長鏈非編碼RNA—TERRA 在哺乳動物中的表觀遺傳調控

Abstract

在哺乳動物中，不僅僅只有蛋白質會與染色質結合去調控基因表現，RNA也會參與其中去調控。先前的研究指出，長鏈非編碼RNA（未轉譯成蛋白質去表現基因的RNA）通過與染色質相互作用來參與表觀遺傳的調控。在這裡，我們研究含有端粒重複序列的RNA（TERRA），其從亞端粒區轉錄到端粒位置。先前的研究指出，TERRA與組蛋白修飾（例如EZH2，SETDB1和SETD2），染色質重塑物（例如ATRX）在生物體內與之進行結合並藉以此調控基因表現。我們通過TERRA可能會去調控組蛋白中(H3K9me3和H3K27me3)的甲基轉移酶，與ATRX在基因體的佔據量來調控基因表現。為了驗證這一假設，我們在小鼠胚胎幹細胞中進行TERRA去除 (Knockdown)的實驗，並對ATRX，,SETDB1和SETD2進行了染色質免疫沉澱（ChIP）的試驗。我們發現ATRX和SETDB1在端粒DNA上的佔據量顯著增加，而SETD2在端粒DNA上的佔據量卻是減少，。此外在TERRA knockdown後，組蛋白H3K9m3和H3K27me3在端粒處的佔據量也隨之增加。 ATRX ChIP-seq分析顯示TERRA knockdown不僅改變了ATRX佔據端粒DNA的量，也改變了在其他基因的佔據量。令人驚訝的是，TERRA knockdown導致rDNA基因和其他重複序列的ATRX富集增加，這意味著TERRA會影響ATRX 與重複DNA的結合。除此之外ATRX在基因的轉錄起始位置有與SETD2富集的現象，並與H3K27me3佔據位點相斥。這可能意味著ATRX對於異染色質與基因間區段有不同方式的調控。最後我們的結果指出TERRA在生物體內會去調控全基因的染色質修飾和異染色質的狀態。

In mammalian cells, not only proteins bind to chromatin to regulate the gene expression but also do RNAs. Lines of evidence show that long noncoding RNAs, which are not translated to proteins, are involved in epigenetic regulation via interacting with chromatin. Here we study Telomeric Repeat-Containing RNA (TERRA), which is transcribed from subtelomeric regions towards to telomeres. Previous studies have shown that TERRA binds to histone modifiers (e.g., SETDB1, and SETD2), and chromatin remodeler (e.g., ATRX) in vivo. We propose that TERRA may regulate gene expression by modulating the occupancies of several histone-methyl-transferases and ATRX. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) for ATRX, SETDB1, and SETD2 after TERRA depletion in mouse embryonic stem cells. We found that the occupancies of ATRX and SETDB1 at telomeric DNA increased, whereas the occupancy of SETD2 at telomeric DNA decreased. The enrichments of histone H3K9m3 and H3K27me3 at telomeres also increased after TERRA depletion. ATRX ChIP-seq analysis shows that TERRA depletion alters ATRX occupancy not only at telomeric DNA but also at intergenic regions. Surprisingly, TERRA depletion leads to an increase of ATRX enrichment at rDNA locus and several repetitive sequences such as satellite repeats, implying that TERRA evicts ATRX from binding to repetitive DNA. Moreover, ATRX enrichment also increased on transcriptional start sites (TSS) with SETD2 where H3K27me3 was depleted. The results suggest that ATRX on TSS and intergenic regions may have different regulation in heterochromatin. Finally, our results support that TERRA regulates the chromatin modifications and heterochromatin status in a genome-wide manner.