Tommy Kaplan | bioRxiv Science

bioRxiv2015

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

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.

Genomics↗

bioRxiv2014

Establishment of regions of genomic activity during the Drosophila maternal to zygotic transition

A conspicuous feature of early animal development is the lack of transcription from the embryonic genome, and it typically takes several hours to several days (depending on the species) until widespread transcription of the embryonic genome begins. Although this transition is ubiquitous, relatively little is known about how the shift from a transcriptionally quiescent to transcriptionally active genome is controlled. We describe here the genome-wide distributions and temporal dynamics of nucleosomes and post-translational histone modifications through the maternal-to-zygotic transition in embryos of the pomace fly Drosophila melanogaster. At mitotic cycle 8, when few zygotic genes are being transcribed, embryonic chromatin is in a relatively simple state: there are few nucleosome free regions, undetectable levels of the histone methylation marks characteristic of mature chromatin, and low levels of histone acetylation at a relatively small number of loci. Histone acetylation increases by cycle 11, but it is not until cycle 13 that nucleosome free regions and domains of histone methylation become widespread. Early histone acetylation is strongly associated with regions that we have previously shown are bound in early embryos by the maternally deposited transcription factor Zelda. Most of these Zelda-bound regions are destined to be enhancers or promoters active during mitotic cycle 14, and our data demonstrate that they are biochemically distinct long before they become active, raising the possibility that Zelda triggers a cascade of events, including the accumulation of specific histone modifications, that plays a role in the subsequent activation of these sequences. Many of these Zelda-associated active regions occur in larger domains that we find strongly enriched for histone marks characteristic of Polycomb-mediated repression, suggesting a dynamic balance between Zelda activation and Polycomb repression. Collectively, these data paint a complex picture of a genome in transition from a quiescent to an active state, and highlight the role of Zelda in mediating this transition.

Developmental Biology↗