The role of chromatin structure in modulating the functions of DNA in transcription, replication, recombination and repair is becoming increasingly apparent. We have shown that the yeast alpha2 repressor positions a nucleosome adjacent to its operator. This organized chromatin structure is propagated; at least four additional nucleosomes are positioned with base pair precision. Mutations in yeast genes known to be necessary for repression by alpha2 also lead to perturbation of the organized chromatin structure. Studies moving the transcription initiation site relative to the alpha2 operator demonstrate that repression is involved with chromatin structure above the level of an individual nucleosome. The entire STE6 gene is packaged in organized nucleosomes; the end of the 4 kbp domain is at the 3' end of the structural gene. The domain appears to exist as repeated close packed dimers interspersed by long (42 bp) linkers. This organization has potentially interesting implications for formation of an unique chromatin structure. Studies defining chromatin features which preclude access of in vivo expressed dam methylase have been completed. Using a methylase which also recognizes GATC but modifies C instead of A, we have adapted a chemical method which gives a positive signal for methylation, facilitating analyses. The Hha methylase which modifies CpG sequences has been expressed in yeast; this offers a modification site every 30-50 bp, on average, allowing chromatin analyses without introduction of restriction sites. Haploid specific genes are repressed in yeast diploids by an a1/alpha2 heterodimer. Three consensus binding sites and two near-sites are upstream of the SST2 gene, as is an organized chromatin structure. To determine which site(s) were important in repression, we disrupted each of the a1/alpha2 binding sites, singly and in combination. Surprisingly, even with all five sites mutated, a reporter gene remained repressible in diploid cells. The cis-acting elements involved in control of a meiosis specific heat shock gene have been defined. In addition to the heat shock element, an upstream repressor sequence and a sequence which flanks it and modulates basal expression are critical for proper repression and induction.
