Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 11 to 14 amino acid residues in defined sequence
Reexamination Certificate
2000-11-08
2003-03-04
Ketter, James (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
11 to 14 amino acid residues in defined sequence
C435S069100, C435S069700, C530S333000, C530S350000
Reexamination Certificate
active
06528620
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compounds which repress DNA transcription. Specifically, the invention relates to a thirteen amino acid polypeptide sequence which is able to autonomously function as a transcription repressor domain through its ability to independently bind mSin3A. The invention further relates to chimeric transcriptional repressors which comprise the thirteen amino acid polypeptide and a DNA-binding molecule.
TECHNICAL BACKGROUND
Precise changes in gene expression are crucial to both normal and disease processes, Gene expression is regulated by DNA-binding transcription factors and the proteins that interact with these DNA-bound factors. Coactivators and corepressors mediate the ability of DNA-bound transcription factors to modulate gene expression—coactivators by increasing the expression of genes, and corepressors by down-regulating the expression of genes.
Transcriptional regulation depends on the assembly of large multiprotein complexes. For example, the preinitiation complex (Kadonaga, J. T.
Cell
92: 307-313 (1998)), chromatin remodeling complexes (Cairns, B. R.
Trends Biochem. Sci.
23:20-25 (1998), Wu, C.
J Biol. Chem.
272:28171-28174 (1997)), and histone deacetylase-containing corepressor complexes (Rundlett et al.,
Proc. Natl. Acad. Sci.
USA 93:14503-14508 (1996), Zhang et al.,
Cell
95:279-289 (1998)) have been shown to be in the 1-2×10
6
dalton size range. Molecular connections between proteins in these molecular machines, and the structural basis of their assembly, are not well understood. Initially, transcription repression domains were defined by structure/function analysis, which revealed that, like activation domains, they are more likely to contain particular amino acids rather than have easily identifiable protein-protein interaction domains. This finding led to the hypothesis that activation and repression domains share similar molecular targets and that the structure of the activation or repression domain in itself was not required for function. Transciptional repressors function by at least three distinct mechanisms: by direct contact with components of the basal transcriptional machinery, e.g. even-skipped (Um et al.,
Mol. Cell. Biol.
15:5007-5016 (1995)), Dr1 (Yeung et al.,
Genes Dev.
8424:2097-2109 (1994)), and MOT1 (Auble et al.,
Genes Dev.
8:1920-1934 (1994)); by tethering histone deacetylase-containing corepressor complexes to the promoter, e.g. the Mad family (Hassig et al.,
Cell
89:341-347 (1997), Laherty et al.,
Cell
89:349-356 (1997)), Rb (Brehm et al.,
Nature
391:597-601 (1998), Magnaghi-Jaulin et al.,
Nature
391:601-605 (1998), and Luo et al.,
Cell
92:463-473 (1998)), and MeCP2 (Jones et al.,
Nat. Genet.
19:187-191 (1998), Nan et al.,
Nature
393:386-389 (1998)); or by tethering corepressors that lack deacetylase activity to the promoter, e.g. hairy (Paroush et al.,
Cell
79:805-815 (1994)) and MAT&agr;2-MCM1 (Kadosh and Struhl,
Cell
89:365-371 (1997)). In each of these cases, little or no structural data are available for the repression domain. In contrast, one theme that has emerged recently from the study of activation domains is that relatively short stretches of amino acids can adopt amphipathic &agr;-helical structures and mediate stable functional interactions between transcriptional activators and coactivators. Kussie et al.,
Science
274:948-953 (1996), Radhakrishnan, et al.,
Cell
91:741-752 (1997), and Uesugi et al.,
Science
277:1310-1313 (1997).
Reversible acetylation of the amino-terminal tails of core histones plays an important role in the regulation of gene expression. In general, regions of chromatin that are hyper-acetylated are transcriptionally active, while hypoacetylated regions are silenced. Grunstein, M.,
Nature
389-352 (1997). The recent discovery that several transcriptional co-activators are histone acetyltransferases and that co-repressor complexes contain histone deacetylases as active components has provided a mechanistic basis for this correlation. Wolffe and Pruss,
Cell
84:817-819 (1996), Hassig et al.,
Curr. Opin. Chem. Biol.
1:300-308 (1997), Grant et al.,
Trends Cell Biol.
8:193-197 (1998), Struhl,
Genes Dev.
12:599-606 (1998), Davie,
Curr. Opin. Genet. Dev.
