Abstract

H2A.Z mono-ubiquitylation has been linked to transcriptional repression, but the mechanisms involved are not well understood. To address this, we developed a biotinylation-based approach to purify ubiquitylated H2A.Z (H2A.Zub) mononucleosomes for biochemical and genome-wide analyses. 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. ChIP-Seq analyses reveal that H2A.Zub-nucleosomes are enriched over non-expressed genes, and suggest that it is the relative ratio of ubiquitylated to non-ubiquitylated H2A.Z, rather than 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 mediating global transcriptional repression through its recruitment of silencing factors and nuclear architectural proteins.