How cells regulate the expression of genes is fundamental to most aspects of biology. My laboratory is interested in understanding the regulation of gene expression at active and inactive regions of the yeast genome. The goal is to learn about mechanisms that regulate Pol II transcription and chromatin structure. We identify proteins that regulate transcription using classic and modern genetic techniques. We then investigate the mechanisms through which these factors influence transcription and chromatin function using a combination of genetic, molecular, and biochemical methods. These methods include mutagenesis, genetic screens, growth assays, RNA-seq, and chromatin immunoprecipitation. Our findings will explain how chromatin dynamics influence gene expression and genome integrity. Many of the factors we study have homologs in human cells, indicating that our discoveries may provide insights into mechanisms that regulate gene expression in higher eukaryotes.

Methylation of histone H3 by Set1 regulates transcription

Our current research addresses complex questions regarding the role of histone methylation in the regulation of transcription. In budding yeast, there is a single lysine methyltransferase, Set1, that catalyzes the mono-, di- and tri-methylation of the fourth residue, lysine 4 (K4), of histone H3. The SET family of lysine methyltransferases is evolutionarily conserved in structure and function. Set1 is a great model to decipher the effects of histone methylation on gene expression and chromatin function because it is the only histone H3K4 specific methyltransferase in S. cerevisiae. Set1 generates three chromatin marks, and each mark is not equivalent to gene regulation. We exploit novel genetic variants of the conserved Set1 protein that differentially affect methylation of lysine 4 of histone H3 (e.g., abolish H3K4 tri-methylation while keeping H3K4 mono-methylation intact). Studies using these Set1 mutants provide insights into the roles of different H3K4 methyl marks in transcription by Pol II. This research is expected to define new paradigms in gene regulation.

K. Williamson, V. Schneider, R. A. Jordan, M. Henderson Pozzi, J. E. Mueller, M. Bryk, Catalytic and functional roles of conserved amino acids in the SET domain of the S. cerevisiae lysine methyltransferase Set1. PLoS One 8 (2013).

Gene silencing at the ribosomal DNA locus

Our research on silent chromatin focuses on the ribosomal DNA locus (rDNA array) that contains ~150-200 repeats of the rRNA genes. We characterize mechanisms that regulate silent chromatin in the rDNA array using Pol II-transcribed genes as reporters or proxies for measuring the strength of silent chromatin. Silent chromatin in the rDNA represses not only Pol II transcription but also genetic recombination. We recently characterized the silencing of transcription and recombination using a library of yeast strains, each with a reporter gene in a different rRNA gene repeat. RNA analysis was conducted to measure steady-state transcript levels from the reporter gene. Chromatin immunoprecipitation experiments were also performed to measure the association of the NAD+-dependent histone deacetylase Sir2 (silencing protein) at the reporter gene. Findings indicate that silent chromatin is not uniform across the rDNA array and suggests that Sir2 plays a primary role in determining the strength of silent chromatin in an rRNA gene repeat within the ribosomal DNA locus. Future studies into the characteristics of rDNA silent chromatin will lead to a better understanding of the epigenetic regulation of highly repeated DNA sequences.

C. Li, J. E. Mueller, M. Bryk, Sir2 represses endogenous Pol II transcription units in the ribosomal DNA non-transcribed spacer. Mol. Biol. Cell 17, 3848–3859 (2006).