8:173-178 (1998). mSin3A and mSin3B were identified as corepressors required for the transciptional and biological activities of the Mad proteins. Ayer et al.,
Cell
80:767-776 (1995); Schreiber-Angus et al.,
Cell
80:777-786 (1995). mSin3A has recently been shown to be a component of a large multi-protein complex(s) that also contains the histone deacetylases HDAC1 and HDAC2 in apparently stoichiometric amounts. The enzymatic activities of the mSin3A-bound HDACs are required for full transcriptional repression by the Mad family proteins. Hassig et al.,
Cell
89:341-347 (1997); Laherty et al.,
Cell
89:349-356 (1997), Zhang et al.,
Cell
89:357-364 (1997). Subsequently, the mSin3A-HDAC complex has been implicated as a corepressor utilized by a diverse and rapidly expanding collection of transcriptional repressors, including RXR, MeCP2, estrogen receptor, RPX, and Pit1. (Jones et al.,
Nat. Genet.
19:187-191 (1998), Struhl,
Genes Dev.
12:599-606 (1998), Laherty et al.,
Mol. Cell
2:33-42 (1998), Heinzel et al.,
Nature
387:43-48 (1997), Nagy et al.,
Cell
89:373-380 (1997).
mSin3A and mSin3B and their
Saccharomyces cerevisiae
orthologue SIN3 each contain four similar domains each suggested to form two amphipathic &agr;-helices separated by a flexible linker. Ayer et al.,
Cell
80:767-776 (1995), Wang et al.,
Mol. Cell Biol.
10:5927-5936 (1990). These regions, termed PAH domains for paired amphipathic &agr;-helix, were originally proposed to function as protein-protein interaction domains. Wang et al.,
Mol. Cell Biol.
10:5927-5936 (1990). Recent experiments have demonstrated this to be the case. For example, Mad proteins interact with PAH2 (Ayer et al.,
Cell
80:767-776 (1995), Schreiber-Angus et al.,
Cell
80:777-786 (1995)), a repression domain of the nuclear hormone corepressor N-CoR interacts with PAH1 (Heinzel et al.,
Nature
387:43-48 (1997), Alland et al.,
Nature
387:49-55 (1997)), and the mSin3 interacting protein SAP30 binds to PAH3. (Laherty et al.,
Mol. Cell
2:33-42 (1998). The four PAH domains of the different Sin3 proteins are highly conserved. For example, PAH2 is 90% similar between mSin3A and mSin3B and it is approximately 70% similar to the PAH2 domain of
S. cerevisiae
SIN3 (Ayer et al.,
Cell
80:767-776 (1995)) and recently identified SIN3 homologues from
Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster,
and
Arabidopsis thaliana.
Within a given protein, the four PAH domains are roughly 45% similar with the hydrophobic positions of the putative amphipathic &agr;-helices being most highly conserved, suggesting that PAH domains may share structural features. Ayer et al.,
Cell
80:767-776 (1995); see also Kasten et al.,
Mol. Cell. Biol.
16:4215-4221 (1996) (demonstrating that human Mad1 can interact with yeast SIN3). With the exception of the Mad family, the domains required for Sin3 binding of the other SIN3 interacting proteins, SAP30, SAP18, N-CoR, UME6, HDAC1, and HDAC2, etc., share no obvious sequence similarity.
The Mad family of basic region-helix-loop-helix-leucine zipper (bHLHZip) proteins functions as transcriptional repressors and antagonize the transcriptional and transforning activity of the Myc proto-oncogenes. Ayer et al.,
Cell
72:211-222 (1993), Koskinen et al.,
Cell Growth Differ.
6:623-629 (1995), Lahoz et al.,
Proc. Natl. Acad. Sci.
USA 91:5503-5507 (1994), Hurlin et al.,
EMBO J.
14:5646-5659 (1995), and Vastrik et al.,
J. Cell Biol.
128:1197-1208 (1995). Currently, four Mad family members have been identified: Mad1, Mxi1, Mad3, and Mad4. Ayer et al.,
Cell
72:211-222 (1993), Hurlin et al.,
EMBO J.
14:5646-5659 (1995), Zervos et al.,
Cell
72:223-232 (1993). These proteins share extensive sequence homology throughout their entire open reading frames, with the
Ayer Donald E.
Billin Andrew N.
Ketter James
Loeb Bronwen M.
Madson & Metcalf
University of Utah Research Foundation
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