Abstract
The nucleosome core regulates DNA-templated processes through the highly conserved nucleosome acidic patch. While structural and biochemical studies have shown that the acidic patch controls chromatin factor binding and activity, few studies have elucidated its functions in vivo. We employed site-specific crosslinking to identify proteins that directly bind the acidic patch in Saccharomyces cerevisiae and demonstrated crosslinking of histone H2A to Paf1 complex subunit Rtf1 and FACT subunit Spt16. Rtf1 bound to nucleosomes through its histone modification domain, supporting its role as a cofactor in H2B K123 ubiquitylation. An acidic patch mutant showed defects in nucleosome positioning and occupancy genome-wide. Our results provide new information on the chromatin engagement of two central players in transcription elongation and emphasize the importance of the nucleosome core as a hub for proteins that regulate chromatin during transcription.
Highlights
Cucinotta et al Introduction The nucleosome, comprised of ~147bp of DNA wrapped around a histone octamer core, has long been regarded as a barrier to transcription in eukaryotes
The nucleosome acidic patch is required for proper nucleosome occupancy and positioning genome-wide We have shown that the nucleosome acidic patch directly interacts with subunits of two transcription elongation factors, Polymerase Associated Factor 1 Complex (Paf1C) and FACT, that regulate nucleosome occupancy and modification
Prompted by our discovery that the acidic patch is important for transcription-coupled histone modifications and transcription elongation [9], we focused our study on proteins involved in these processes
Summary
Cucinotta et al Introduction The nucleosome, comprised of ~147bp of DNA wrapped around a histone octamer core, has long been regarded as a barrier to transcription in eukaryotes. One crucial process regulated by the nucleosome acidic patch is the monoubiquitylation of a highly conserved lysine near the C-terminus of H2B (H2B K123 in S. cerevisiae, H2B K120 in H. sapiens) [5,9,14,15] In both yeast and humans, H2B ubiquitylation (H2Bub) is associated with transcription elongation and promotes Pol II processivity [16,17,18]. Whether transcription elongation factors implicated in H2B K123ub directly engage the acidic patch or affect H2B K123ub through other mechanisms remained unclear To address this question, we chose to apply an approach for investigating direct binding to the acidic patch in the context of cellular chromatin. Our data show that the nucleosome acidic patch is important for chromatin architecture and directly interacts with transcription elongation factors that regulate histone modifications
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