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GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes.[ChIP-seq]

ABSTRACT: Promoter-proximal pausing of RNA polymerase II (RNA-Pol) is a rate-limiting step primed for rapid and synchronous induction of genes involved in critical physiological processes. Its underlying mechanisms are not fully understood. Using cytogenetic, genomic and genetic approaches, we provide evidence to support a direct role of a prevalent upstream GAGA binding factor (GAF) in transcriptional pausing of Hsp70 and many developmental regulators in Drosophila. For GAF target genes, the abundance of paused RNA-Pol and GAF is closely correlated. Promoters with higher GAF occupancy show stronger reduction of paused RNA-Pol in Gaf mutants. In addition, nucleosome organization is preferentially affected in the upstream region by GAF in a dosage-dependent manner. Genetic assays using a dominant eye phenotype caused by GAF overexpression suggests that GAF cooperates with nucleosome remodeler NURF, pausing factor NELF and an upstream binding factor BAB1. Thus, GAF plays a critical role in a regulatory network to facilitate transcriptional pausing through modulation of upstream nucleosomes.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE70632 | GEO | 2016/07/19

SECONDARY ACCESSION(S): PRJNA289241

REPOSITORIES: GEO

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GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes. [MNase-seq]

Project description:Promoter-proximal pausing of RNA polymerase II (RNA-Pol) is a rate-limiting step primed for rapid and synchronous induction of genes involved in critical physiological processes. Its underlying mechanisms are not fully understood. Using cytogenetic, genomic and genetic approaches, we provide evidence to support a direct role of a prevalent upstream GAGA binding factor (GAF) in transcriptional pausing of Hsp70 and many developmental regulators in Drosophila. For GAF target genes, the abundance of paused RNA-Pol and GAF is closely correlated. Promoters with higher GAF occupancy show stronger reduction of paused RNA-Pol in Gaf mutants. In addition, nucleosome organization is preferentially affected in the upstream region by GAF in a dosage-dependent manner. Genetic assays using a dominant eye phenotype caused by GAF overexpression suggests that GAF cooperates with nucleosome remodeler NURF, pausing factor NELF and an upstream binding factor BAB1. Thus, GAF plays a critical role in a regulatory network to facilitate transcriptional pausing through modulation of upstream nucleosomes.

2016-07-19 | GSE70633 | GEO

GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing [MNase]

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2015-04-04 | E-GEOD-58956 | biostudies-arrayexpress

GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing [MNase]

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Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation

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GAGA-Associated Factor Fosters Loop Formation in the Drosophila Genome

Project description:The role of genome organization in the control of gene expression persists as a central mystery of modern biology. Most efforts have focused on the role of boundary elements and CTCF in the compartmentalization of the genome into a series of topological associating domains (TADs). These compartments have been suggested to enable long-range DNA-DNA associations via loop extrusion processes. However, there is emerging evidence for long-range focal contacts, whereby specific DNA sequences associated with promoters and distal enhancers interact to form chromatin loops. One such class of DNAs, tethering elements, binds GAGA-associated factor (GAF). Previous studies provided evidence that GAF possesses amyloid properties in vitro, enabling the formation of loops bridging separate DNA molecules. In this study we investigated the possibility that GAF also functions as a looping factor in Drosophila development. We employed a combination of Micro-C assays, proteasomal degradation, and genome editing to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is particularly important for long-range interactions of GAF bound to distant GAGA-rich tethering elements. By contrast, the C-terminal low complexity domain (poly Q) plays only a minor role in loop formation. The most striking effects of GAF mutants are observed for long-range promoter-promoter interactions that coordinate the activities of distant paralogous genes.

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Pioneer factor GAF cooperates with PBAP and NURF to regulate transcription [CUT&RUN]

Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.

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