Sarah Tripplehorn-Arndt & Hainer LabsÂ
Investigating the recruitment and function of the nucleosome remodeler Chd1
In eukaryotes, a complex interplay exists between chromatin and transcription. While chromatin poses a formidable barrier to transcription, active transcription also leads to changes in chromatin structure. A class of proteins termed nucleosome remodelers plays a fundamental role in selectively removing, replacing, and repositioning nucleosomes across the genome, including over important transcription units such as promoter regions and gene bodies. The nucleosome remodeler Chd1 positions nucleosomes across actively transcribed regions of the genome, preventing transcription initiation factors from inappropriately accessing cryptic promoters within gene bodies and dictating key mammalian cell fate decisions. However, much is unknown about how Chd1 is recruited to chromatin and how its remodeling functions are regulated. I hypothesize that Chd1 activity is dependent on the transcription elongation factor Rtf1 to function at sites of active transcription, and that Rtf1 regulates the recruitment of Chd1 in Saccharomyces cerevisiae. Rtf1, a subunit of the highly conserved Polymerase Associated Factor 1 Complex (Paf1C), is required for the co-transcriptional deposition of histone modifications and is involved in maintaining Paf1C on RNA Polymerase II. Previous work from the Arndt lab demonstrated a loss of Chd1 occupancy on chromatin in yeast strains containing deletion mutations in RTF1, suggesting that Rtf1 and Chd1 cooperate co-transcriptionally in yeast. To identify critical residues involved in the Rtf1-Chd1 interaction, I performed a yeast two-hybrid assay. Specific residues near the N-terminus of Rtf1 are critical for binding Chd1, and these highly conserved Rtf1 residues are also important in preventing cryptic initiation from an intragenic reporter locus in vivo. Discovery of the biochemical and functional implications of these key N-terminal residues has led me to develop yeast strains expressing epitope-tagged Chd1 and epitope-tagged Rtf1 point mutants from their endogenous loci. With these integrated point mutants, I can separate the Rtf1-Chd1 dependent functions from other highly critical functions of Rtf1 and selectively probe the consequences of disrupting the Rtf1-Chd1 interaction. My future work will address whether N-terminal Rtf1 point mutants are sufficient to prevent Chd1 chromatin occupancy and disrupt Chd1 chromatin remodeling activities genome-wide.
Friday, October 2, 2020
12 PM
A219B or via ZoomÂ