TERC藉由WDR82與CCAR1進行表觀遺傳的調控

Abstract

端粒酶RNA組成（TERC）作為端粒酶延長端粒的模板RNA，除作為端粒酶延長端粒的模板外，亦有研究指出其具備非典型功能，包括促進發炎反應、調控細胞凋亡及基因表現。在我們實驗室先前的研究中，透過iDRiP-MS分析並鑑定與TERC交互的蛋白，並找出了多種TERC可能參與的細胞功能，在與TERC交互的蛋白中包括CCAR1與Wdr82，這兩者皆屬於與TERC交互的前三十名。細胞分裂週期與細胞凋亡調節因子1（CCAR1）是一種核受體輔因子，可與媒介體複合體結合，對於RNA聚合酶II（RNA Pol II）在轉錄起始時的招募至轉錄起始點相當重要。WD重複結構域蛋白82（Wdr82）是SET1A複合體的成員，該複合體負責在轉錄起始位點（TSS）進行H3K4三甲基化，有助於SET1複合體招募至RNA Pol II。 為了研究TERC如何影響基因表現，我們在降低TERC表現量的小鼠胚胎幹細胞中，對CCAR1、Wdr82、RNA Pol II Ser5以及H3K4me3進行了Cut&Run定序。TERC缺失導致TSS處CCAR1和RNA Pol II Ser5的結合增加，而Wdr82與H3K4me3在TSS處的結合則減少。在定序資料中，我們挑選了在TSS處具有TERC結合位點的基因Ticrr進行報導基因實驗。並發現在TERC表現量降低後，Ticrr的啟動子活性下降，顯示TERC透過調控轉錄來控制基因表現。 最後，為了繪製TERC與其相關蛋白（WDR82、CCAR1及TERT）之間的結合區域圖譜，我在細胞中表現了帶有SFB標籤的目標蛋白。經由紫外線交聯RNA與蛋白質之後，純化帶標籤的蛋白，並對其結合的TERC RNA進行反轉錄與定序。我們將此方法命名為 UV-RIPT-seq（UV RNA免疫沉澱與定向RNA定序）。UV-RIPT-seq的分析結果顯示，CR4/CR5結構域是WDR82與CCAR1的主要結合區。此外，在假結構域（pseudoknot domain）與H/ACA結構域中也偵測到額外的結合訊號。有趣的是，在TERT的UV-RIPT-seq資料中也觀察到數個相同的結合位點，暗示WDR82與CCAR1可能與TERT競爭或干擾其與TERC的結合。這些發現突顯了TERC在端粒延長以外的重要功能，並暗示其與WDR82和CCAR1的交互作用可能參與基因表現的表觀遺傳調控。

The Telomerase RNA component (TERC) serves as the RNA template for telomerase to elongate telomeres. Emerging studies have shown that TERC has non-canonical functions such as promoting inflammatory responses, influencing apoptosis, and regulating gene expression. Previously, we performed iDRiP-MS to identify TERC-interacting proteins and revealed a diverse set of TERC-associated proteins, including CCAR1 and Wdr82, both of which ranked among the top thirty interactors in the TERC interactome. Cell division cycle and apoptosis regulator 1 (CCAR1), a cofactor of nuclear receptors, interacts with the mediator complex, which is crucial for the recruitment of RNA Polymerase II (RNA Pol II) during transcription initiation. WD repeat domain 82 (WDR82) is a component of the SET1A complex, which is responsible for H3K4 trimethylation at transcriptional start sites (TSS), promoting the recruitment of the SET1 complex to RNA Pol II. Cut&Run sequencing for CCAR1, WDR82, RNA Pol II CTD phosphoserine 5 (RNA Pol II ser5), and H3K4me3 in TERC knockdown mouse embryonic stem cells revealed that TERC depletion led to increased CCAR1 and RNA Pol ll Ser5 occupancies at TSS, while WDR82 and H3K4me3 occupancies were decreased at TSS. Among TERC target genes with altered epigenetic profiles upon TERC depletion, Ticrr, which contains TERC binding sites at TSS, was selected for a reporter assay. Following TERC depletion, Ticrr promoter activity was reduced, suggesting that TERC regulates gene expression by modulating transcription. To map the interaction sites between TERC and its associated proteins (WDR82, CCAR1, and TERT), I expressed SFB-tagged versions of these proteins in cells. Following UV crosslink of RNA-protein complex, the tagged proteins were purified, and the bound TERC RNA was reverse transcribed and sequenced. We named this method UV-RIPT-seq (UV RNA immunoprecipitation for targeted RNA sequencing). The UV-RIPT-seq analysis revealed that the CR4/CR5 domain is the primary binding site for WDR82 and CCAR1. Additional interactions were also detected in the pseudoknot domain and H/ACA domain. Interestingly, several of these binding sites were also observed in the TERT UV-RIPT-seq data, suggesting that WDR82 and CCAR1 may compete with or interfere with TERT binding to TERC. These findings highlight the important roles of TERC beyond its classical function in telomere elongation, suggesting that its interaction with WDR82 and CCAR1 may contribute to the epigenetic regulation of gene expression.