The Kcnq1ot1 long non-coding RNA affects chromatin conformation and expression of Kcnq1, but does not regulate its imprinting in the developing heart.
The Kcnq1ot1 long non-coding RNA affects chromatin conformation and expression of Kcnq1, but does not regulate its imprinting in the developing heart.
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DOI：
10.1371/journal.pgen.1002956
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发表时间：
2012-09
期刊：
影响因子：
4.5
通讯作者：
Engel N
中科院分区：
文献类型：
--
作者：
Korostowski L;Sedlak N;Engel N
虽然许多问题所提出的发现印记已经回答，我们还没有占组织或阶段特异性印记。Kcnq 1印迹域表现出复杂的组织特异性表达模式共存的全域顺式作用控制元件。父系表达的反义非编码RNA Kcnq 1 ot 1的转录使胚胎中的一些邻近基因沉默，而其他基因不受影响。Kcnq 1在心脏发育早期被标记，但在妊娠中期后变为双等位基因。为了探索这种现象和Kcnq 1 ot 1的作用，我们使用等位基因特异性测定和染色体构象研究野生型小鼠和Kcnq 1 ot 1提前终止突变的小鼠。我们发现，Kcnq 1印记在早期心脏的建立和维持独立的Kcnq 1 ot 1的表达，从而排除了Kcnq 1 ot 1在抑制Kcnq 1的作用，即使在沉默其他基因的域。单至双等位基因转换的确切时间是菌株依赖性的，其中CAST/EiJ等位基因比C57 BL/6 J等位基因更早被激活并获得更高的水平。出乎意料的是，Kcnq 1 ot 1本身也切换到双等位基因表达，特别是在心脏，这表明组织特异性的印记损失可能是常见的胚胎发生过程中。母系Kcnq 1 ot 1转录比父系ncRNA短，其激活依赖于绕过母系甲基化启动子的替代转录起始位点。在母体染色体上产生Kcnq 1 ot 1不会沉默Cdkn 1c。我们发现，在后来的发展阶段，然而，Kcnq 1 ot 1具有调节Kcnq 1水平的作用，因为它的缺乏导致Kcnq 1的过度表达，伴随着异常的三维结构的染色质的事件。因此，我们的研究揭示了Kcnq 1印迹结构域内的调节机制，该结构域专门在心脏中对Kcnq 1起作用，Kcnq 1是心脏发育和功能至关重要的基因。我们还发现了一种新的机制，通过这种机制，反义非编码RNA通过调节染色质的灵活性和增强子的访问来影响转录。印记基因只表达一个拷贝，活性等位基因取决于亲本来源。这些基因通常以组的形式组织，并在长距离内受到调控。这些领域的失调可能导致发育性疾病和癌症。父系表达的Kcnq 1 ot 1非编码RNA从Kcnq 1基因的内含子中以反义方向出现，并使几个邻近基因沉默。Kcnq 1基因最初是印记的，但在心脏发育过程中变为双等位基因。为了理解这种行为的机制，我们进行了表达和构象的Kcnq 1域在此过渡期间的研究，发现早期阶段的印记Kcnq 1是不依赖于转录的Kcnq 1 ot 1，从而揭示了存在一个独特的机制沉默的父亲Kcnq 1等位基因。令人惊讶的是，Kcnq 1 ot 1在与Kcnq 1相同的时间范围内失去了其在心脏中的印记表达。缺乏Kcnq 1 ot 1确实导致Kcnq 1在心脏发育后期过度表达，这一事件的特征是染色质灵活性增加，允许Kcnq 1启动子建立异位调节接触。因此，我们发现了新的调节机制，这将有助于我们了解印记结构域的组织特异性以及它们如何在疾病中出错。
Although many of the questions raised by the discovery of imprinting have been answered, we have not yet accounted for tissue- or stage-specific imprinting. The Kcnq1 imprinted domain exhibits complex tissue-specific expression patterns co-existing with a domain-wide cis-acting control element. Transcription of the paternally expressed antisense non-coding RNA Kcnq1ot1 silences some neighboring genes in the embryo, while others are unaffected. Kcnq1 is imprinted in early cardiac development but becomes biallelic after midgestation. To explore this phenomenon and the role of Kcnq1ot1, we used allele-specific assays and chromosome conformational studies in wild-type mice and mice with a premature termination mutation for Kcnq1ot1. We show that Kcnq1 imprinting in early heart is established and maintained independently of Kcnq1ot1 expression, thus excluding a role for Kcnq1ot1 in repressing Kcnq1, even while silencing other genes in the domain. The exact timing of the mono- to biallelic transition is strain-dependent, with the CAST/EiJ allele becoming activated earlier and acquiring higher levels than the C57BL/6J allele. Unexpectedly, Kcnq1ot1 itself also switches to biallelic expression specifically in the heart, suggesting that tissue-specific loss of imprinting may be common during embryogenesis. The maternal Kcnq1ot1 transcript is shorter than the paternal ncRNA, and its activation depends on an alternative transcriptional start site that bypasses the maternally methylated promoter. Production of Kcnq1ot1 on the maternal chromosome does not silence Cdkn1c. We find that in later developmental stages, however, Kcnq1ot1 has a role in modulating Kcnq1 levels, since its absence leads to overexpression of Kcnq1, an event accompanied by an aberrant three-dimensional structure of the chromatin. Thus, our studies reveal regulatory mechanisms within the Kcnq1 imprinted domain that operate exclusively in the heart on Kcnq1, a gene crucial for heart development and function. We also uncover a novel mechanism by which an antisense non-coding RNA affects transcription through regulating chromatin flexibility and access to enhancers. Imprinted genes express only one copy and the active allele depends on the parental origin. These genes are usually organized in groups and are subject to regulation over long distances. Misregulation in these domains can lead to developmental diseases and cancer. The paternally expressed Kcnq1ot1 non-coding RNA emerges from an intron of the Kcnq1 gene in antisense direction and silences several neighboring genes. The Kcnq1 gene is initially imprinted but becomes biallelic during development of the heart. To understand the mechanisms underlying this behavior, we performed expression and conformational studies of the Kcnq1 domain during this transition and found that the early stage imprinting of Kcnq1 is not dependent on transcription of Kcnq1ot1, thus revealing the existence of a distinct mechanism of silencing of the paternal Kcnq1 allele. Surprisingly, Kcnq1ot1 loses its imprinted expression specifically in the heart in the same time frame as Kcnq1. Absence of Kcnq1ot1 does lead to overexpression of Kcnq1 in later cardiac development, an event characterized by increased chromatin flexibility allowing the Kcnq1 promoter to establish ectopic regulatory contacts. Thus, we have uncovered novel regulatory mechanisms that will help our understanding of tissue specificity in imprinted domains and how they can go awry in disease.
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