
function is desired to be combined with that of hFAST-1. An hFAST-1
fusion protein can be produced by using standard recombinant DNA
techniques to combine the sequences of the desired first and second
protein segments into an expression vector, which is introduced
into a cell or cell line that is subsequently induced to express
the fusion protein. The fusion protein may either be used within
the cell or cell line containing the vector or it can be isolated
and optionally purified from the cell or cell line, or from the
culture medium, using standard cell homogenization, extraction, and
protein purification methods.
[0037] Antibodies can be prepared which specifically bind to
epitopes of an hFAST-1 protein, polypeptide, or fusion protein.
Such antibodies can be immunoglobulins of any class, i.e., IgG,
IgA, IgD, IgE, or IgM. The antibodies can be obtained by
immunization of a mammal such as a mouse, rat, rabbit, goat, sheep,
primate, human, or other suitable species. The antibodies can be
whole immunoglobulins or fragments thereof, provided that specific
binding for hFAST-1 epitopes is maintained. Antibodies to hFAST-1
can be the result of genetic engineering, e.g., interspecies or
chimeric antibodies. The antibodies can be polyclonal antibodies
which are obtained from the serum of an immunized animal, i.e.,
antiserum. The antibodies can also be monoclonal antibodies, formed
by immunization of a mammal with an hFAST-1 antigen, fusion of
lymph or spleen cells from the immunized mammal with a myeloma cell
line, and isolation of specific hybridoma clones, as is known in
the art.
[0038] hFAST-1 antibodies can, if desired, be purified by any
method known in the art, e.g., affinity purification using a column
with hFAST-1 antigen as the affinity ligand. The antibodies can be
eluted from the column, for example, using a buffer with a high
salt concentration.
[0039] Antibodies which specifically bind to hFAST1 proteins,
polypeptides, or fusion proteins provide a detection signal at
least 5-, 10-, or 20-fold higher than a detection signal provided
with other proteins when used in Western blots or other
immunochemical assays. Preferably, antibodies which specifically
bind to hFAST-1 epitopes do not detect other proteins in
immunochemical assays and can immunoprecipitate a hFAST-1 protein,
polypeptide, or fusion protein from solution.
[0040] The invention also provides isolated subgenomic
polynucleotides which encode hFAST-1 protein and polypeptides.
Subgenomic polynucleotides contain less than a whole chromosome.
Preferably, the polynucleotides are intron-free. One subgenomic
polynucleotide encodes the hFAST-1 protein shown in SEQ ID NO:2.
hFAST1 polynucleotide molecules can also comprise a contiguous
sequence of at least 10, 11, 12, 15, 19, 20, 25, 30, 32, 35, 37,
40, 45, 50, 60, 70, 74, 80, 90, or 100 nucleotides selected from
SEQ ID NO: 1. Optionally, a subgenomic polynucleotide can comprise
the nucleotide sequence of SEQ ID NO:1.
[0041] The complement of the nucleotide sequence shown in SEQ ID
NO:1 is a contiguous nucleotide sequence which forms Watson-Crick
base pairs with a contiguous nucleotide sequence shown in SEQ ID
NO:1 and is also a subgenomic polynucleotide, which can be used to
provide hFAST-1 antisense oligonucleotides. A double-stranded
polynucleotide which comprises the nucleotide sequence shown in SEQ
ID NO:1 is also a subgenomic polynucleotide.
[0042] Isolated and purified oligonucleotides which encode at least
13 contiguous amino acids of hFAST1 protein as shown in SEQ ID
NO:2, or which comprise at least 19 contiguous nucleotides of SEQ
ID NO:1, are also included as subgenomic polynucleotides.
[0043] The hFAST-1 gene can be isolated by the method described in
Example 1. The gene is isolated when it is obtained free from
unrelated polynucleotide sequences, leaving only coding,
non-coding, and regulatory sequences associated with the expression
of hFAST-1 protein.
[0044] hFAST-1 subgenomic polynucleotides can be isolated and
purified free from other nucleotide sequences using standard
nucleic acid purification techniques. For example, restriction
enzymes and probes can be used to isolate polynucleotide fragments
which comprise nucleotide sequences encoding hFAST-1 protein.
Isolated and purified subgenomic polynucleotides are in
preparations which are free or at least 90% free of other
molecules. Optionally, hFAST-1 subgenomic polynucleotides can
contain sequences from non-coding regions of the hFAST-1 gene, such
as introns, or sequences from a promoter region or transcription
terminator region.
