Mono-allelic epigenetic regulation of bi-directional polycistronic transcription initiation by RNA Polymerase II in Trypanosoma brucei.
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ABSTRACT:
ORGANISM(S): Trypanosoma brucei
PROVIDER: GSE273238 | GEO | 2024/07/29
REPOSITORIES: GEO
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Project description:Protein coding genes in the trypanosome genome are organized in polycistronic transcription units (PTUs). How RNA Polymerase II (Pol II) initiates PTU transcription has not been resolved and the current model favors initiation driven by chromatin epigenetic marks, rather than core-promoters. Here, we identify sequence-specific core promoters by functional characterization of ChIP-Seq Pol II accumulation-peaks. Sequences located between oppositely orientated PTUs contained two independent and distinct core promoters, driving unidirectional transcription. Detailed analysis of one promoter identified 75 bp, necessary and sufficient to fully drive reporter expression, and containing short functional motifs. Analysis of further promoters suggested activity and initiation is regulated and both, focused and dispersed transcription patterns were found. Thus, in contrast to the model of unregulated and promoter-independent transcription initiation, sequence-specific promoters determine the initiation of RNA Pol II transcription of protein coding genes in trypanosomes. These findings resolve the discrepancy between trypanosomes and other eukaryotes in how Pol II initiates protein-coding gene transcription.
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Project description:Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
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Project description:Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.
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2021-10-07 | GSE133655 | GEO
Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
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Project description:RNA Polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes such as small nuclear RNA (snRNA) genes is not understood at the structural level. Here we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol II preinitiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE) located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc called wing-1 and wing-2 bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.
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