There has been extraordinary progress in molecular biology during the 50-year span that began with the discovery of the DNA double helix and culminated with the nearly complete specification of our genetic inheritance at level of DNA sequence. The bulk of the eukaryotic genome is packaged into nucleosome particles, each of which comprises an octamer with two copies of each of four core histones--H2A, H2B, H3, and H4--which wrap nearly two turns of DNA. Nucleosomes can be differentiated both by numerous post-translational histone modifications and by incorporation of histone variants, which can replace canonical histones to form nucleosomes with special roles and properties. In contrast to our understanding of genomes, the inheritance of differences in gene expression between cells and tissues and how they are mediated by histones and other chromatin proteins is poorly understood. To better understand inheritance that does not depend on DNA sequence, we apply genomic tools to the study of proteins of the epigenome: histones, transcription factors, nucleosome remodelers, and RNA polymerase II (RNAPII).
Recent studies in our lab have focused on understanding the nucleosome dynamics of chromatin, and its relationship to gene expression and epigenetic inheritance. We have developed powerful genome-wide strategies and tools for measuring nucleosome dynamics and mapping epigenomic features. We use tools such as salt fractionation, a method to extract classically ‘active’ chromatin; CATCH-IT, a novel metabolic labeling strategy to directly measure nucleosome turnover; ORGANIC, a method of mapping native chromatin at single base-pair resolution that applies to transcription factors and remodelers as well as nucleosomes; INTACT, a cell-type specific nuclear purification method to determine chromatin differences between tissues; 3’NT, a method of determining the last base added onto nascent RNAPII transcripts; and TMP-Seq, a high-resolution genome-wide assay to detect torsional states. Using these tools we attempt to understand how RNAPII, nucleosome remodelers, transcription factors, and histone variants all affect and are affected by dynamic chromatin structure.
Transcribing through Nucleosomes
Nucleosomes are barriers to transcription in vitro, but is this the case in vivo? To address this question, we used 3’NT to comprehensively map the positions of elongating and arrested RNAPII at nucleotide resolution. We find that the nucleosomes are barriers to RNAPII elongation at essentially all genes, with the nucleosome downstream of the transcriptional start site the strongest barrier. Depletion of the histone variant H2A.Z from a nucleosome strengthens the barrier to RNAPII, which suggests that H2A.Z has evolved to reduce nucleosome barrier strength. One potential mechanism for overcoming the nucleosome barrier to transcription is to mobilize nucleosomes by ATP-dependent remodelers. We find that the Chd1 remodeler is recruited to promoters of mouse genes where it causes nucleosomes to turn over during transcription and allows RNAPII to escape into the gene body. Another potential mechanism for overcoming the nucleosome barrier to transcription is the DNA torsional stress created by RNA polymerase transit, which can unwrap and destabilize nucleosomes. We find that inhibiting topoisomerases results in both increased torsion measured at high resolution and increased turnover of nucleosomes, confirming this mechanism in vivo. Furthermore, compounds that intercalate between the bases, and thus potentially generate torsional stress, also enhance nucleosome turnover associated with transcription, suggesting an epigenetic mechanism for cell killing by widely used chemotherapeutic drugs. Our findings provide a mechanistic framework for transcription through a nucleosome in vivo.
Epigenetic Inheritance of Centromeres
While histone variants such as H2A.Z seem to help modulate transcription, another class of histone variants in which we have a long-standing interest mediates chromosome segregation. Centromere-specific histone H3 variants, collectively called cenH3 (also known as CENP-A in vertebrates or Cse4 in fungi) determine the location of the kinetochore that attaches to microtubules to segregate chromosomes in mitosis and meiosis. Nucleosomes containing cenH3 have a unique structure and wrap DNA to form positive supercoils, in contrast to conventional nucleosomes that form negative supercoils, raising the possibility that positive supercoiling may play a role in the epigenetic inheritance of centromeres. We are interested in how the special properties of cenH3 nucleosomes allow the centromere to be stably inherited and yet evolve into centromeres as diverse as the point centromeres of budding yeast with a single cenH3 nucleosome, to the holocentric centromeres of the nematode worm Caenorhabditis, in which the entire chromosome appears to act as a centromere. Remarkably, we find that the holocentric centromeres of Caenorhabditis resemble dispersed point centromeres of budding yeast, with single cenH3 nucleosomes at 707 discrete sites flanked by well-positioned canonical nucleosomes. Many of these sites coincide with transcription factor hotspots that bind multiple transcription factors at low affinity without the specific binding motifs of any of them. This raises the possibility that transcription factor binding helps to maintain cenH3 sites in the absence of cenH3, which turns over during the cell cycle, by preventing the encroachment of conventional nucleosomes.