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).
We have introduced genomic tools to probe the dynamic structure of the chromatin landscape and explore its relationship to gene regulation and centromere function. These tools include salt fractionation, a method to extract classically ‘active’ chromatin; CATCH-IT, a metabolic labeling strategy to directly measure nucleosome turnover; INTACT, a cell-type-specific nuclear purification method to determine chromatin differences between tissues; ORGANIC, a method for mapping native chromatin at base-pair resolution; 3'NT, a method for determining the last base added onto a nascent chain within the active site of RNA polymerase II (RNAPII); TMP-seq, a method to map DNA torsion genome-wide; MNase X-ChIP-seq, a high-resolution cross-linked chromatin immunoprecipitation (ChIP) protocol for large insoluble complexes, ChEC-seq, an in situ alternative to ChIP; MINCE-seq, a metabolic labeling strategy for observing changes in nucleosomes and transcription factors (TFs) during DNA replication; and CUT&RUN, an in situ alternative to ChIP using antibody-targeted tethered MNase to release DNA adjacent to proteins of interest, which has low background, high resolution, requires about one tenth the sequencing depth of ChIP, and permits profiling with low cell numbers.
Transcription through Nucleosomes
We have used 3'NT to address a long-standing question in the transcription field: How do RNA polymerases overcome nucleosome barriers in vivo? By comprehensively mapping the positions of elongating and arrested RNAPII using 3'NT, we found that nucleosomes are barriers to RNAPII elongation at essentially all genes, with the nucleosome downstream of the transcriptional start site the strongest barrier. The histone variant H2A.Z is enriched in this nucleosome, and we found that it acts to reduce nucleosome barrier strength. One potential mechanism for overcoming the nucleosome barrier to transcription is to mobilize nucleosomes by ATP-dependent remodelers. Using MNase X-ChIP-seq, we found 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. Using TMP-seq, we found that inhibiting topoisomerases results in both increased torsion measured at high resolution and increased turnover of nucleosomes, confirming this mechanism in vivo. We applied H4S47C-anchored cleavage mapping to identify asymmetric nucleosomes flanking budding yeast promoters that are evidently intermediates in nucleosome remodeling. We also found that compounds used in standard chemotherapy that intercalate between the bases and potentially generate torsional stress also enhance nucleosome turnover associated with transcription, suggesting a chromatin-based mechanism for cell killing by these drugs. Taken together, our findings provide a mechanistic framework for transcription through a nucleosome in vivo.
We have used MINCE-seq to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. We found that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. The most conspicuous changes are at promoters that have high levels of RNAPII stalling and DNA accessibility and show specific enrichment for the BRM remodeler. Enhancer chromatin is also disrupted during replication, suggesting a role for TF competition in nucleosome re-establishment. Thus, the characteristic nucleosome landscape emerges from a uniformly packaged genome by the action of TFs, RNAPII, and remodelers minutes after replication fork passage. MINCE-seq thus provides a first glimpse into the dynamic processes that establish and maintain the chromatin landscape every cell generation.
A class of histone variants in which we have a long-standing interest mediates chromosome segregation. Centromere-specific histone H3 variants, called cenH3, CENP-A (in mammals), or Cse4 (in yeast), mark the location of the kinetochore, which attaches to microtubules to segregate chromosomes in mitosis and meiosis. We previously showed that cenH3 nucleosomes of budding yeast wrap DNA to form positive supercoils, in contrast to conventional nucleosomes, which form negative supercoils. More recently, we precisely characterized this nucleosome in vivo and in vitro. We used ORGANIC and V-plot analysis (Figure 1) to show that the ~120 bp budding yeast centromere consists of a particle containing cenH3 and H2A wrapped by the ~90 percent AT-rich ~80 bp central DNA segment (CDEII). This supports a hemisome model in which a core containing one each of the four histones is wrapped by CDEII. We also produced stable cenH3-H4-H2A-H2B hemisomes in vitro by reconstitution with a 78 bp CDEII DNA duplex. To precisely delineate the organization of the particle wrapped by CDEII, we applied H4S47C-anchored cleavage mapping, which converts histone H4 into a cleavage reagent, thus revealing the precise position of histone H4 in every nucleosome in the genome. We found that a single core structure is compatible with centromere cleavage patterns and distances; in this structure, oppositely oriented cenH3-H4-H2A-H2B hemisomes occupy one of two rotationally phased positions on each of the 16 yeast centromeres at similar frequencies within the population. In contrast, H4S47C-anchored cleavage mapping of centromeric nucleosomes in fission yeast indicates these nucleosomes have two H4 molecules, suggesting conventional octameic nucleosomes or possibly tetrasomes or hexasomes.
We are also asking how the special properties of cenH3 nucleosomes might allow the centromere to evolve into diverse centromere types. These range from the point centromeres of budding yeast, with a single cenH3 nucleosome, to the regional centromeres of fission yeast and many other single-cell eukaryotes that encompass a few kilobases of often unique sequence, to the repeat-rich centromeres of most animals and plants that span megabases of DNA, and to holocentric centromeres, in which attachment to the spindle apparatus spans the entire length of the chromosome. In fission yeast, cenH3 nucleosomes inhabit the central domain that is nearly devoid of H3 nucleosomes, but they are not consistently positioned and are variably spaced, with no preferred positions for assembly of inner kinetochore proteins. In contrast in repeat-based centromeres of plants and animals, satellite sequences position the cenH3 nucleosomes, which are translationally and rotationally phased, possibly contributing to kinetochore stability. Applying new experimental and computational tools, we have begun to elucidate the molecular organization of animal and plant centromeres embedded in homogeneous satellite repeats, which have proven intractable to current mapping strategies. Our approach revealed that a unique chromatin complex occupies young dimeric α-satellite arrays that dominate functional human centromeres, with two positioned CENP-A (cenH3) nucleosomes separated by a linker that contains the CENP-B box, bound by CENP-B. CENP-T appears to occupy this linker, forming a complex with two CENP-A nucleosomes and CENP-C.
Together with the Harmit Malik lab (HHMI, Fred Hutchinson Cancer Research Center), we found that evolution of holocentricity in insects accompanies complete loss of cenH3. In nematodes, we found that single cenH3 nucleosomes are scattered at ~700 discrete sites flanked by well-positioned canonical nucleosomes. These sites coincide with sites that bind multiple TFs at low affinity, which raises the possibility that TF binding helps to maintain centromeric sites in the absence of cenH3 by preventing the encroachment of conventional nucleosomes.