Transcriptional repression serves widely in gene regulation in eukaryotes. Locus control, silencing, sex chromosome dosage compensation, imprinting and other epigenetic effects involve chromatin as a means of repression. In approaching the molecular mechanism of repression in the yeast model, we have determined the nucleosomal organization of several yeast domains: the two HM loci, two a-cell specific genes, and the recombination enhancer. Each of these as a common feature, close packed dimeric nucleosomes, and a unique feature, quantized linker length of DNA between these dimers. Based on the specific repressors that nucleate the structures and co-repressors that interact with histone tails, I postulate specific architecture for each of the three types of domains we have defined. The first half of this proposal deals with testing the model structures by quantitative determination of the composition of chromatin for each unique strain, definition of the kinetics and stabilities of proposed interactions between repressors, co-repressors and chromatin, and morphological and physicochemical study of the domains assembled in vivo and in vitro. The recombination enhancer discovered by Haber and characterized structurally by our laboratory, differentially controls the recombination potential of the left arm of chromosome III in the two yeast haploid cell types. It facilitates interactions between the MATa locus and HMLalpha in mating type interconversion. In the second part of this proposal, we address the mechanisms of long range communication among these three elements by identification of proteins that interact with the recombination enhancer and study of the structures of all three elements during induced synchronous mating type switching. We will also study the silent loci to address the seeming paradox of a chromatin structure that precludes transcription yet is perfectly appropriate for recombination or transposon integration. The proposed research takes a wide range of experimental approach to two fundamental questions in gene regulation, the chromosomal organization of extensive repressed domains and long range communication between chromatin domains.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052311-07
Application #
6498685
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
1996-02-09
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
7
Fiscal Year
2002
Total Cost
$281,340
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Ercan, Sevinc; Reese, Joseph C; Workman, Jerry L et al. (2005) Yeast recombination enhancer is stimulated by transcription activation. Mol Cell Biol 25:7976-87
Simpson, Robert T; Ducker, Charles E; Diller, John D et al. (2004) Purification of native, defined chromatin segments. Methods Enzymol 375:158-70
Ercan, Sevinc; Simpson, Robert T (2004) Global chromatin structure of 45,000 base pairs of chromosome III in a- and alpha-cell yeast and during mating-type switching. Mol Cell Biol 24:10026-35
Ducker, C E; Simpson, R T (2000) The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome. EMBO J 19:400-9
Gavin, I M; Kladde, M P; Simpson, R T (2000) Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene. EMBO J 19:5875-83
Ravindra, A; Weiss, K; Simpson, R T (1999) High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa. Mol Cell Biol 19:7944-50
Weiss, K; Simpson, R T (1998) High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha. Mol Cell Biol 18:5392-403
Simpson, R T (1998) Chromatin structure and analysis of mechanisms of activators and repressors. Methods 15:283-94
Wu, C; Weiss, K; Yang, C et al. (1998) Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching. Genes Dev 12:1726-37
Gavin, I M; Simpson, R T (1997) Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure. EMBO J 16:6263-71

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