Variant Chromatin Structures-Targeting and Functions

PROJECT SUMMARY My research investigates variant chromatin structures that decorate the chromosomal landscape of eukaryotic cells, focusing on how these structures are targeted to specific genomic sites and what these structures do in cells. The bulk of the chromatin consists of canonical nucleosome, which has a core of two H2A, H2B, H3 and H4 histones wrapped by ~150 base pairs of DNA. However, variant chromatin particles exist, and they create unique features along the DNA, bookmarking the genome and demarcating functional domains. One project is centered around the histone variant H2A.Z, which is a prominent landmark associated with promoters. H2A.Z is required for optimal transcription response and early animal development. But exactly how H2A.Z modulates transcription is unclear. My lab has made significant progress in understanding H2A.Z’s targeting mechanism. One breakthrough was the development of a biochemical approach that utilizes native yeast nucleosomes as a substrate for H2A.Z incorporation. This innovation revealed sequence motifs within nucleosomes crucial for H2A.Z targeting. Our proposed studies aim to explore the interplay between H2A.Z and the transcription machinery. We posit that H2A.Z nucleosomes, positioned just downstream of promoters, serves as a ‘speedbump’ to RNA polymerase, moderating its transition from initiation to elongation. To test this, we will deploy an array of biochemical and genomic techniques, including a novel in vitro transcription assay that utilizes native nucleosomes. Our research will extend to examine how H2A.Z’s role in transcriptional repression within gene coding regions and the interplay between histone modifications and transcription. The second project investigates the R-octasome, a nucleosome-like particles but with a core made up of four H3 and H4. We recently solved the cryo-EM structure of the R-octasome and detected its existence in yeast. However, the biological function of R-octasomes is unknown. Preliminary data suggest that R-octasomes are associated with the telomeric repeats. Using a mutant that is disruptive to R-octasome formation, our data suggest that its integrity is important for subtelomeric gene silencing. We hypothesize that R-octasomes are integral components of telomeric heterochromatin. A combination of genetic and biochemical tools will be used to pinpoint how R-octasomes contribute to heterochromatin formation. We will interrogate the biophysical properties of R-octasomes, especially on its role in higher-order structure formation. Numerous studies have established correlations between variant chromatin structures and genomic functions. However, pinpointing their mechanistic roles remains challenging. Unlike the histone modification field, where ‘reader’ proteins recognizing histone codes is well established, variant chromatin structures, like H2A.Z, do not appear to be ‘read’ by conventional recognition domains. Rather, they exert their influence by modulating the biophysical and biochemical properties of chromatin in a more nuanced way. Our research aims to investigate variant chromatin structures in the native context and to develop new methodologies to assess their functions.