Integrator is a genome-wide attenuator of non-productive transcription

ABSTRACTTermination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the Cleavage and Polyadenylation (CPA) and Integrator (INT) complexes originally found to act at the ends of protein-coding and snRNA genes, respectively. Here we monitor CPA- and INT-dependent termination activities genome-wide, including over 8000 previously unannotated transcription units (TUs), that produce unstable RNA. We verify the global activity of CPA, that occurs at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is, however, largely sequence-independent and restricted to a ~3 kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity; it dampens transcriptional output from weak promoters and it provides quality-control of RNAPII complexes, that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, attenuation of non-productive transcription.

CDK9-dependent RNA polymerase II pausing controls transcription initiation

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

Structure of transcribed chromatin is a sensor of DNA damage

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme.

RNA Polymerase Trafficking in Bacillus subtilis Cells

ABSTRACT To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the σA and β′ subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis.

Transcriptional Pausing Caused by NELF Plays a Dual Role in Regulating Immediate-Early Expression of the junB Gene

ABSTRACT Human 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.