[0045] In order to clone, replicate, modify, express, or otherwise
manipulate hFAST-1 subgenomic polynucleotides, sequences of hFAST-1
can be incorporated into a recombinant construct. A recombinant
construct can be a linear or circular polynucleotide, e.g., a viral
DNA or RNA or a plasmid. Optionally, a recombinant construct is
capable of transferring desired nucleotide sequences into a
prokaryotic or eukaryotic cell. The construct can be a vector and
can contain additional nucleotide sequences such as replication
origins, promoters, transcription terminators, and reporter genes
to facilitate replication, insertion into the host cell genome,
expression, or detection of the vector. For example, an expression
vector can comprise a promoter capable of activating expression in
a host cell.
[0046] Vectors or recombinant constructs can be prepared by
standard recombinant DNA techniques. Vectors or other recombinant
constructs containing either native or modified hFAST-1 sequences
or fragments thereof can optionally contain sequences from other
proteins so as to create fusion proteins or can contain reporter
gene sequences. Protein sequences which serve as portions of fusion
proteins or as reporter genes can be from any human or non-human
protein. Any reporter gene, such as the genes for green fluorescent
protein (GFP), luciferase, chloramphenicol acetyltransferase, or
.beta.-galactosidase, can be incorporated into such vectors or
constructs in order to facilitate determination of the level or
localization of expression of hFAST-1 proteins or polypeptides. The
expression of such reporter genes can be detected, for example, as
fluorescence or as enzyme activity or by standard
immunocytochemical techniques.
[0047] hFAST-1 subgenornic polynucleotides can be incorporated into
an expression vector which is then used to transfect an appropriate
cell line, and used to produce hFAST-1 protein, polypeptides, or
fusion proteins containing all or a portion of hFAST-1. Using the
sequence information for hFAST-1 shown in SEQ ID NOS:1 and 2,
variants of hFAST-1 can be constructed which retain all or a
portion of the biological activity of hFAST-1.
[0048] A vector can be introduced into a suitable host cell by
standard transfection techniques, to produce a recombinant host
cell. Transfection with an hFAST-1 vector can be either transient
or stable, as required by the particular needs of an hFAST-1
expression protocol.
[0049] The recombinant host cell is a cell or cell line which is
suitable for transfection by the vector and for expression of the
hFAST-1 protein or polypeptide. Many different cell types are
suitable as the recombinant host cell. Examples of such cells are
the cells of bacteria, yeast, insects, amphibians, and mammals,
such as a mouse, rat, primate, human, or other suitable species.
Recombinant host cells can also be tumor cells grown either in cell
culture or in an animal, such as a nude mouse.
[0050] The orientation of hFAST-1 coding sequences in a recombinant
construct or an expression vector relative to promoter and
transcription terminator sequences can be as found in the native
hFAST-1 gene or can be inverted so as to allow the production of
hFAST-1 antisense oligonucleotides. If coding sequences are
utilized from the sense strand of the gene, i.e., the strand which
encodes the amino acid sequence of SEQ ID NO:2, expression of the
encoded amino acid sequence will result. If sequences from the
complementary (antisense) strand are utilized, then upon
transcription from the promoter, an RNA will be produced which is
complementary to native mRNA encoding hFAST-1. Antisense hFAST-1
oligonucleotides can be used to decrease expression of hFAST-1.
Optionally, the recombinant construct or vector can also comprise a
transcription terminator, in which case the inverted
hFAST-1-derived sequence is located between the promoter and
transcription terminator.
[0051] The invention also provides recombinant constructs and
double-stranded DNA fragments which can be used, for example, in
binding or transcriptional activating assays, using hFAST-1. A
recombinant construct of the invention can comprise a reporter gene
under the control of an activin response element. The activin
response element comprises an hFAST-1 binding motif as shown in SEQ
ID NO:4. Optionally, the recombinant construct can comprise a
vector, as described above. Any reporter gene which produces a
detectable product can be used. For example, the reporter gene can
encode a non-human protein, such as green fluorescent protein,
luciferase, chloramphenicol acetyltransferase, or
.beta.-galactosidase.
[0052] Double-stranded DNA fragments of the invention can comprise
an activin response element. The activin response element includes
an hFAST-1 binding motif, as shown in SEQ ID NO:4. Optionally, the
double-stranded DNA fragment can be covalently attached to an
insoluble polymeric support, such as a tissue culture plate, slide,
or nylon membrane.
