Mutations in SET1 affect ribosomal DNA chromatin structure, transcriptional silencing, and histone H3 methylation in S. cerevisiae.
Catalina Alfonso, Mary Bryk
Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX, 77843-2128, USA
Transcriptional silencing of RNA pol II genes in the rDNA of S. cerevisiae requires Set1, a member of COMPASS that catalyzes the methylation of K4 of histone H3. Yeast strains containing a gene encoding a variant of histone H3 that cannot be methylated at lysine 4 exhibit silencing defects similar to those of a set1 null mutant. We want to understand how K4-methylated H3 regulates rDNA silencing. By examining rDNA chromatin structure, we hope to gain insight into how H3 methylation regulates gene silencing. Using micrococcal nuclease (MNase) accessibility experiments, we have observed differences in cleavage in the rDNA nontranscribed spacer in a set1 null compared to a WT strain. One possibility is that loss of H3 K4 methylation from rDNA chromatin alters the binding of proteins that regulate transcriptional silencing. Set1 belongs to a family of transcription regulators that share a conserved SET domain. A truncated SET1 gene, encoding a 300-aa portion of Set1 (set-007) including the SET domain, is necessary and sufficient for rDNA silencing and H3 methylation. By studying sequence alignments and structural data of SET proteins, we identified conserved residues in Set1 that may be required for H3 methylation and rDNA silencing. We have made several aa-substitution mutants in the truncated SET1 gene. Strains containing some of the mutants, including H1017L and Y967A, show defects in rDNA silencing and H3 methylation. Results of the mutant analysis will be presented.
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