Bondra Eliana R, Rine Jasper
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, United States.
bioRxiv. 2023 May 11:2023.05.08.539937. doi: 10.1101/2023.05.08.539937.
In heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Rap1, and Sir proteins. Despite a vast understanding of the establishment and maintenance of Sir-silenced chromatin, the mechanism of gene silencing by Sir proteins has remained a mystery. Utilizing high resolution chromatin immunoprecipitation, we found that Rap1, the native activator of the bi-directional α promoter, bound its recognition sequence in silenced chromatin and its binding was enhanced by the presence of Sir proteins. In contrast to prior results, various components of transcription machinery were not able to access α in the silenced state. These findings disproved the long-standing model of indiscriminate steric occlusion by Sir proteins and led to investigation of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a novel role for promoter-bound Rap1 in the maintenance of silent chromatin through interactions with the Sir complex. We also found that promoter-bound Rap1 activated α when in an expressed state, and aided in the transition from transcription initiation to elongation. Highlighting the importance of epigenetic context in transcription factor function, these results point toward a model in which the duality of Rap1 function was mediated by local chromatin environment rather than binding-site availability.
The coarse partitioning of the genome into regions of active euchromatin and repressed heterochromatin is an important, and conserved, level gene expression regulation in eukaryotes. Repressor Activator Protein (Rap1) is a transcription factor that promotes the activation of genes when recruited to promoters, and aids in the establishment of heterochromatin through interactions with silencer elements. Here, we investigate the role of Rap1 when bound to a promoter in silent chromatin and dissect the context-specific epigenetic cues that regulate the dual properties of this transcription factor. Together, our data highlight the importance of protein-protein interactions and local chromatin state on transcription factor function.
在异染色质中，它是通过位点特异性DNA结合因子（包括转录激活因子Rap1）与Sir蛋白之间的相互作用形成的。尽管对Sir沉默染色质的建立和维持有了广泛的了解，但Sir蛋白导致基因沉默的机制仍然是个谜。利用高分辨率染色质免疫沉淀技术，我们发现双向α启动子的天然激活因子Rap1在沉默染色质中结合其识别序列，并且Sir蛋白的存在增强了它的结合。与先前的结果相反，转录机器的各种组分在沉默状态下无法接近α。这些发现推翻了长期以来认为Sir蛋白通过无差别空间阻碍发挥作用的模型，并促使人们对Sir沉默染色质中的转录激活因子Rap1进行研究。通过使用一种监测沉默丧失事件的高灵敏度检测方法，我们确定了启动子结合的Rap1通过与Sir复合物相互作用在维持沉默染色质中的新作用。我们还发现，处于表达状态时，启动子结合的Rap1会激活α，并有助于从转录起始过渡到延伸。这些结果突出了表观遗传背景在转录因子功能中的重要性，表明Rap1功能的双重性是由局部染色质环境而非结合位点可用性介导的。
将基因组粗略划分为活性常染色质区域和抑制性异染色质区域是真核生物中一种重要且保守的基因表达调控水平。阻遏激活蛋白（Rap1）是一种转录因子，当被招募到启动子时促进基因激活，并通过与沉默元件相互作用协助异染色质的建立。在这里，我们研究Rap1与沉默染色质中的启动子结合时的作用，并剖析调节该转录因子双重特性的特定背景表观遗传线索。总之，我们的数据突出了蛋白质-蛋白质相互作用和局部染色质状态对转录因子功能的重要性。
