The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development [H3K27ac Cut & Run]. The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development [H3K27ac Cut & Run]

Chromatin remodeling is central to the dynamic changes in gene expression that drive cell fate determination. During development, the sets of enhancers that are accessible for use change globally as cells transition between stages. While transcription factors and nucleosome remodelers are known to work together to control enhancer accessibility, it is unclear how the short stretches of DNA that they individually unmask yield the kilobase-sized accessible regions characteristic of active enhancers. Here, we performed a genetic screen to investigate the role of nucleosome remodelers in control of dynamic enhancer activity. We find that the Drosophila SWI/SNF complex, BAP, is required for repression of a temporally dynamic enhancer, brdisc. Contrary to expectations, we find that the BAP-specific subunit Osa is dispensable for mediating changes in chromatin accessibility between early and late stages of wing development. Instead, we find that Osa is required to constrain the levels of brdisc activity when the enhancer is normally active. Genome-wide profiling reveals that Osa directly binds brdisc as well as thousands of other developmentally dynamic regulatory sites, including multiple genes encoding components and targets of the Notch signaling pathway. Transgenic reporter analyses demonstrate that Osa is required for activation and for constraint of different sets of target enhancers in the same cells. Moreover, Osa loss results in hyperactivation of the Notch ligand Delta and development of ectopic sensory structures patterned by Notch signaling early in development. Together, these findings indicate that proper constraint of enhancer activity is necessary for regulation of dose-dependent developmental events. Overall design: anti-H3K27ac CUT&RUN from third larval wandering wing discs in Osa-GFP and yw females

染色质重塑（Chromatin Remodeling）是驱动细胞命运决定的基因表达动态变化的核心调控过程。在个体发育过程中，可被利用的增强子（Enhancer）集合会随细胞在不同发育阶段间的转换发生全局性改变。尽管已知转录因子（Transcription Factor）与核小体重塑因子（Nucleosome Remodeler）可协同调控增强子可及性，但目前仍不清楚二者各自暴露的短片段DNA，如何形成活性增强子所特有的千碱基级可及区域。本研究通过遗传筛选（Genetic Screen），探究核小体重塑因子在动态增强子活性调控中的具体作用。我们发现，果蝇（Drosophila）来源的SWI/SNF复合物（SWI/SNF Complex）BAP，对于抑制一个时序动态型增强子brdisc是必需的。与预期相悖的是，我们发现BAP特异性亚基Osa，在翅发育的早期与晚期阶段之间介导染色质可及性变化时并非必需。反之，我们发现当该增强子处于正常激活状态时，Osa的核心功能是限制brdisc的活性水平。全基因组图谱分析（Genome-wide Profiling）结果显示，Osa可直接结合brdisc以及数千个其他发育动态调控位点，其中包括多个编码Notch信号通路（Notch Signaling Pathway）组分与下游靶点的基因。转基因报告基因分析（Transgenic Reporter Assay）结果表明，在同一细胞中，Osa对于不同靶标增强子集合的激活与活性限制均不可或缺。此外，Osa缺失会导致Notch配体Delta的过度激活，以及发育早期由Notch信号通路调控的异位感觉结构的形成。综上，这些研究结果表明，对增强子活性进行适度限制，对于调控剂量依赖性发育事件是至关重要的。整体实验设计：针对Osa-GFP与yw雌性果蝇的三龄幼虫爬行期翅盘，开展抗H3K27ac CUT&RUN实验。