Gene body H2B monoubiquitylation regulates gene-selective RNA Polymerase II pause release and is not rate limiting for transcription elongation

Abstract

Histone H2B monoubiquitylation (H2Bub1) is localized preferentially to transcribed regions of genes and spreads concomitantly with the progression of RNA polymerase II (Pol II). In mammalian cells, H2Bub1 levels are highly correlated with transcription elongation rates, consistent with the general belief that H2Bub1 facilitates the elongation process. Yet, a causative role of H2Bub1 in regulating elongation rates within live cells remains to be proven. Using our recently developed 4sUDRB-seq method, we examined the impact of H2Bub1 downregulation, through silencing of its cognate E3 ubiquitin ligase RNF20, on genomewide transcription elongation rates. Surprisingly, H2Bub1 downregulation had no measurable effect on global elongation rates. Instead, it led to upregulation of over 1,000 genes by altering their Pol II pause release times; notably, those genes are characterized by the presence of H2Bub1 in relatively close proximity to the paused Pol II. Conversely, another set of genes was downregulated upon partial H2Bub1 depletion, and in those genes H2Bub1 appeared to be required for efficient recruitment of Pol II to the promoter region. Overall, our data shed new light on the molecular mechanisms by which H2Bub1 regulates gene expression and imply that the role of H2Bub1 in transcription elongation should be reconsidered.\n\nAUTHOR SUMMARYTranscription elongation is an important component of the gene expression process. Numerous factors and chromatin modifications, including some that are misregulated in various human diseases, have been suggested to regulate transcription elongation. New methods to measure genomewide transcription elongation rates now enable, for the first time, to determine how a specific factor affects transcription elongation and what is the outcome of its misregulation. Using such method, we examined the role of one specific chromatin modification, histone H2B monoubiquitination (H2Bub1), in regulating transcription. Strikingly, although H2Bub1 is widely believed to serve as a regulator of transcription elongation, its downregulation did not affect genomewide elongation rates. Instead, we found that H2Bub1 regulates the expression of distinct subsets of genes by either promoting recruitment of the transcription machinery or, conversely, favoring the pausing of this machinery shortly after initiation of transcription. Our findings demonstrate that the use of genomewide elongation rate measurements can redefine the true roles of putative transcription elongation factors. Furthermore, they provide a new understanding of the functions of H2Bub1 and its impact on gene expression patterns, which is of particular interest because H2Bub1 is often downregulated in human cancer.