TERRA作為一個表觀遺傳學的調控者維持全基因組中DNA G-四聯體的形成

Abstract

G-四聯體DNA是由富含鳥嘌呤的序列形成的非典型的二級結構，在基因組中廣泛存在。 G4結構被認為與轉錄、翻譯和基因組穩定性有關。長鏈非編碼端粒RNA (TERRA)，是由次端粒區域轉錄至端粒末端的一種非編碼RNA。 TERRA含有UUAGGG重複序列，使其能夠形成G-四聯體結構並與G-四聯體蛋白相互作用（例如ATRX，RTEL1和BLM DNA解旋酶），進而抑制G4形成，維持基因組穩定性。在本研究中，我們表明了TERRA透過其富含G序列調控G-四聯體蛋白並維持DNA G-四聯體結構形成。 我們先前的G4 ChIP-seq顯示TERRA降解後，減少轉錄起始位點（TSS）的G-四聯體形成，並伴隨著ATRX覆蓋率的增加。抑制ATRX緩解了TERRA knockdown所造成細胞中基因表達的變化，表示ATRX介導了TERRA knockdown所引起的基因表達失調。為了研究TERRA調控BLM在小鼠基因組中的分佈和其與DNA G-四聯體結構形成的關聯性，我們對BLM進行了染色質免疫沉澱測序。TERRA knockdown增加了BLM在端粒、染色質間端粒重複序列和一些特殊重複序列中的富集程度。此外， BLM富集程度的增加與G-四聯體結構形成減少相關。免疫染色結果證實TERRA knockdown導致BLM和RTEL1在染色質上的覆蓋率增加，並減少DNA G-四聯體結構的形成。然而，將富含G序列的DNA寡核苷酸送入細胞後可以減緩這些反應。此外，引入小片段的TERRA RNA可以降低染色質上BLM和RTEL1的覆蓋率並增加DNA G4結構的形成。基於這些發現，我們提出TERRA RNA藉由其富含G序列阻止G-四聯體蛋白到染色質上，防止G-四聯體結構被解開。總結來說，我們的研究揭示了TERRA具有阻止G4解旋酶募集到基因組上以維持G4結構形成的角色。

G-quadruplex (G4) DNA, a non-B form secondary structure formed with Guanine-rich sequences, widely exists in the genome. G4 structures are thought to be involved in the regulation of transcription, translation and genome stability. Telomeric repeat containing RNA (TERRA), a long non-coding RNA, is transcribed from subtelomeric region toward telomeric end. TERRA containing UUAGGG repetitive sequences enables folding to G-quadruplex and interacts with G4 regulators, including ATRX, RTEL1 and BLM DNA helicases that function in suppressing G4 formation to maintain genome stability. Here, we show that TERRA regulates G4 binding proteins and maintains G4 formation through its G-rich sequences. Our previous G4 ChIP-seq analysis revealed a decrease in G4 formation at transcription start sites (TSS) after TERRA depletion, accompanied by elevated ATRX occupancy. Here I found that ATRX is increased in the chromatin bound fraction upon TERRA depletion. Silencing ATRX mitigates the effect of altered gene expression in TERRA knockdown cells, suggesting that ATRX mediates the dysregulation of gene expression caused by TERRA depletion. To investigate whether TERRA mediates the BLM occupancy in the genome, BLM chromatin immunoprecipitation (ChIP) sequencing was performed. TERRA depletion increased BLM abundance at telomeres, interstitial telomeric repeats, and some repetitive sequences. Additionally, such increased BLM occupancy was associated with G4 reduction. Immunostaining results support that TERRA depletion results in elevated abundance of BLM and RTEL1 on chromatin and a reduction of G4 formation, whereas introducing G-rich DNA oligos counteracts these effects. Moreover, the delivery of TERRA RNA oligos reduces BLM and RTEL1 occupancies on chromatin and increases DNA G4 structures. Based on these findings, we propose that TERRA RNA sequesters G4 binding proteins through its G-rich sequences, thereby preventing DNA G4 from unwinding. Overall, our findings uncover the role of TERRA in regulating the occupancies of G4 binding proteins and preserving G4 formation on the genome.