MOLECULAR MECHANISMS OF TRANSCRIPTIONAL REPRESSION

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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.