Project description:Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report the existence of genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associated with Hmo1, previously shown to mediate the architecture of S phase-transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions. The present finding of the existence of extra-telomeric functions for Ten1 in the regulation of RNA polymerase II in cooperation with Stn1 and Cdc13 has profound repercussions on future studies both on telomeric and transcription pathways.
Project description:The wheat Pol II enzyme was purified, and a transcription initiation complex was assembled on the potato spindle tuber viroid (PSTVd) RNA template. The transcription initiation complex was characterized using LC-MSMS.
Project description:Pol II has been recognized as a passively regulated holoenzyme. However, whether Pol II plays specific regulatory roles remain unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, Specific Degradation of Disassociated Subunits (SDDS), we demonstrated that dRPB3 functions as a regulatory component of Pol II to enable the preferential control of 3’ end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3’ end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that Pol II gains specific regulatory functions by trapping disassociated subunits.
Project description:Nucleosomes restrict the access of transcription factors to chromatin. RSC is a SWI/SNF-family chromatin-remodeling complex from yeast that repositions and ejects nucleosomes in vitro. Here, we examined these activities and their importance in vivo. We utilized array-based methods to examine nucleosome occupancy and positioning at more than 200 locations in the genome following the controlled destruction of the catalytic subunit of RSC, Sth1. Loss of RSC function caused pronounced and general reductions in transcription from Pol I, II, and III genes. At Pol III genes, Sth1 loss conferred a general gain in nucleosome density and an accompanying reduction in RNA Pol III occupancy. In contrast, we observed primarily single nucleosome changes, including movement, at Pol II promoters. Importantly, a greater number of changes were observed near the transcription start sites of RSC-occupied promoters than non-occupied promoters. These changes are distinct from those due to general loss of transcription. Thus, RSC action affects both nucleosome density and positioning in vivo, but applies these remodeling modes differently at Pol II and Pol III genes. Keywords: ChIP-chip, nucleosome, mononucleosome, RSC, transcription
Project description:Telomeres cap and protect chromosome ends. Drosophila telomeres consist of repetitive sequences dominated by retrotransposons. Telomeric sequences are transcribed and participate in a negative feedback loop in which they are processed into self-targeting piRNA on the one hand and are serve as the sole source of the transposase responsible for telomere maintenance on the other hand. We show that the tight regulation of the expression of telomeric sequences in the germline is regulated by the NSL complex. As a consequence, both primary biogenesis from telomeric piRNA clusters as well as telomere stability are impaired in cells following NSL complex depletion.
Project description:The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.
Project description:In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo. ChIP-seq with antibody against pol II in wild type and Pcf11 mutants: Pcf11-2, Pcf11-9 and Pcf11-13 grown at 25C and 37C along with input samples
Project description:Inhibition of transcriptional elongation plays an important role in gene regulation in metazoans, including C. elegans, which lacks Negative Elongation Factor homologs. Here we combine genomic and biochemical approaches to dissect a novel role of C. elegans AF10 homolog, ZFP-1, in transcriptional control. We show that ZFP-1 and its interacting partner DOT-1.1 have a global role in negatively modulating the level of Pol II transcription on essential widely expressed genes. Moreover,the ZFP-1/DOT-1.1 complex contributes to progressive Pol II stalling on essential genes during development and to rapid Pol II stalling during stress response. The slowing down of Pol II transcription by ZFP-1/DOT-1.1 is associated with an increase in H3K79 methylation and a decrease in H2B monoubiquitination, which promotes transcription. We propose a model where recruitment of ZFP-1/DOT-1.1 and deposition of H3K79 methylation at highly expressed genes initiates a negative feedback mechanism for modulation of their expression. GRO-seq (Global Run-On sequening) for nascent transcript detectiong on WT and zfp-1(ok554) mutant nematode (C. elegans) larvae in L3 stage, performed in duplicate per condition (4 samples total).
Project description:ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including S. pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.
Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.