[0053] Any polynucleotide or oligonucleotide of this invention can
be labeled using standard methods to facilitate detection. For
example, polynucleotides or oligonucleotides can be radiolabeled
with .sup.32P or covalently linked to a fluorescent or biotinylated
molecule.
[0054] The invention provides methods for screening for test
compounds which decrease or augment TGF-.beta. activity. Compounds
which decrease or augment TGF-.beta. activity can be used to modify
or regulate transcriptional activation associated with the
TGF-.beta. signaling pathway. Such compounds can be applied
therapeutically, for example, to alter the growth of tumor cells or
to alter normal or abnormal developmental processes.
[0055] Test compounds can be selected from natural substances
secreted, extracted, isolated, or purified from microbes, plants,
or animals, or can be synthetic agents. The test compounds can be
pharmacologic agents already known in the art or can be compounds
previously unknown to have any pharmacological activity.
[0056] In one embodiment of the invention, a test compound is
contacted with a first protein and a second protein. The first
protein comprises all or a portion of Smad2, or a naturally
occurring biologically active variant thereof, which is capable of
binding to hFAST-1. The second protein comprises all or a portion
of hFAST-1, or a naturally occurring biologically active variant
thereof, which is capable of binding to the portion of the Smad2
protein. Contacting can occur in vitro. The first and second
proteins can be produced recombinantly, isolated from human cells,
or synthesized by standard chemical methods. The binding sites can
be located on fill-length proteins, fusion proteins, or
polypeptides. If desired, the test compound can be contacted with
one of the two proteins prior to contacting with the other protein.
Optionally, the step of contacting can also be performed by
contacting a test compound with a cell which expresses the first
and second proteins. The cell can be a normal human cell, for
example, a breast, colon, thymus, or muscle cell, or can be a
related cell line.
[0057] Binding or dissociation of the first and second proteins in
the presence of the test compound can be determined by measuring
any of the following amounts: (a) the first protein which is bound
to the second protein, (b) the second protein which is bound to the
first protein, (c) the first protein which is not bound to the
second protein, or (d) the second protein which is not bound to the
first protein. The amount of a complex formed by the first and
second proteins can also be determined. The first or second protein
can be radiolabeled or labeled with fluorescent or enzymatic tags
and can be detected, for example, by scintillation counting,
fluorometric assay, monitoring the generation of a detectable
product, or by measuring the apparent molecular mass of the bound
or unbound proteins by gel filtration or electrophoretic mobility.
Either the first or second protein can be bound to a solid support,
such as a column matrix or a nylon membrane.
[0058] A test compound which decreases the amount of (a) or (b) or
which increases the amount of (c) or (d) is a candidate compound
for inhibiting the action of TGF-.beta.. Preferably, the test
compound decreases the amount of (a) or (b) or increases the amount
of (c) or (d) by at least 30-40%, more preferably by at least
40-60%, 50-70%, 60-80%, 70-90%, 75-95%, or 80-98%.
[0059] In another embodiment, test compounds can be screened for
their ability to decrease or augment TGF-.beta. related activity. A
cell is contacted with a test compound and with TGF-.beta.. The
cell comprises all or a portion of Smad2 protein, or a biologically
active variant thereof, which is capable of binding to hFAST-1. The
cell also comprises all or a portion of hFAST-1 protein, or a
biologically active variant thereof which is capable of binding to
Smad2. The cell also comprises hSmad4 protein.
[0060] Smad2, hFAST-1, and hSmad4 proteins or polypeptides can be
supplied to the cell, for example, by transfecting the cell with
DNA constructs which encode these proteins or polypeptides.
Alternatively, cell types which normally contain one or more of the
proteins or polypeptides can be used, such as normal breast, colon,
thymus, or muscle cells, or related cell lines.
[0061] The cell also comprises a vector. The vector comprises a
reporter gene under the control of an ARE. The ARE comprises a DNA
motif (hFAST-1 binding domain) as shown in SEQ ID NO:4. By
measuring the level of transcription or expression of the reporter
gene using standard methods, the effect of the test compound can be
determined. A test compound which increases the amount of reporter
gene transcription or expression is a potential drug for augmenting
TGF-.beta. activity, and a test compound which decreases the amount
of reporter gene transcription or expression is a potential drug
for decreasing TGF-.beta. activity. Preferably, the test compound
increases or decreases the amount of transcription or expression of
the reporter gene by at least 30-40%, more preferably by at least
40-60%, 50-70%, 60-80%, 70-90%, 75-95%, or 80-98%.
