Ubiquitylated H2A.Z nucleosomes are associated with nuclear architectural proteins and global transcriptional silencing

Abstract

H2A.Z mono-ubiquitylation has been linked to transcriptional repression, but the mechanisms associated with this process are not well understood due to the lack of reagents specific for this modified form of H2A.Z. To address this, we developed a biotinylation-based approach to specifically purify ubiquitylated H2A.Z (H2A.Zub) mononucleosomes and characterize their biochemical composition and genomic distribution. We observe that H2A.Zub nucleosomes are enriched for the repressive histone post-translational modification H3K27me3, but depleted of H3K4 methylation and other modifications associated with active transcription. Consistent with these findings, ChIP-Seq analyses reveal that H2A.Zub-nucleosomes are enriched over non-expressed genes. However, the genomics data also suggest that it is the relative ratio of ubiquitylated to non-ubiquitylated H2A.Z, rather than the absolute presence or absence of H2A.Z ubiquitylation, that correlates with gene silencing. Finally, we observe that H2A.Zub-eniched mononucleosomes preferentially co-purify with transcriptional silencing factors as well as proteins involved in higher order chromatin organization such as CTCF and cohesin. Collectively, these results suggest an important role for H2A.Z ubiquitylation in the genome-wide regulation of chromatin and transcription through its recruitment of transcriptional silencing factors and nuclear architectural proteins.3 Introduction:

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins

“…For example, it has been demonstrated that H2A.Z and H3.3 containing mononucleosomes are enriched for activating histone PTMs such as K4 methylated H3 (Viens et al, 2006;Sarcinella et al, 2007;Won et al, 2015), whereas macroH2A containing mononucleosomes are enriched for repressive histone PTMs like K9 methylated H3 (Won et al, 2015). In addition, this method has also been used to dissect the cross-talk between macroH2A1 and H2B acetylation (Chen et al, 2014;Ruiz and Gamble, 2018), and H2A.Z ubiquitylation with H3K27me3 (Ku et al, 2012;Surface et al, 2016;Ng et al, 2019). Lastly, the incorporation of specific core or variant histones into chromatin has been demonstrated by the nucleosome-immunoprecipitation method (Kanda et al, 1998;Wiedemann et al, 2010;Lau et al, 2011;Ruiz and Gamble, 2018).…”

The Role of the Histone Variant H2A.Z in Metazoan Development

“…H2A.Z is also located at centromeres and, in mouse trophoblast stem cells, its abundance at centromeres fluctuates, such that the highest concentration occurs at mitosis when it is required for chromosome segregation [ 82 ]. Monoubiquitylation of H2A.Z is also associated with heterochromatin [ 85 ].…”