HENIKOFF LAB -- When RNA Polymerase encounters a nucleosome

When RNA Polymerase encounters a nucleosome

When Daniel Zilberman was a postdoc in the lab, he discovered that the universal histone variant, H2A.Z, which is generally enriched on nucleosomes flanking promoters, is mutually antagonistic with DNA methylation in Arabidopsis1. We next asked whether a similar relationship exists in mammals, using a mouse B-cell lymphoma model, where chromatin states can be monitored during tumorigenesis. In collaboration with the lab of my colleague Bob Eisenman, graduate student Melissa Conerly found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during MYC-induced transformation of pre-B cells and, subsequently, during lymphomagenesis2. In addition, we found that H2A.Z and DNA methylation are generally anticorrelated around TSSs in both wild-type and MYC-transformed cells, as expected for the opposite effects of these chromatin features on promoter competence. Depletion of H2A.Z over TSSs both in cells that are induced to proliferate and in cells that are developing into a tumor suggested that progressive loss of H2A.Z during tumorigenesis results from the advancing disease state. These changes were accompanied by increases in chromatin salt solubility. Surprisingly, ~30% of all genes showed a redistribution of H2A.Z from around TSSs to bodies of active genes during the transition from MYC-transformed to tumor cells, with DNA methylation lost from gene bodies where H2A.Z levels increased. No such redistributions were observed during MYC-induced transformation of wild-type pre-B cells. Our results implied that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression.

            Other studies in the lab on H2A.Z have been spearheaded by graduate student Chris Weber. A single octameric nucleosome can contain two H2A.Z histones (homotypic) or one H2A.Z and one canonical H2A (heterotypic). To elucidate the function of H2A.Z, Chris generated high-resolution maps of homotypic and heterotypic Drosophila H2A.Z nucleosomes3. Although homotypic and heterotypic H2A.Z nucleosomes mapped throughout most of the genome, homotypic nucleosomes were enriched and heterotypic nucleosomes were depleted downstream of active promoters and intron-exon junctions. The distribution of homotypic H2A.Z nucleosomes resembled that of classical active chromatin and showed evidence of disruption during transcriptional elongation. Both homotypic H2A.Z nucleosomes and classical active chromatin were depleted downstream of paused polymerases. These results suggested that H2A.Z enrichment patterns result from intrinsic structural differences between heterotypic and homotypic H2A.Z nucleosomes that follow disruption during transcriptional elongation.

            In addition to understanding the effect of transcription on H2A.Z composition, Chris wanted to understand how H2A.Z might affect transcription. This required knowing precisely where all RNAPII is, but current methods did not provide the necessary data. Taking advantage of the fact that engaged RNAPII is stable and insoluble, Chris developed a simple method, “3’NT”, for determining the position of the base added onto the growing nascent RNA transcript4. 3’NT does not use antibodies, tags or harsh treatments, and maps all RNAPII regardless of whether it is actively transcribing or is stall and backtracked. Together with postdoc Srinivas Ramachandran, Chris found that the entry site of the first (+1) nucleosome is a barrier to RNAPII for essentially all genes, including those undergoing regulated pausing farther upstream. In contrast to the +1 nucleosome, gene body nucleosomes are low barriers and cause RNAPII stalling both at the entry site and near the dyad axis. The extent of the +1 nucleosome barrier correlates with nucleosome occupancy but anti-correlates with enrichment of H2A.Z. Importantly, depletion of H2A.Z from a nucleosome position results in a higher barrier to RNAPII. Our results suggest that nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z. This study provides 1) a simple method for mapping transcription as defined by the base incorporated at the active site of RNA polymerase, 2) detection and quantification of the nucleosome barrier to transcription in vivo and 3) insights into what H2A.Z has likely evolved to do, namely to modulate the nucleosome barrier to transcription.

Postdoc Srinivas Ramachandran used H4S47C-anchored cleavage mapping to determine whether half-nucleosomes can be found at genomic locations other than the centromere5. He discovered a subset of +1 and -1 nucleosomes around transcription start sites that fail to show H4S47C-anchored cleaveage on one half of the nucleosome, but both halves of the nucleosome were protected from MNase digestion with increased cleavage at the dyad, indicating these are not hemisomes but asymmetric octameric nucleosomes that  have lost H4-DNA contact on one side of the dyad axis. These nucleosomes are sites of enrichment for H2A.Z and the RSC remodeling complex, suggesting that the asymmetry may result from an intermediate in chromatin remodeling. Partial depletion of RSC resulted in a reduction in occupancy at asymmetric +1 nucleosomes and a reduction in gene expression at well-expressed genes, suggesting that RSC facilitates transcription, and its action on the nucleosome causes it to be asymmetric. RSC is thought to bind nucleosomes and unwrap DNA to the dyad, while binding both the histone octamer and nucleosomal DNA in the absence of ATP6. Asymmetric nucleosomes may represent this intermediate state. Activation by ATP would mobilize the DNA and possibly evict the nucleosome facilitating transcription.

References

1              Zilberman, D., Coleman-Derr, D., Ballinger, T. & Henikoff, S. Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456, 125-129, doi:10.1038/nature07324 (2008).

2              Conerly, M. L. et al. Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis. Genome Res 20, 1383-1390, doi:10.1101/gr.106542.110 (2010).

3              Weber, C. M., Henikoff, J. G. & Henikoff, S. H2A.Z nucleosomes enriched over active genes are homotypic. Nat Struct Mol Biol 17, 1500-1507, doi:10.1038/nsmb.1926 (2010).

4              Weber, C. M., Ramachandran, S. & Henikoff, S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell 53, 819-830, doi:10.1016/j.molcel.2014.02.014 (2014).

5              Ramachandran, S., Zentner, G. E. & Henikoff, S. Asymmetric nucleosomes flank promoters in the budding yeast genome. Genome Res 25, 381-390, doi:10.1101/gr.182618.114 (2015).

6              Ramachandran, S. & Henikoff, S. Nucleosome dynamics during chromatin remodeling in vivo. Nucleus 7, 20-26, doi:10.1080/19491034.2016.1149666 (2016).