[0062] In another embodiment of the invention, a two hybrid method
can be used to evaluate the binding of all or portions of hFAST-1
with other proteins such as Smad2. A cell can be contacted with a
test compound to screen for drugs which have the ability to
decrease or augment TGF-.beta. activity.
[0063] The cell comprises two fusion proteins, which can be
provided to the cell by means of expression constructs. The first
fusion protein comprises either a DNA binding domain or a
transcriptional activating domain and all or a portion of an hFAST1
protein or a naturally occurring biologically active variant of
hFAST-1. The portion of hFAST-1 consists of a contiguous sequence
of amino acids selected from the amino acid sequence shown in SEQ
ID NO:2 and is capable of binding to Smad2. The portion of hFAST-1
can be selected so that it comprises neither a DNA binding domain
nor a transcriptional activation domain. The second fusion protein
comprises either a DNA binding domain or a transcriptional
activating domain and all or a portion of Smad2 or a naturally
occurring biologically active variant of Smad2. The portion of
Smad2 is that portion which is capable of binding to hFAST-1. If
the first fusion protein comprises a transcriptional activating
domain, the second fusion protein comprises a DNA binding domain.
On the other hand, if the first fusion protein comprises a DNA
binding domain, the second fusion protein comprises a
transcriptional activating domain.
[0064] The cell also comprises a reporter gene comprising a DNA
sequence to which the DNA binding domain specifically binds. When
the portion of hFAST-1 and the portion of Smad2 bind, the DNA
binding domain and the transcriptional activating domain will be in
close enough proximity to reconstitute a transcriptional activator
capable of initiating transcription of the detectable reporter gene
in the cell. The expression of the reporter gene in the presence of
the test compound is then measured. A test compound which decreases
expression of the reporter gene is a potential drug for increasing
TGF-.beta. activity. A test compound which decreases the expression
of the reporter gene is a potential drug for decreasing TGF-.beta.
activity. Preferably, the test compound increases or decreases
reporter gene expression by at least 30-40%. More preferably, the
test compound increases or decreases reporter gene expression by at
least 40-60%, 50-70%, 60-80%, 70-90%, 75-95%, or 80-98%.
[0065] Many DNA binding domains and transcriptional activating
domains can be used in this system, including the DNA binding
domains of GAL4, LexA, and the human estrogen receptor paired with
the acidic transcriptional activating domains of GAL4 or the herpes
virus simplex protein VP16 (See, e.g., G. J. Hannon et al., Genes
Dev. 7, 2378, 1993; A. S. Zervos et al., Cell 72, 223, 1993; A. B.
Votjet et al, Cell 74, 205, 1993; J. W. Harper et al., Cell 75,
805, 1993; B. Le Douarin et al., Nucl. Acids Res. 23, 876, 1995). A
number of plasmids known in the art can be constructed to contain
the coding sequences for the fusion proteins using standard
laboratory techniques for manipulating DNA (see Example 1, infra).
Suitable detectable reporter genes include the E. coli lacZ gene,
whose expression can be measured colorimetrically (e.g., Fields and
Song, supra), and yeast selectable genes such as HIS3 (Harper et
al., supra, Votjet et al., supra; Hannon et al., supra) or URA3 (Le
Douarin et al., supra). Methods for transforming cells are also
well known in the art. See, e.g., Hinnen et al., Proc. Natl. Acad.
Sci. U.S.A. 75, 1929-1933, 1978.
[0066] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples, which are provided
herein for purposes of illustration only and are not intended to
limit the scope of the invention.
EXAMPLE 1
[0067] Example 1 describes the isolation of the hFAST-1 gene.
[0068] Sequences corresponding to xFAST-1, but outside the forkhead
domain, were used to search the National Center for Biotechnology
Information (NCBI) nucleotide sequence database `dbest` using the
BLAST program `tblastn`. An EST sequence (accession # AA218611) was
identified based on its homology with the Smad interaction domain
of xFAST-1. Primers were designed to extend the EST sequence using
a RACE method. Briefly, nested PCR was performed using CLONTECH's
Marathon-ready Human Colorectal Adenocarcinoma cDNA as the initial
template and a set of EST-specific primers in combination with the
AP1 or AP2 primers provided with the Marathon-ready cDNA. After two
rounds of PCR amplification, the PCR products were gel-purified and
sequenced using Thermo Sequenase (Amersham).
