SUMMARY The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II-associated Factor 1 (PAF1) to possess an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II’s C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a regulator of promoter-proximal pausing by Pol II.

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the Trr/MLL3/4-COMPASS family1–3. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr-COMPASS eclose and develop to a productive adulthood. Parallel experiments with a Trr allele that augments enzyme product specificity reveal that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of mammalian MLL3 and MLL4 catalytic SET domain in embryonic stem cells does not disrupt self-renewal capability of the ES cells. Trr catalytic mutant alleles manifest subtle developmental phenotypes when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that metazoan development can occur in the context of Trr/COMPASS with H3K4me1 enzymatic deficiency, and points to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways.

Summary Although it is established that some general transcription factors are inactivated at mitosis, many details of Mitotic Inhibition of Transcription (MIT) and its underlying mechanisms are largely unknown. We have identified Mitotic Transcriptional Activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA Polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MIT. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MIT whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

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