Silencing is an epigenetic form of transcriptional regulation in eukaryotes in which certain regions of chromosomes, e.g., certain genes, are made into transcriptionally inactive chromatin structures. [For reviews, see Loo, S. et al. (1995) Annu. Rev. Cell Dev. Biol. 11: 519-548 and Pillus, L. and Grunstein, M. Chromatin Structure and Gene Expression (ed. Elgin, S. C. R.) (1995) IRL Press, Oxford University Press, pp. 123-146.] Silencers, which are specialized regulatory sites in DNA, and various proteins, including general DNA-binding proteins and silencing proteins, are responsible for silencing. Silencing is related, in part, to the (degree of) acetylation of histones, especially histone H4.
Silencing appears to involve at least three distinct phases or processes: establishment, maintenance, and inheritance. Establishment refers to the genetic switch from active to silenced (inactive) chromatin. Maintenance refers to the continuation of the silenced state of the chromatin. Inheritance refers to the propagation of the silenced state as the chromatin is replicated.
In Saccharomyces cerevisiae, a number of proteins and genes involved in silencing have been identified. Silencing in yeast is integral to yeast mating-type biology.
Yeast mating-type is determined by the allele present at the mating-type locus, MAT, located near the center of chromosome III. Cells of Saccharomyces cerevisiae can be one of three types: a haploid, .alpha. haploid, or a/.alpha. diploid. Cells with the a allele at the MAT locus are of the a mating-type. Cells with the .alpha. allele at the MAT locus are of the .alpha. mating-type. The MATa and MAT.alpha. alleles encode regulatory proteins which control genes specifying the functional differences between a and .alpha. cell types. Opposite type haploid cells can mate with one another to form diploid cells. The simultaneous expression of MATa and MAT.alpha. alleles leads to disruption of normal haploid cell functions, including the ability to mate.
In addition to the transcriptionally active MAT locus, S. cerevisiae cells also contain silenced (i.e., transcriptionally inactive) copies of the a and .alpha. genes at two other loci, HML and HMR, which are located near opposite ends of chromosome III. Upon transposition of an a and .alpha. allele from HML or HMR to MAT, yeast switch from a to .alpha., or vice versa. In haploid cells the silent mating-type loci are repressed. If these loci are not repressed, the simultaneous expression of a and .alpha. leads to the non-mating phenotype of a/.alpha. diploid cells.
Repression of the silent mating-type loci involves regulatory sites adjacent to HML and HMR [Hawthorne, D. (1963) Genetics, 48:1727-1729; Kassir, Y. et al. (1983) Mol. Cell. Biol. 3:871-880; Strathern, J. et al. (1979) Cell 18:309-319; Abraham, J. et al. (1984) J. Mol. Biol. 176:307-331; Feldman, J. et al. (1984) J. Mol. Biol. 178: 815-834]. The HML-flanking silencers are HM-E and HML-I; the HMR-flanking silencers are HMR-E and HMR-I. The E and I silencers comprise elements corresponding to an autonomously replicating sequence (ARS) consensus sequence, and Rap1p- and Abf1-protein binding sites [Brand, A. et al. (1987) Cell 51:709-719].
Four SIR (silent information regulator) genes silence HML and HMR [Haber, J. et al. (1979) Genetics 93:13-35; Klar, A. et al. (1979) Genetics 93:37-50; Rine, J. et al. (1987) Genetics 116:9-22; Rine, J. et al. (1979) Genetics 93:877-901].
SIR1 encodes the Sir1p protein, which is thought to play a role in establishment of silencing at the silent mating-type loci, HML and HMR; Sir1p is not involved in maintenance or propagation of silencing. Mutant sir1 cells are deficient in establishing silencing; however, after silencing is established, these mutant cells maintain and propagate the silenced state at HML and HMR [Pillus, L. et al. (1989) Cell 59:637-647].
SIR2 encodes the Sir2p protein, which is thought to be required for silencing. Cells with sir2 mutations are not only deficient in silencing, but also have an elevated level of recombination in ribosomal DNA [Gottlieb, S. et al. (1989) Cell 56: 771-776]. Sir2P also appears to play a role in acetylation of histones. Evidence suggests that Sir2p is, or regulates, a deacetylase or that it inhibits a histone acetyltransferase. Overproduction of Sir2p results in decreased acetylation of core histones H2B, H3 and H4 [Braunstein, M. et al. (1993) Genes Dev. 7:592-604].
SIR3 encodes the Sir3p protein, which is also required for silencing. Cells with sir3 mutations are deficient in silencing, and have increased rates of mitotic recombination [Palladino, F. et al. (1993) Cell 75:543-555]. Sir3p can form a stable complex with a protein (Rap1p) involved in DNA repair [Paetkau, D. et al. (1994) Genes Dev. 8:2035-2045]; hence Sir3p is thought to be involved in DNA repair as well as in silencing. Increasing SIR3 gene dosage leads to increased silencing [Renauld, H. et al. (1993) Genes Dev. 7:1133-1145]. Cells without Sir3p are completely deficient in silencing [Aparicio, O. et al. D. (1991) Cell 66:1279-1287; Rine, J. et al. (1987) Genetics 116:9-22].
SIR4 encodes the Sir4p protein, also absolutely necessary for silencing. Cells with sir4 null mutations are deficient in silencing, and have a fourfold increase in chromosomal loss. SIR4 is postulated to encode a structural component of chromosomes [Palladino, F. et al. S. (1993) Cell 75:543-555].
