There has been extraordinary progress in molecular biology, beginning with the discovery of the DNA double helix and culminating with the nearly complete specification of our genetic inheritance at the level of DNA sequence. However, the inheritance of differences between cells and tissues and the inheritance of centromeres remain poorly understood. To understand these enigmatic modes of inheritance, which do not strictly depend on DNA sequence, we develop in vivo genomic tools for studying DNA-binding proteins of the epigenome, including histones, transcription factors, nucleosome remodelers, and RNA polymerases. These tools have helped us elucidate the processes that are responsible for establishing and maintaining gene expression states and centromeres.
Development of Tools for Studying Chromatin Dynamics
Several of our recent studies have focused on understanding chromatin dynamics and its relationship to gene expression and epigenetic inheritance. We have developed a series of powerful genome-wide tools for measuring dynamics and mapping epigenomic features. These include CATCH-IT, a novel 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 single–base pair resolution that we have applied to nucleosomes, chromatin remodelers, and transcription factors; 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 high-resolution genome-wide assay to detect torsional states; and a modified high-resolution cross-linked chromatin immunoprecipitation (X-ChIP) protocol for large insoluble complexes. We are continuing to develop enabling epigenomic tools, including a genome-wide method for efficient time-resolved transcription factor mapping that provides base pair resolution without chromatin solubilization or antibodies.
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 our high-resolution X-ChIP method, 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. 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 doxorubicin and other anthracycline compounds, standard chemotherapeutic drugs that have been used in the clinic for 40 years. Taken together, our findings provide a mechanistic framework for transcription through a nucleosome in vivo, and we anticipate additional insights into this process from new mapping methods that we are developing.
Another class of histone variants in which we have a long-standing interest mediates chromosome segregation. Centromere-specific histone H3 variants, called cenH3, CENP-A, or Cse4, 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. We have now 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. From a chromatin perspective, the cenH3 hemisome over CDEII is an odd particle indeed: it is precisely constrained in position to the base pair, but shows full reflectional and rotational flexibility. We have since applied H4S47C-anchored cleavage mapping to identify other unusual nucleosomes, leading to the discovery of asymmetric nucleosomes flanking budding yeast promoters that are evidently intermediates in nucleosome remodeling.
We are also asking 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 holocentric centromeres, in which attachment to the spindle apparatus spans the entire length of the chromosome. 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 transcription factors at low affinity, which raises the possibility that transcription factor binding helps to maintain centromeric sites in the absence of cenH3 by preventing the encroachment of conventional nucleosomes. We are applying new experimental and computational tools that we have developed to elucidate the molecular organization of human and other centromeres embedded in homogeneous satellite repeats, which have proven intractable to current mapping strategies.
Some of this work has been supported in part by a grant from the National Institutes of Health.
As of November 14, 2014