Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA Polymerase II in trypanosomes
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ABSTRACT:
PROVIDER: PRJNA541456 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13260277_1.fastq.gz | Fastqsanger.gz | |||
| SRR13260277_2.fastq.gz | Fastqsanger.gz | |||
| SRR13260278_1.fastq.gz | Fastqsanger.gz | |||
| SRR13260278_2.fastq.gz | Fastqsanger.gz | |||
| SRR13260279_1.fastq.gz | Fastqsanger.gz |
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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.
2021-02-01 | GSE130798 | GEO
Project description:Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II, while a smaller subset can be transcribed by RNA polymerase I. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4, complementing the tandem arrays with a single ectopic H4 gene. The resulting ‘histoneH4’ strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4K4 or H4K14 mutants. Both proteomic and transcriptomic analysis of H4K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.
2025-03-11 | PXD061709 | Pride
Project description:Genome-wide transcription studies are revealing increasing examples of ‘dispersed promoters’ that unlike ‘focused promoters’, lack well-conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well-defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation and occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site-specific and genome-wide assays, we identified GT-rich promoters that can drive unidirectional transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.
2017-05-31 | GSE98061 | GEO
Project description:Understanding the precise functions and relationship of BRD2 with other bromodomain and extraterminal motif (BET) proteins is central for the application of BET-specific and pan inhibitors. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at low levels of H3K4me3-modified promoters and Pol II elongation by suppressing R-loops. Single and double depletion revealed that BRD2 and BRD3, but not BRD4, redundantly and independently function in Pol II transcription at different promoters and cooperatively occupy enhancers. Interestingly, we found that depletion of BRD2 affects the expression of different genes during differentiation processes, priming with promoter regulation in ES cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state.
2024-05-22 | PXD030774 | Pride
Project description:Trypanosoma brucei, a member of the Excavates supergroup, falls in an evolutionarily ancient branch of eukaryotes. We have mapped nucleosome positions in T. brucei and identified a map that differs from that of other eukaryotes in several important ways. Unlike in other eukaryotes, the RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined -1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent Variant Surface Glycoprotein (VSG) gene arrays showed extensive arrays of well-positioned nucleosomes, which may act to repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within arrays of silent VSG genes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions. Our results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor.
2017-03-13 | GSE90593 | GEO
Project description:CTCF blocks anti-sense transcription initiation at divergent gene promoters
| PRJNA725532 | ENA
Project description:The nuclear envelope serves as important mRNA surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualised nuclear export in trypanosomes by probing large, endogenous mRNA by intramolecular multi-colour single molecule FISH (smFISH). In addition, unspliced mRNAs were visualised by co-probing two adjacent introns or intergenic regions. We found that the initation of nuclear export requires neither the completion of transcription nor trans-splicing. Nevertheless, the inhibition of trans-splicing blocked cytoplasmic transport of the of unspliced mRNAs and only a small fraction reached the nucleus-distant cytoplasm. Most of the unspliced transcripts remained at the nuclear periphery, within transport and in nuclear periphery granules (NPGs) localised at the cytoplasmic site of nuclear pores that resemble stress granules in composition. Our work shows that, in striking contrast to other eukaryotes, trypanosomes can start nuclear export while the mRNA is still transcribed, but unspliced transcripts remain ‘stuck’ in nuclear pores, probably awaiting processing or decay. Our data indicate that trypanosomes regulate the completion of nuclear export rather than the start.
2018-10-26 | PXD010143 | Pride
Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.
2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress
Project description:Mono-allelic epigenetic regulation of bi-directional polycistronic transcription initiation by RNA Polymerase II in Trypanosoma brucei.
| PRJNA1140653 | ENA
Project description:We describe the results of a genome-wide analysis of human cells that suggests most protein coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9/14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at post-initiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcriptional initiation, where mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.
2008-06-27 | E-TABM-277 | biostudies-arrayexpress