The Sir2, Sir3, and Sir4 proteins are also required for telomeric silencing, another type of silencing in S. cerevisiae which has many similarities to mating-type loci silencing. Null mutations in SIR2, SIR3, or SIR4 result in total loss of silencing, both mating-type loci and telomeric silencing. Sir1p, unlike the proteins encoded by SIR2, SIR3, and SIR4, is not necessary for silencing at the silent mating-type loci and apparently has no function at telomeres. Mutations in the SIR genes do not affect cell viability.
Evidence indicates that local chromatin structure is involved in regulation of silencing. The positively charged N-terminal regions of histones H3 and H4 are believed to facilitate silencing via both compaction of chromatin and through specific interactions with silencing proteins. Intact N-termini of histones H3 and H4 are required for complete silencing of HML and HMR [Thompson, J. et al. (1994) Nature 369:245-247; Johnson, L. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6286-6290; Kayne, P. et al. (1988) Cell 55:27-39; Megee, P. et al. (1990) Science 247:841-845; Park, E. et al. (1990) Mol. Cell. Biol. 10:4932-4934]. The positive charges of the N-termini of histones H3 and H4 appear to be important to their role in silencing. The DNA-binding properties of histones H3 and H4 are altered if the lysines in the N-termini are acetylated [Hong, L. et al. (1993) J. Biol. Chem. 268:305-314].
The N-termini of histones H3 and H4 are often highly acetylated in transcriptionally active regions [Hebbes, T. et al. (1988) EMBO J. 7: 1395-1402; Lee, D. et al. (1993) Cell 72:73-84], and unacetylated in transcriptionally inactive (silenced) regions [Pillus, L. et al. in Chromatin Structure and Gene Expression (ed. Elgin, S. C. R.) (1995) IRL Press, Oxford University Press pp. 123-146]. Different patterns of acetylation are seen in heterochromatin versus euchromatin, as shown by immunological reagents directed to differentially acetylated lysines in the N-terminal regions of histone H4 [Turner, B. et al. (1992) Cell 69:375; Bone, J. et al. (1994) Genes Dev. 8: 96]. The H4 histones of the inactive X chromosome (Barr bodies) in female mammals are not acetylated [Jeppesen, P. et al. (1993) Cell 74: 281].
It is thought that acetylated histones bind DNA less tightly, making DNA more accessible to proteins involved in gene expression. Four nuclear histone acetylating enzymes and five deacetylating enzymes were recently reviewed [Pennisi, E. (1997) Science 275: 155-157]. Histone acetylases include: Tetrahymena, S. cerevisiae, and human HAT A (Gcn5p); human PCAF; human p300/CBP; and human, Drosophila, and S. cerevisiae TAF.sub.II 230/250. Histone deacetylases include: human, Drosophila, S. cerevisiae, and possibly Xenopus, mouse and nematode DHAC1 (RPD3); S. cerevisiae HDA1; S. cerevisiae HOS1; S. cerevisiae HOS2; and S. cerevisiae HOS3.
Because acetylation of histones affects gene expression, it is thought to affect cell cycle and proliferation, and therefore malfunctions of these processes can result in cancer. For example, trapoxin, a potential anti-cancer agent, inhibits cell growth and makes cancer cells revert to their normal, differentiated state. Trapoxin also inhibits histone deacetylation [Taunton, J. et al. (1996) Science 272: 408]. Blocking the removal of acetyl groups from histones is believed to be responsible for trapoxin's anti-tumor effect by restoring normal gene expression, e.g., transcription of a tumor-suppressor gene.
The present invention identifies novel genes encoding silencing proteins in S. cerevisiae. It further provides the proteins encoded by these genes. It also provides methods of using these genes and proteins in a screen for drugs useful in the treatment of mammalian, particularly human, diseases. It specifically provides a method of using mutant strains of S. cerevisiae in a method for screening agents for their ability to affect Tip60 expression and/or activity [Kamine, J. et al. (1996) Virology 216:357-366] and for their ability to affect the 5' translocation partner (protein) of the MOZ-CBP (monocytic leukemia zinc finger protein-CREB binding protein) chimeric oncogene [Borrow, J. et al. (1996) Nature Genet. 14:33-41].
The HIV-1 protein Tat is necessary for HIV replication and is a strong transactivator of HIV gene expression. Tip60, a 60 kDa protein which interacts with the cysteine-rich region of Tat, is postulated to be a cofactor of Tat-dependent regulation of gene expression in HIV [Kamine, J. et al. (1996) Virology 216:357-366]. Overexpression of Tip60 results in a fourfold increase of Tat transactivation of the HIV-1 promoter in transient expression assays. Nothing is known about the regulation of Tip60 in vivo.
The translocation t(8;16)(p11;p13) found in 4-7 patients per 1,000 patients with acute myeloid leukemia (AML) fuses the MOZ gene to the gene encoding the CREB-binding protein [Borrow, J. et al. (1996) Nature Genet. 14:3314 41]. The CREB-binding protein is a transcriptional coactivator which connects the basal transcriptional machinery to other transcription factors. Moz (monocytic leukemia zinc finger protein) is a putative chromatin-bound acetyltransferase. Moz contains two C4HC3 (four cysteines, one histidine, three cysteines) zinc fingers, a C2CH (two cysteines, one cysteine, one histidine) zinc finger and an acetyltransferase consensus domain. The Moz-CBP oncoprotein may mediate leukemogenesis via abnormal acetylation of chromatin at promoters which require CBP bridging. This abnormal acetylation may thereby abnormally acetylate genes whose expression is inappropriate for normal hematopoiesis.
There is as long-felt need in the art for methods of screening agents for their usefulness in the treatment of AIDS and acute myeloid leukemia. Such needed methods are quick, inexpensive and easy to carry out compared to existing methods which do not focus on the genetic basis of these diseases, including tests on animal models.