[0069] To ensure the correctness of the sequence, the sequences of
multiple independent PCR products from cDNA and genomic DNA were
determined. Multiple stop codons in all three reading frames were
identified at both 5' and 3' ends of the PCR products and used to
derive a long ORF defining hFAST-1. The first in-frame methionine
in this ORF was assumed to be the initiation site for translation,
and the sequences surrounding this methionine matched the Kozak
consensus (Kozak, 1992).
[0070] A sequence alignment between hFAST-1 and xFAST-1 was carried
out using the MACAW multiple alignment software (v2.01). The
results are shown in FIG. 1. The coding sequence of the hFAST-1
gene is shown in SEQ ID NO:1. The corresponding amino acid sequence
is shown in SEQ ID NO:2.
[0071] The hFAST-1 and xFAST-1 genes are considerably divergent.
There are only two regions of significant similarity between
xFAST-1 and hFAST-1, corresponding to the presumptive DNA-binding
forkhead domain and the carboxyl terminal Smad-binding domain (FIG.
1). A prominent nuclear localization domain (hFAST-1 residues
22-30) was conserved at the amino-terminal end of the forkhead
domain of both proteins.
EXAMPLE 2
[0072] Example 2 demonstrates expression of hFAST-1.
[0073] RT-PCR was performed with Platinum Taq DNA polymerase
(GibcoBRL) and primers NT2-11 (5'-CTGGAAAGACTCCATTCG-3'; SEQ ID NO:
5) and NT2-8 (5'-CACAGAGGCCTCTCAGAAG-3'; SEQ ID NO: 6). These
primers span an intron and thereby allow discrimination of
mRNA-derived PCR products from those derived from genomic DNA or
unprocessed RNA. The cDNA templates were prepared from total RNA of
different normal tissues using SuperScript II reverse transcriptase
(GibcoBRL) and random hexamers as primers (Thiagalingam et al.,
1996).
[0074] The hFAST-1 gene appeared to be expressed in all normal
human tissues tested, including those of breast, colon, thymus, and
muscle, as well as in several cancer cell lines FIG. 2A).
EXAMPLE 3
[0075] Example 3 demonstrates chromosomal mapping of the hFAST-1
gene.
[0076] A genomic clone containing hFAST-1 was obtained by screening
a bacterial artificial chromosome (BAC) library. This clone was
used in fluorescence in situ hybridization (FISH) analyses of human
metaphase spreads, revealing that the hFAST-1 gene resided at
chromosome 8q24.
[0077] For chromosomal mapping of the hFAST-1 gene, two independent
BAC clones containing the hFAST-1 gene were labeled with
biotin-16-dUTP by nick translation. Human prometaphase chromosome
spreads were fixed on slides and pretreated with RNase and pepsin.
Multicolor FISH was performed as described (Lengauer et al., 1997).
Hybridization signals were detected with FITC Avidin-DCS (Vector),
and chromosomes were counterstained with DAPI. The resulting
banding pattern and hybridization signals were evaluated by
epifluorescence microscopy with a Nikon Eclipse E800.
[0078] Fifty randomly selected prometaphases were evaluated for
each clone, and each of them showed hybridization signals on the
distal long arm of both chromatids at chromosomal region 8q24.3.
The chromosomal location was confirmed by double hybridization of
hFAST-1 sequences and a centromere probe specific for chromosome 8
(Dunham et al., 1992). Fine-mapping of hFAST-1 to the 8q24.3 band
was confirmed by fractional length measurements (Lichter et al.,
1990).
EXAMPLE 4
[0079] This example demonstrates sequence analysis of hFAST-1 in
colon cancer cells.
[0080] Many studies have shown that TGF-.beta. responsiveness is
abrogated during tumorigenesis (Fynan and Reiss, 1993). To
determine whether the hFAST-1 gene was commonly altered in cancers,
its sequence was examined in 45 colorectal cancer cell lines
passaged in vitro or as xenografts in nude mice. For this purpose,
the structure and sequence of the gene were determined from PCR
analyses of genomic DNA and cDNA, revealing two small introns, at
codons 58/59 and 93/94, respectively. Genomic DNA was PCR-amplified
with primers NT2-12 (5'-CCCCCTTCCATCCGAATG-3'; SEQ ID NO:7) and
NT2-3 (5'-GAGCTGCTGTGTCGCAGAC-3'; SEQ ID NO: 8). This amplification
resulted in a 1750 bp PCR product containing the entire coding
region of hFAST-1 plus its two introns. After gel purification, the
PCR products were sequenced using Thermo Sequenase (Amersham).
