The long non-coding RNA Kcnq1ot1 controls maternal p57 expression in muscle cells by promoting H3K27me3 accumulation to an intragenic MyoD-binding region
The long non-coding RNA Kcnq1ot1 controls maternal p57 expression in muscle cells by promoting H3K27me3 accumulation to an intragenic MyoD-binding region
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DOI：
10.1186/s13072-019-0253-1
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发表时间：
2019-01-16
影响因子：
3.9
通讯作者：
Maione, Rossella
中科院分区：
文献类型：
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作者：
Andresini, Oriella;Rossi, Marianna Nicoletta;Maione, Rossella
细胞周期抑制剂p57（kip2）通过协调多种细胞类型的细胞增殖和分化，在哺乳动物的发育中起着关键作用。P57 （kip2）的表达受到多种表观遗传机制的精细调控，包括父本印迹。kcnq10t1是一种长链非编码RNA (LncRNA)，其基因定位于p57（Kip2）印迹结构域，由父本等位基因独家表达，并通过染色质水平调控参与邻近印迹基因的顺式沉默。鉴于我们之前的证据表明，在肌肉分化过程中，肌生成因子和印迹控制元件在调控母体p57（Kip2）等位基因中的功能相互作用，我们研究了kcnqot1在控制肌肉细胞中p57（Kip2）表达中发挥印迹独立作用的可能性。我们发现，siRNA导致kcnq10t1缺失导致分化过程中母体和功能性p57（Kip2）等位基因的上调，这表明该LncRNA之前未被披露的作用。一致地，染色质寡亲和沉淀试验表明，kcnq10t1不仅与已知的基因座的父本印迹控制区相互作用，而且还与一个新的p57（Kip2）调控区域的母本和父本等位基因相互作用，该区域位于基因内部，含有两个肌肉特异性因子MyoD的结合位点。此外，kcnq10t1缺失后的染色质免疫沉淀试验表明，LncRNA是H3K27me3积累所必需的，H3K27me3是一种由组蛋白甲基转移酶EZH2催化的染色质修饰，在母体p57（kip2）基因内区域积累。最后，分化后，通过ChIP和RNA免疫沉淀分析，MyoD与该区域的结合及其与kcnq10t1的物理相互作用与EZH2和H3K27me3从染色质上的缺失以及p57（Kip2）的去抑制有关。这些发现强调了Kcnq1ot1存在独立于印记的作用，为仍然神秘的LncRNA的生物学提供了新的见解。此外，它们扩大了我们对分化过程中p57（Kip2）严格和精细调控的分子机制的认识，并可能在几种病理条件下观察到它的异常沉默。
BackgroundThe cell-cycle inhibitor p57(kip2) plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57(kip2) expression is finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57(Kip2) imprinting domain, is expressed exclusively from the paternal allele and participates in the cis-silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57(Kip2) allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57(Kip2) expression in muscle cells.ResultsWe found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57(Kip2) allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57(Kip2) regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57(kip2) intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57(Kip2) de-repression.ConclusionsThese findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of p57(Kip2) during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.