Project description:Transcriptional cofactors communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression, which is a hallmark of all multicellular organisms. However, the extent to which different cofactors display intrinsic specificity for distinct promoters is unclear. Testing intrinsic COF – CP preferences requires the systematic assessment of transcriptional activation for many CPs in the presence or absence of a given COF in an otherwise constant standardized reporter system. We therefore combined a plasmid-based high-throughput reporter assay, Self-Transcribing Active Core Promoter-sequencing (STAP-seq), with the specific recruitment of individual COFs to create a high-throughput activator bypass-like assay. Using this assay, we tested whether 23 different individually tethered Drosophila melanogaster COFs could activate transcription from any of 72,000 CP candidates in S2 cells. We selected COFs that represent different functional classes and enzymatic activities: histone-acetyl transferases (Nejire - the fly ortholog of CBP/P300, Mof, Gcn5 - ortholog of pCAF, Atac2, Tip60, Br140), three components of the COMPASS-like histone H3K4 methyltransferase complexes (Lpt, Trr and Trx), nucleosome remodeling ATP-ase (Brm), two Chromo or Chromo-shadow domain (Chro and Mof) and three Bromodomain (Brd8, Brd9 and fs(1)h - ortholog of Brd4) -containing COFs, three subunits of the Mediator Complex (MED15, MED24 and MED25), three subunits of the TFIID general transcription factor (Taf4, Tbp and Trf2) and three less well-characterized COFs (EMSY, Gfzf and Pzg). We used the strong transcriptional activator P65 as a positive and GFP as a negative control. In addition, we tested 6 COFs and the 2 controls in two other Drosophila cell lines: ovarian somatic cells (OSC) and Kc167 cells. We uncovered five classes of core promoters, distinguished by their differential response to specific cofactors and by the presence of known sequence elements, such as the TATA-box, Down-stream Promoter Element, or TCT motif. Moreover, the five promoter types show distinct chromatin properties at endogenous loci. The observed compatibilities between cofactors and promoters can explain how different enhancers specifically activate distinct sets of genes, alternative promoters within the same genes, or even distinct transcription starting sites within the same promoters. Thus, cofactor–promoter compatibilities may underlie distinct transcriptional programs.

Project description:Development and homeostasis of all multicellular organisms rely on differential cell-type-specific gene expression that is regulated by non-coding genomic enhancer elements. Enhancers function through the transcription-factor-mediated recruitment of cofactors, a structurally and functionally diverse set of proteins that activate RNA polymerase II transcription at promoters. Despite the important roles of enhancers and cofactors in transcriptional regulation, it is still not clear if all cofactors are required for all enhancers or if different enhancers have distinct cofactor dependencies and whether differential cofactor dependencies could be used to functionally categorize enhancers. Here, we quantified enhancer activities along the entire human genome using the massively parallel enhancer-activity assay STARR-seq in HCT116 cells, following the rapid auxin-inducible degradation of eight different cofactors. We identified groups of enhancers with distinct cofactor requirements, including enhancers whose activity is insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4, respectively. In particular, Mediator seemed dispensable for P53-responsive enhancers and MED14-depleted cells in which transcription globally failed were still able to induce endogenous P53 target genes such as p21. Similarly, BRD4 was not required for genes with a CCAAT- and TATA-box proximal enhancer, including histone genes and LTR12 family retrotransposons, and for the induction of TATA-box containing heat-shock genes. Taken together, we show that different types of enhancers with distinct cofactor dependencies exist, including enhancer types that function in the absence of well-known cofactors and are employed to regulate specific gene classes and transcriptional programs. This represents the first functional categorization of enhancers by their cofactor dependencies that improves our understanding of alternative ways to activate transcription, which can aid in developing more precise interventions to modulate gene expression.

Project description:Development and homeostasis of all multicellular organisms rely on differential cell-type-specific gene expression that is regulated by non-coding genomic enhancer elements. Enhancers function through the transcription-factor-mediated recruitment of cofactors, a structurally and functionally diverse set of proteins that activate RNA polymerase II transcription at promoters. Despite the important roles of enhancers and cofactors in transcriptional regulation, it is still not clear if all cofactors are required for all enhancers or if different enhancers have distinct cofactor dependencies and whether differential cofactor dependencies could be used to functionally categorize enhancers. Here, we quantified enhancer activities along the entire human genome using the massively parallel enhancer-activity assay STARR-seq in HCT116 cells, following the rapid auxin-inducible degradation of eight different cofactors. We identified groups of enhancers with distinct cofactor requirements, including enhancers whose activity is insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4, respectively. In particular, Mediator seemed dispensable for P53-responsive enhancers and MED14-depleted cells in which transcription globally failed were still able to induce endogenous P53 target genes such as p21. Similarly, BRD4 was not required for genes with a CCAAT- and TATA-box proximal enhancer, including histone genes and LTR12 family retrotransposons, and for the induction of TATA-box containing heat-shock genes. Taken together, we show that different types of enhancers with distinct cofactor dependencies exist, including enhancer types that function in the absence of well-known cofactors and are employed to regulate specific gene classes and transcriptional programs. This represents the first functional categorization of enhancers by their cofactor dependencies that improves our understanding of alternative ways to activate transcription, which can aid in developing more precise interventions to modulate gene expression. "

Project description:Development and homeostasis of all multicellular organisms rely on differential cell-type-specific gene expression that is regulated by non-coding genomic enhancer elements. Enhancers function through the transcription-factor-mediated recruitment of cofactors, a structurally and functionally diverse set of proteins that activate RNA polymerase II transcription at promoters. Despite the important roles of enhancers and cofactors in transcriptional regulation, it is still not clear if all cofactors are required for all enhancers or if different enhancers have distinct cofactor dependencies and whether differential cofactor dependencies could be used to functionally categorize enhancers. Here, we quantified enhancer activities along the entire human genome using the massively parallel enhancer-activity assay STARR-seq in HCT116 cells, following the rapid auxin-inducible degradation of eight different cofactors. We identified groups of enhancers with distinct cofactor requirements, including enhancers whose activity is insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4, respectively. In particular, Mediator seemed dispensable for P53-responsive enhancers and MED14-depleted cells in which transcription globally failed were still able to induce endogenous P53 target genes such as p21. Similarly, BRD4 was not required for genes with a CCAAT- and TATA-box proximal enhancer, including histone genes and LTR12 family retrotransposons, and for the induction of TATA-box containing heat-shock genes. Taken together, we show that different types of enhancers with distinct cofactor dependencies exist, including enhancer types that function in the absence of well-known cofactors and are employed to regulate specific gene classes and transcriptional programs. This represents the first functional categorization of enhancers by their cofactor dependencies that improves our understanding of alternative ways to activate transcription, which can aid in developing more precise interventions to modulate gene expression.

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Transcriptional cofactors display core promoter class-specificity in human

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