Complete sequence determination of the coding sequence plus the two
introns in the 45 tumors revealed no variations from the wild-type
sequence other than three polymorphisms (one silent change at codon
150, one serine to threonine substitution at codon 113, and one
threonine to serine substitution at codon 125).
EXAMPLE 5
[0081] This example demonstrates interaction of hFAST-1 with
Smad2.
[0082] To determine whether hFAST-1, like its Xenopus counterpart,
would bind to Smad2, .sup.35S-labeled proteins were generated
through in vitro transcription and translation of an hFAST-1 cDNA
clone. A plasmid (pGST-Smad2/MH2) expressing the carboxyl terminus
of Smad2 (codons 183-467, comprising the MH2 domain (Riggins et
al., 1997)) fused to GST was constructed as previously described
(Zawel et al., 1998). Full-length hFAST-1 was PCR-amplified with
primers NT2/flag-TNT1 (5'-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGA
CTACAAGGACGACGATGACAAGGGGCCCTGCAGCGGCTCC-3'; SEQ ID NO:9) and
primer NT2-3. A C-terminal fragment of hFAST-1 was amplified with
primers NT2/flag-TNT2
(5'-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGACTACAAG
GACGACGATGACAAGCCCCTTCCTGGCCCCACGAG-3'; SEQ ID NO:10) and primer
NT2-3. As a control, the entire ORF of PIG3 (Polyak et al., 1997)
was also PCR-amplified.
[0083] These PCR products were used as templates in an in vitro
transcription and translation (TNT) reaction using TNT.RTM.T7
Coupled Reticulocyte Lysate System (Promega). The .sup.35S-labeled
TNT products were incubated with the GST-Smad2/MH2 fusion protein
coupled to agarose beads for 2 hours at 40.degree. C. in EBC buffer
(50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40). After five
washes with EBC buffer at room temperature, the agarose beads were
collected by brief centrifugation and the bound proteins eluted by
boiling in SDS-sample buffer. The eluted proteins were separated in
a 4-20% Tris-glycine gel and autoradiography was performed.
[0084] The labeled proteins were incubated with agarose beads
linked to the carboxyl-terminal MH2 domain of human Smad2,
previously shown to bind xFAST-1 (Chen et al., 1997; Liu, 1997).
Both full length hFAST-1 and a C-terminal fragment of hFAST-1
containing residues 221 to 365 bound efficiently and specifically
to the MH2 domain of Smad2, demonstrating that Smad2-binding is a
conserved property of FAST-1 proteins (FIG. 2B).
EXAMPLE 6
[0085] This example demonstrates hFAST-1 mediated transcriptional
activation.
[0086] In order to determine whether hFAST-1 could function in vivo
as a signal transducer for TGF-.beta., an expression vector was
constructed in which hFAST-1 was under the control of the CMV
promoter (pCMV-hFAST-1). To construct the vector, normal human
colon cDNA was used as the template to PCR-amplify the hFAST-1 ORF
with primers NT2-exp5' (5'-TATGCGGCCGCCACCATGGGGCCCTGCAGCG-3'; SEQ
ID NO:11) and NT2-exp3' (5'-TATGCGGCCGCGAGCTGCTGTGTCGCAGAC-3'; SEQ
ID NO:12). The PCR product was cloned into the Not1 site of pCI-neo
(Promega) and the recombinant plasmid (pCMV-hFAST-1) sequenced to
ensure its integrity. Transfection was carried out as described
(Zhou et al., 1998).
[0087] pCMV-hFAST-1 was transfected into the mink lung epithelial
cell line MvLu1 together with the AR3-lux reporter containing three
copies of the activin response element (ARE) from the Xenopus Mix.2
promoter (Chen et al., 1996; Hayashi et al., 1997). The plasmid
pAR3-lux was provided by J. Wrana (The Hospital for Sick Children,
Toronto). AR3-lux was activated over 30-fold by hFAST-1, and this
response was completely dependent on TGF-.beta. exposure (FIG. 3A).
