Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724, USA.
Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, 11724, USA.
Nat Commun. 2024 Oct 30;15(1):9384. doi: 10.1038/s41467-024-53417-9.
Transcriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) in Schizosaccharomyces pombe is a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4, promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (Clr4), which undergoes liquid droplet formation in vitro, along with Swi6 the effector of transcriptional gene silencing. Our data suggests that phase separation is exquisitely sensitive to non-coding RNA (ncRNA) which promotes self-association of Clr4, chromatin association, and di-, but not tri- methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2, down-regulating centromeric transcription and small RNA (sRNA) production. The deubiquitinase Ubp3 counteracts both activities.
RNAi 介导的转录沉默悖论地依赖于转录，但从转录到沉默的转变是如何实现的仍然不清楚。裂殖酵母中的隐密基因调控复合物（CLRC）是一种需要 RNAi 招募的 cullin-环 E3 连接酶，用于沉默。我们发现 E2 泛素缀合酶 Ubc4 与 CLRC 相互作用，并单泛素化组蛋白 H3K9 甲基转移酶 Clr4，促进从共转录基因沉默（H3K9me2）到转录基因沉默（H3K9me3）的转变。Clr4 的泛素化发生在一个固有无序区域（Clr4）中，该区域在体外形成液滴，与转录基因沉默的效应物 Swi6 一起。我们的数据表明，相分离对非编码 RNA（ncRNA）非常敏感，促进 Clr4 的自我缔合、染色质结合，以及二甲基化，但不是三甲基化。Ubc4-CLRC 还靶向转录共激活因子 Bdf2，下调着丝粒转录和小 RNA（sRNA）的产生。去泛素酶 Ubp3 拮抗这两种活性。
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