Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14835, USA.
G3 (Bethesda). 2022 Nov 4;12(11). doi: 10.1093/g3journal/jkac223.
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.
果蝇 32kDa 边界元件相关因子（BEAF）结合在几千个主要管家基因的启动子区域。BEAF 既参与染色质域边界活性，又参与启动子功能，但分子机制仍不清楚。在这里，我们表明 BEAF 与 PBAP 的多溴亚基（Pbro）物理相互作用，PBAP 是一种 SWI/SNF 类染色质重塑复合物。BEAF 还与 Pbro 和其他 PBAP 亚基表现出遗传相互作用。我们使用 RNAi 介导的敲低后在培养的 S2 细胞中进行精确的 RNA 运行测序和微球菌核酸酶测序，研究这种相互作用对基因表达和染色质结构的影响。我们的结果与这种相互作用在基因激活中起着微妙作用的观点一致。在 BEAF 敲低后，不到 5%的 BEAF 相关基因受到显著影响。大多数基因下调，伴随着启动子核小体耗竭区域的填充和+1 核小体的轻微上游移位。Pbro 敲低导致几百个基因下调，并与 BEAF 敲低相关，但与启动子近端 GAGA 因子结合的相关性更好。微球菌核酸酶测序支持 BEAF 结合在管家基因启动子附近，而 Pbro 在调节基因中更为重要。然而，RNA 聚合酶 II 启动子近端暂停的普遍但轻微减少以及两种蛋白质敲低后核小体耗竭区域核小体占有率的增加是相似的。我们讨论了冗余因子保持 BEAF 相关启动子活性并掩盖 BEAF 与 PBAP 的 Pbro 亚基在 S2 细胞中的相互作用在 S2 细胞中作用的可能性。我们确定 Facilitates Chromatin Transcription（FACT）和 Nucleosome Remodeling Factor（NURF）为候选冗余因子。