A similar TGF-.beta.-dependent activity of hFAST1 was observed in
human HaCaT cells, another TGF-.beta. responsive line.
[0088] In contrast to the AR3-lux reporter, cotransfection of the
hFAST-1 expression vector had no effect on the activation of the
TGF-.beta. responsive reporters p3TP-lux or SBE4-lux. Expression of
an activin receptor whose kinase was engineered to be
constitutively active even in the absence of ligand (Attisano et
al., 1996), also conferred high levels of AR3-lux activity in the
presence of co-transfected hFAST-1 (FIG. 3B).
[0089] Human HCT116 cells were employed to examine other
requirements for FAST-1-dependent activation of AR3-lux. The
endogenous TGF-.beta. receptor type II (RII) gene is mutated in
these cells (Markowitz et al., 1995; Parsons et al., 1995), but
TGF-.beta. responses can be restored by exogenous expression of the
RII gene (Wang et al., 1995; Zhou et al., 1998). The TGF-.beta. RII
expression vector has been described by Zhou et al. (1998). FIG. 3C
shows that co-expression of the RII receptor was required for the
TGF-.beta.- and hFAST-1-dependent activation of AR3-lux.
[0090] To demonstrate that the activation of AR3-lux was dependent
on the DNA-binding forkhead domain of hFAST-1, an hFAST-1
expression vector was generated in which a single residue within
the forkhead domain was altered (arginine substituted for histidine
at residue 83). Crystallographic studies of the HNF-3.gamma.
forkhead domain had shown that this histidine contacted DNA and
would be expected to be critical for its activity (Clark et al.,
1993). The results in FIG. 3C show that this arginine substitution
totally abrogated hFAST-1 activity.
[0091] Finally, the hypothesis was tested that Smad4 is required
for the hFAST-1 activation of AR3-lux. The 5-18 cell line is a
derivative of HCT116 cells in which both alleles of Smad4 were
disrupted by targeted homologous recombination (Zhou et al., 1998).
Transfection of hFAST-1 into these cells resulted in little AR3-lux
activity compared to the parental line (FIG. 3C). Thus the
transcriptional activity of hFAST-1, even when overexpressed, was
dependent on an intact endogenous Smad4 gene.
EXAMPLE 7
[0092] This example demonstrates sequence-specific DNA binding of
hFAST-1.
[0093] Forkhead proteins are known to bind DNA in a specific
fashion, with the loose consensus sequence (G/A)(T/C)(C/A)AA(C/T)A
(Kaufmann and Knochel, 1996; SEQ ID NO:13). The xFAST-1 protein was
discovered on the basis of its binding to the ARE within the
promoter of the activin-inducible Mix.2 gene, and the responsible
sequences were mapped to a six bp sequence (AAATGT) which was
repeated twice within the ARE but which was not very similar to the
forkhead consensus (Chen et al., 1996). To define the DNA sequences
which could bind to hFAST-1, oligonucleotides were selected which
could bind to the protein from a random pool. The oligonucleotides
were degenerate in a 20 bp central region and were flanked on each
side by 20 bp regions of known sequence. The hFAST-1-DNA complexes
were separated by EMSA and the recovered DNA amplified by PCR.
Following three rounds of selection and amplification, recovered
oligonucleotides were cloned and individually tested for binding to
hFAST-1 in EMSA.
[0094] To produce a GST-fusion protein (FAST-FL) containing the
full length hFAST-1, the entire ORF of hFAST-1 was PCR-amplified
and cloned into the BamH1 site of pGEX2TK (Pharmacia). A GST-fusion
protein (FAST-FH) containing only the forkhead domain of hFAST-1
was constructed similarly. GST-fusion proteins containing the MH1
or MH2 domains of Smad2 were produced as previously described
(Zawel et al., 1998). Proteins produced in bacteria from these
vectors were purified with glutathione-agarose and used to select
random oligonucleotides as previously described (Zawel et al.,
1998). In brief, following binding to 1 .mu.g of GST-FAST-1
proteins (or following "mock" reactions without added protein),
EMSA was performed and the location of the DNA-protein complexes
within the gels was approximated based on the mobility of complexes
generated with an ARE-derived probe (Chen et al., 1996). Gel slices
were homogenized, incubated at 65 oC for 30 min, and then passed
through Spin-X columns (Costar). Recovered oligonucleotides were
extracted with phenol-chloroform, precipitated with ethanol,
re-amplified, and subjected to the next round of binding. Following
completion of the third selection-amplification cycle, PCR products
were cloned into pZERO2.1 (Invitrogen).
