Structural Epigenomics

Nucleosomes are disrupted during transcription and other active processes, but the structural intermediates during nucleosome disruption in vivo are unknown. To identify intermediates, Srinivas Ramachandran mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by sequencing1. At the first nucleosome position downstream of the transcription start site, we identified unwrapped intermediates, including hexasomes that lack either proximal or distal contacts. Inhibiting topoisomerases or depleting histone chaperones increased unwrapping, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased unwrapping. Our results indicate that positive torsion generated by elongating RNAPII causes transient loss of histone-DNA contacts. Using this mapping approach, we found that nucleosomes flanking human CTCF insulation sites are similarly disrupted. We also identified diagnostic subnucleosomal particle remnants in cell-free human DNA data as a relic of transcribed genes from apoptosing cells. Thus identification of subnucleosomal fragments from nuclease protection data represents a general strategy for structural epigenomics.

In yeast, nucleosome depleted regions (NDRs) wider than ~150 bp have been reported to contain “fragile nucleosomes” that have a nucleosome-like MNase footprint and increased sensitivity to MNase2. However, ChIP-seq experiments failed to detect histones at these sites and chemical cleavage mapping did not detect DNA contacts with H3 or H43,4, casting doubt on whether histones were present at these sites. To resolve this issue, Sandipan Brahma used CUT&RUN with an antibody to the catalytic subunit of the RSC (remodeling the structure of chromatin) remodeling complex followed by native chromatin immunoprecipitation of CUT&RUN complexes (CUT&RUN.ChIP) to show that RSC is bound at NDRs wider than ~150 bp and is bound to histones at a fraction of these sites5.These histones are present as partially unwrapped nucleosomal intermediates, suggesting that nucleosomes bind transiently to these sites but are engulfed, unwrapped and displaced by RSC. Partial unwrapping appears to expose sites and facilitate binding of general regulatory factors, which may remain bound after nucleosome displacement. At narrow NDRs (<120 bp) histones are not present.

References

  1. Ramachandran, S., Ahmad, K. & Henikoff, S. Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates. Mol Cell 68, 1038-1053 e1034, doi:10.1016/j.molcel.2017.11.015 (2017).
  2. Kubik, S. et al. Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast. Mol Cell 60, 422-434, doi:10.1016/j.molcel.2015.10.002 (2015).
  3. Chereji, R. V., Ocampo, J. & Clark, D. J. MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers. Mol Cell 65, 565-577 e563, doi:10.1016/j.molcel.2016.12.009 (2017).
  4. Chereji, R. V., Ramachandran, S., Bryson, T. D. & Henikoff, S. Precise genome-wide mapping of single nucleosomes and linkers in vivo. Genome Biol 19, 19, doi:10.1186/s13059-018-1398-0 (2018).
  5. Brahma, S. & Henikoff, S. RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast. Mol Cell 73, 238-249 e233, doi:10.1016/j.molcel.2018.10.046 (2019).