Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2023 Oct 3;120(40):e2304343120. doi: 10.1073/pnas.2304343120. Epub 2023 Sep 28.
In heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Repressor Activator Protein (Rap1), and Sir proteins. Despite an 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 bidirectional 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 role of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a 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.
异染色质是通过特定于位置的 DNA 结合因子（包括转录激活因子 Repressor Activator Protein（Rap1）和 Sir 蛋白）之间的相互作用形成的。尽管人们已经了解了 Sir 沉默染色质的建立和维持机制，但 Sir 蛋白的基因沉默机制仍然是一个谜。利用高分辨率染色质免疫沉淀技术，我们发现 Rap1 是双向α启动子的天然激活剂，它在沉默的染色质中结合其识别序列，并且 Sir 蛋白的存在增强了其结合。与之前的结果相反，转录机制的各种成分在沉默状态下无法进入α。这些发现否定了 Sir 蛋白通过无差别空间位阻进行非特异性沉默的长期模型，并促使我们研究转录激活因子 Rap1 在 Sir 沉默染色质中的作用。我们使用一种高度敏感的检测失活事件的检测方法，发现结合在启动子上的 Rap1 通过与 Sir 复合物相互作用，在维持沉默染色质方面发挥作用。我们还发现，当处于表达状态时，结合在启动子上的 Rap1 激活了α，并有助于从转录起始到延伸的转变。这些结果强调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介导的，而不是结合位点的可用性。