[0095] Sixty bp probes corresponding to single clones were
generated for EMSA by colony PCR using the following
.sup.32P-labeled primers: 5'-TAGTAAACACTCTATCAATTGG-3' (SEQ ID
NO:14) and 5'-GTCCAGTATCGTTTACAGCC-3' (SEQ ID NO:15). To determine
the oligonucleotide sequences contained within single clones,
inserts were amplified by colony PCR using M13 forward and reverse
primers and the PCR products sequenced using Thermo Sequenase and
an SP6 primer. To test binding to PCR products derived from clones,
1.0-1.5 .mu.g protein (.about.1 .mu.M final concentration) and 50
ng of DNA (end-labeled to 2.times.10.sup.6 dpm/.mu.g) were used. To
test binding to chemically synthesized oligonucleotides (rather
than those generated through PCR), complementary oligonucleotides
were synthesized and labeled with .gamma..sup.32P-ATP and T4
polynucleotide kinase prior to annealing. The sequence of the FBE
oligonucleotide was 5'-CGGATTGTGTATTGGCTGTAC-3' (SEQ ID NO:16), and
the sequence of the control oligonucleotides (FBE*), containing two
alterations of the FBE consensus, was 5'-CGGATTCTGTATCGGCTGTAC-3'
(SEQ ID NO:17). The sequence of the ARE oligonucleotide was
5'-TATCTGCTGCCCTAAAATGTGTATTCCA TGGAAATGTCTGCCCTTCTCTCCGTAC-3' (SEQ
ID NO:18). For binding to oligonucleotides, 0.3-0.5 .mu.g of
protein (.mu.0.4 .mu.M final concentration) and 0.5 ng of DNA
(end-labeled to 2.times.10.sup.8 dpm/.mu.g) was used.
[0096] The inserts from 22 of 23 recovered clones bound to hFAST-1
(FIG. 4A). Comparison of the sequences of clones exhibiting hFAST-1
binding revealed a striking consensus (FIG. 4B). All clones
contained two invariant three base elements separated by two G or T
residues. The inferred consensus was TGT(G/T)(G/T)ATT (FIG. 4B; SEQ
ID NO:4). To test whether this 8 bp consensus could indeed mediate
hFAST-1 binding, an oligonucleotide containing a single copy of it
was synthesized and tested in EMSA. This oligonucleotide (FBE, for
FAST-1 binding element) bound efficiently to purified full length
hFAST-1 protein and also (though less well) to the forkhead domain
of hFAST-1 (FIG. 4C). FBE did not bind to similarly purified Smad2
proteins (FIG. 4C). An oligonucleotide in which two of the
consensus positions were altered exhibited no binding to hFAST-1,
documenting the specificity of the interaction (FIG. 4C).
[0097] The 8 bp consensus TGT(G/T)(G/T)ATT (SEQ ID NO:4) defined
here was not related to the consensus ((G/A)(T/C)(C/A)AA(C/T)A; SEQ
ID NO:13) inferred from the study of other forkhead proteins
(Kaufmann and Knochel, 1996). Interestingly, the ARE element from
the Mix.2 promoter contains a perfect match (TGTGTATT) to the
consensus defined here. This 8 bp sequence overlapped one of the
two repeats (AAATGT) which Chen et al. (Chen et al., 1996)
suggested might be responsible for xFAST-1 binding, but it is
likely that the TGTGTATT sequence was actually responsible for this
binding. Chen et al. performed an informative experiment with a
variant of the ARE which did not bind xFAST-1 complexes.
Importantly, one of the three altered residues in this non-binding
variant coincidentally affected the second base of the 8 bp
consensus noted above, changing it to TCTGTATT (changed residue
underlined). To specifically test whether the FBE was the critical
element of the ARE for binding to FAST-1, we synthesized two 50 bp
oligonucleotides, one comprising the entire sequence of the ARE
((Chen et al., 1996; SEQ ID NO:10) and one comprising the identical
sequence except for a single base substitution within the FBE
(TCTGTATT instead of TGTGTATT). Only the wild type ARE sequence
bound to FAST-1 (FIG. 4D).
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Sequence CWU 1
1
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