Activation domains and Coactivator direct genomic localization of fused DNA binding domains, defining the subset of activated promoters
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae BY4741 Saccharomyces cerevisiae
PROVIDER: GSE234433 | GEO | 2023/12/22
REPOSITORIES: GEO
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Project description:Activation domains (ADs) within transcription factors (TFs) induce gene expression by recruiting coactivators to specific regulatory regions. Within the prevailing model, TF-coactivator recruitment is independent of DNA binding, which is consistent with direct AD-coactivator interactions seen outside cells. However, this independence was not yet tested within the genomic context. Here, we targeted two Med15-interacting ADs to hundreds of budding yeast promoters through fusions with multiple DNA binding domains (DBDs), gradually controlling their abundances using libraries of synthetic promoters. Genomic profiling revealed that AD identity influences DNA binding locations and that transcription induction and Med15 recruitment are restricted to a subset of DBD-bound promoters displaying flexible expression, multiple-TFs binding, and fuzzy nucleosome architecture. Further, when fused to a DBD, Med15 redirected binding towards promoters of fuzzy nucleosomes, overcoming DBD-based preferences. Our results demonstrate that ADs and their recruited coactivators posses an inherent preference for genomic localization and, therefore, define the subset of induced promoters.
2023-12-19 | GSE234431 | GEO
Project description:Activation domains and Coactivator direct genomic localization of fused DNA binding domains, defining the subset of activated promoters
| PRJNA981283 | ENA
Project description:Activation domains and Coactivator direct genomic localization of fused DNA binding domains, defining the subset of activated promoters [RNA-seq]
| PRJNA981303 | ENA
Project description:Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.
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Project description:BACKGROUND:Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1? (peroxisome proliferator-activated receptor [PPAR]-? coactivator-1?), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1? target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1? target genes in heart failure has not been defined. METHODS AND RESULTS:Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1? target metabolic gene promoters in the pressure-overload-induced heart failure model. PGC-1?-cKO (cardiac-specific PGC-1? knockout) mice showed phenotypic similarity to the pressure-overload-induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1? target genes, even under basal conditions. However, the protein levels of PGC-1? were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1? was consistently reduced both in the pressure-overload model and PGC-1?-cKO mice. In vitro DNA binding assays using an endogenous PGC-1? target gene promoter sequence confirmed that PGC-1? recruits polymerase II to the promoter. CONCLUSIONS:These results suggest that PGC-1? promotes the recruitment of polymerase II to the PGC-1? target gene promoters. Downregulation of PGC-1? target genes in the failing heart is attributed, in part, to a reduction of the PGC-1? occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.
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Project description:Coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase recruited by several transcription factors, methylates a large variety of proteins and plays a critical role in gene expression. We report, in this paper, four crystal structures of isolated modules of CARM1. The 1.7 A crystal structure of the N-terminal domain of CARM1 reveals an unexpected PH domain, a scaffold frequently found to regulate protein-protein interactions in a large variety of biological processes. Three crystal structures of the CARM1 catalytic module, two free and one cofactor-bound forms (refined at 2.55 A, 2.4 A and 2.2 A, respectively) reveal large structural modifications including disorder to order transition, helix to strand transition and active site modifications. The N-terminal and the C-terminal end of CARM1 catalytic module contain molecular switches that may inspire how CARM1 regulates its biological activities by protein-protein interactions.
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Project description:BackgroundH1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown.ResultsSequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones.ConclusionsLinker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.
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Project description:This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single-cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique.
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Project description:Mammalian RNA polymerase II preinitiation complexes assemble adjacent to a nucleosome whose proximal edge (NPE) is typically 40 to 50 bp downstream of the transcription start site. At active promoters, that +1 nucleosome is universally modified by trimethylation on lysine 4 of histone H3 (H3K4me3). The Pol II preinitiation complex only extends 35 bp beyond the transcription start site, but nucleosomal templates with an NPE at +51 are nearly inactive in vitro with promoters that lack a TATA element and thus depend on TFIID for promoter recognition. Significantly, this inhibition is relieved when the +1 nucleosome contains H3K4me3, which can interact with TFIID subunits. Here, we show that H3K4me3 templates with both TATA and TATA-less promoters are active with +35 NPEs when transcription is driven by TFIID. Templates with +20 NPE are also active but at reduced levels compared to +35 and +51 NPEs, consistent with a general inhibition of promoter function when the proximal nucleosome encroaches on the preinitiation complex. Remarkably, dinucleosome templates support transcription when H3K4me3 is only present in the distal nucleosome, suggesting that TFIID-H3K4me3 interaction does not require modification of the +1 nucleosome. Transcription reactions performed with an alternative protocol retaining most nuclear factors results primarily in early termination, with a minority of complexes successfully traversing the first nucleosome. In such reactions, the +1 nucleosome does not substantially affect the level of termination even with an NPE of +20, indicating that a nucleosome barrier is not a major driver of early termination by Pol II.
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