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Project description:The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.
| S-EPMC9635645 | biostudies-literature
Project description:We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells. We examine the effect of the interaction between BEAF and polybromo on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells.
2022-03-05 | GSE197579 | GEO
Project description:We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells. We examine the effect of the interaction between BEAF and polybromo on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells.
2022-03-05 | GSE197574 | GEO
Project description:The Drosophila BEAF Insulator Protein Interacts with the Polybromo Subunit of the PBAP Chromatin Remodeling Complex
| PRJNA811123 | ENA
Project description:The Drosophila BEAF Insulator Protein Interacts with the Polybromo Subunit of the PBAP Chromatin Remodeling Complex [Mnase-seq]
| PRJNA811119 | ENA
Project description:The Drosophila BEAF Insulator Protein Interacts with the Polybromo Subunit of the PBAP Chromatin Remodeling Complex [PRO-seq]
| PRJNA811127 | ENA
Project description:In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.
| S-EPMC4113433 | biostudies-literature
Project description:Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. Here we report a further characterization of BEAF. We show that the BESS domain alone is sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, we document the dynamics of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.
| S-EPMC5021357 | biostudies-literature
Project description:Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct conserved forms of the SWI/SNF class of complexes are characterized by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex or with Bap170 and Bap180 to form the PBAP complex. osa mutations reproduce only a subset of the developmental phenotypes caused by mutations in subunits of the core complex. To test whether the PBAP complex performs the remaining functions, we generated mutations in bap170 and bap180. Surprisingly, we found that Bap180 is not essential for viability, although it is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein, but a mutant form that retains this function is sufficient for both survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of bap170 bap180 double mutants, we found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we generated mutants lacking Bap170, Bap180, and Osa in the germ line to demonstrate that the core Brahma complex can function in oogenesis without any of these accessory subunits.
| S-EPMC2519717 | biostudies-literature
Project description:Tonalli A (TnaA) is a Drosophila melanogaster protein with an XSPRING domain. The XSPRING domain harbors an SP-RING zinc-finger, which is characteristic of proteins with SUMO E3 ligase activity. TnaA is required for homeotic gene expression and is presumably involved in the SUMOylation pathway. Here we analyzed some aspects of the TnaA location in embryo and larval stages and its genetic and biochemical interaction with SUMOylation pathway proteins. We describe that there are at least two TnaA proteins (TnaA130 and TnaA123) differentially expressed throughout development. We show that TnaA is chromatin-associated at discrete sites on polytene salivary gland chromosomes of third instar larvae and that tna mutant individuals do not survive to adulthood, with most dying as third instar larvae or pupae. The tna mutants that ultimately die as third instar larvae have an extended life span of at least 4 to 15 days as other SUMOylation pathway mutants. We show that TnaA physically interacts with the SUMO E2 conjugating enzyme Ubc9, and with the BRM complex subunit Osa. Furthermore, we show that tna and osa interact genetically with SUMOylation pathway components and individuals carrying mutations for these genes show a phenotype that can be the consequence of misexpression of developmental-related genes.
| S-EPMC3631182 | biostudies-literature