Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.
Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.
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长链非编码RNA（lncRNA）介导的转录调控越来越被认为是发育和疾病过程中重要的基因调控机制。 LncRNA 正在成为染色质状态的关键调节因子；然而它们与染色质相互作用的性质和程度仍有待充分揭示。我们之前已经发现 Ppp1r1b-lncRNA 是小鼠和人类心肌细胞和骨骼肌细胞肌原性分化的重要表观遗传调节因子。我们进一步证明，Ppp1r1b-lncRNA 功能是通过与生肌分化转录因子 TBX5 和 MyoD1 启动子处的染色质修饰复合物多梳抑制复合物 2 (PRC2) 的相互作用介导的。在此，我们采用通过 RNA 纯化 (ChIRP) 进行无偏染色质分离和高通量测序来绘制小鼠肌肉成肌细胞系中全基因组 Ppp1r1b-lncRNA 染色质占用的谱图。我们以高置信度（p 值 < 1E-5）和富集分数 ≥ 10）总共发现了 99732 个与 Ppp1r1b-lncRNA 结合位点相对应的真实峰。 Ppp1r1b-lncRNA结合位点平均长度为558 bp，广泛分布在基因组的编码区和非编码区。这些真实峰中大约 46% 被映射到基因元件，其中 1180 个被映射到经过实验验证的启动子序列。重要的是，启动子定位的结合位点在肌源性转录因子和心脏发育中富集，同时表现出与近端启动子的已知基序和RNA Pol-II转录起始的局部相互作用，包括TATA-box、转录起始基序、CCAAT-box和GC-box，支持Ppp1r1b-lncRNA在肌源性调节因子转录起始中的作用。值得注意的是，映射到基因内含子的 Ppp1r1b-lncRNA 结合位点中近 40% 富含转录因子同源盒家族，并表现出富含 TA 的基序序列，表明潜在的基序特异性 Ppp1r1b-lncRNA 结合内含子。最后，在 Ppp1r1b-lncRNA 占据位点中以高置信度检测到超过 136521 个增强子序列。在这些增强子中，3390 个（12%）在胎儿心脏和肌肉中表现出细胞类型/组织特异性富集。总之，我们的研究结果为全基因组 Ppp1r1b-lncRNA：染色质相互作用组提供了进一步的见解，这可能决定其在肌原分化和潜在的其他细胞和生物过程中的功能。
Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.
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影响因子：
14.9
作者：
Kanehisa M;Araki M;Goto S;Hattori M;Hirakawa M;Itoh M;Katayama T;Kawashima S;Okuda S;Tokimatsu T;Yamanishi Y
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