Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities—RNA cleavage and RNA polymerase II dephosphorylation—are structurally and functionally integrated into the INTAC complex.

c Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression.T he largest subunit of RNA polymerase II (Pol II), Rpb1, contains a C-terminal domain (CTD) consisting of 52 heptad repeats of the YSPTSPS consensus sequence in humans (1). The CTD is phosphorylated within these repeats, including at serines 2, 5, and 7 (Ser2, Ser5, and Ser7, respectively) (2). The CTD serves as a phosphorylation-regulated platform for the recruitment of transcription factors, RNA processing factors, and chromatin modifiers, which affect mRNA synthesis, cotranscriptional processing, and histone modifications during the transcription cycle (3).The CTD undergoes a cycle of phosphorylation and dephosphorylation during the transcription cycle of initiation, elongation, and termination (4). During transcription initiation and early transcription, Ser5 of the CTD is phosphorylated by the cyclin-dependent kinase 7 (CDK7) subunit of the basal transcription factor TFIIH (2, 5). The positive transcription elongation factor, P-TEFb (comprised of CDK9 and cyclin T), regulates transcription elongation through phosphorylation of the CTD at Ser2 (6). P-TEFb also phosphorylates negative elongation factor (NELF) (7) and DRB sensitivity-inducing factor (DSIF) (8) during the transition to productive elongation.In the budding yeast Saccharomyces cerevisiae, there are two complexes for CTD Ser2 phosphorylation: the Bur1/Bur2 complex and the Ctk1/Ctk2/Ctk3 (CTDK) complex. The Bur complex implements CTD phosphorylation early in the transcription cycle, while the Ctk complex implements CTD phosphorylation during the elongation phase of RNA Pol II (9). CDK9 was long considered to be the only CTD Ser2 kinase in metazoans, but recently the Drosophila dCdk12/dCyclin K complex was shown to be the major CTD Ser2 kinase implementing Ser2 phosphorylation during the elongation stage, analogous to the S. cerevisiae Ctk1/2 complex (10). In ...

CDK7 phosphorylates the RNA polymerase II (pol II) CTD and activates the P-TEFb-associated kinase, CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3′-ends upon CDK7 inhibition. We also noted CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5′-ends and H3K36me3 was displaced toward gene 3′-ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated vs. P-TEFb-phosphorylated CTD (vs. unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent “CTD code” that regulates chromatin marks in addition to RNA processing and pol II pausing.

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.