MyoD结合的骨骼肌特异长非编码RNA（linc-MCD）调控MyoD功能的分子机制

ENCODE project demonstrates that eukaryotic genome encodes a substantial number of long non-protein coding RNA (lncRNAs), which significantly advance the knowledge about human genome function. As growing body of convincing evidence show the involvement of lncRNAs in the epigenetic regulation of gene transcription, the scientists have recently paid more attention and raised particular interest in the expressional regulation of genes. Although it has long been appreciated that cell lineage specific transcriptional factor play critical role in the cell lineage commitment and differentiation, we actually know little about how the functional specificity of the transcriptional factors is achieved.Based on current information of lncRNA functional studies, especially more and more tissue specific lncRNAs have been identified, it is conceivable that tissue or cell lineage specific lncRNAs may concert with transcriptional factors to regulate their functional specificity on target gene expression. Recently, we have identified a skeletal muscle specifically expressed intergenic lncRNA which is required for muscle cell differentiation, hereafter named as Linc-MCD. Our experimental data demonstrate that Linc-MCD binds MyoD and possibly involves its functional specificity on the target gene expression. Therefore, we have a great interest to further investigate the molecular mechanism of the Linc-MCD in regulating functional specificity of MyoD during muscle cell differentiation as we have proposed in this proposal. Significantly, the function of the Linc-MCD/MyoD interaction will be further corroborated in vivo using Linc-MCD transgenic and knockout mice. Our findings from the proposed work will not only provide a paradigm for the investigations of the functional specificity of other transcriptional factors, but also provide an additional mechanism of lncRNA function in regulating gene transcription in which lncRNA functions to regulate gene expression by directly binding with transcriptional factor.

在个体发育和细胞分化过程中，细胞谱系特异的转录因子通过调控靶基因的表达决定细胞命运，而这类转录因子是如何调控靶基因的特异性表达的分子机制仍不是十分清楚。近年来,随着细胞特异性表达的长非编码RNA的不断发现和其在基因表达调控中的表观遗传学功能的发现，提示长非编码RNA可能与特异性转录因子相互协同调控靶基因表达的特异性。本实验室前期研究发现一个骨骼肌组织特异表达的lncRNA（Linc-MCD）与转录因子MyoD结合，影响MyoD调控靶基因的特异性，并通过增加染色质重塑酶Brg1的募集促进细胞分化。本项目的核心是深入探讨与MyoD结合的Linc-MCD调控MyoD功能及其靶基因特异性的分子机制，并采用Linc-MCD转基因和敲除鼠在体内进一步验证Linc-MCD对MyoD功能的重要性及其对骨骼肌发育与疾病的调控作用。本项目将为其他转录因子调控靶基因特异性的分子机理研究提供参照。

细胞分化过程中，转录因子对靶基因调控的特异性是如何实现的？长非编RNA在转录因子调控靶基因的特异性方面是否起到重要作用？本项目以骨骼肌细胞分化为研究系统，探讨骨骼肌细胞谱系特异表达的长非编码RNA Linc-MCD（Linc-RNA for Muscle Cell Differentiation）与转录调控因子MyoD相互作用对靶基因调控特异性的功能及其分子机制，揭示长非编码RNA是否参与调控转录因子靶基因特异性。在项目执行期间，采用Linc-MCD基因敲除小鼠和骨骼肌损伤再生模型，揭示了Linc-MCD显著促进骨骼肌成体干细胞分化、调控骨骼肌组织损伤再生修复，因此将Linc-MCD重新命名为Linc-RAM（Linc-RNA Activator for Myogenesis）。采用RIP和EMSA实验揭示了Linc-RAM直接结合MyoD，分析了Linc-RAM与MyoD相互作用的结构域；揭示了Linc-RAM与MyoD相互作用调节染色质重塑复合物Baf60c/Brg1组装进而调控骨骼肌细胞分化相关基因表达的分子机制，文章发表在Nature Communications (2017)。在此基础上，进一步揭示了在骨骼肌细胞增殖分化过程中Linc-RAM基因自身表达调控的分子机制，文章发表在RNA Biology (2018)。培养博士研究生毕业1名，硕士研究生毕业1名。