Molecular and biological function of long non-coding RNA transcripts divergent to lung developmental genes

 描述(由申请人提供)：本提案的总体目标是研究长非编码RNA(LncRNAs)在调节肺细胞分化和发育中的分子和功能作用。最近的研究表明，在哺乳动物细胞中发现的数千个转录本对应于lncRNAs。与编码蛋白质的RNA类似，lncRNAs可以是组织特有的，在发育过程中受到高度调控，在疾病中也会发生变化。最近，人们认识到lncRNA在表观基因调控或转录后事件中的作用。相当数量的lncRNAs是通过转录因子和发育调节因子基因启动子的差异转录而产生的。这些不同转录的lncRNA/mRNA对显示出相似的表达模式。我们的目标是明确与肺发育基因差异转录的lncRNAs的作用机制，并评价它们在肺细胞分化和发育中的功能。我们有初步证据表明，小鼠GATA6AS在ESCs分化过程中与GATA6类似上调，与组蛋白修饰蛋白共免疫共沉淀，其下调降低了GATA6mRNA的表达。在支持性研究中，我们发现人NKX2-1AS1类似于NKX2-1在原代肺上皮细胞中表达，而不是在间充质细胞中表达，并且定位于肺癌细胞的核仁和细胞质中。下调NKX2-1AS1不会影响NKX2-1mRNA的表达，但会导致细胞增殖减少和细胞周期基因下调。我们假设肺发育基因座转录的lncRNA/mRNA对的协同作用调节肺上皮细胞的分化。我们将在小鼠胚胎干细胞中测试这一假设，在培养中分化为内胚层和肺/甲状腺前体细胞，并在小鼠胚胎中分化。在目标1中，我们将分析这些lncRNAs在体外培养的胚胎干细胞中对肺细胞命运指定和分化的时机和效率的影响，并通过RNA测序来鉴定在胚胎小鼠肺中表达的其他lncRNAs，而在甲状腺和肝脏原基中不表达。在目标2中，我们将通过下拉实验和质谱分析鉴定相互作用的蛋白质，从而确定Gata6AS、Nkx2-1AS和其他肺lncRNAs的分子作用。在目标3中，我们将通过微阵列测量近端或远端基因的mRNA表达的变化，相应的蛋白质和lncRNAs与染色质的结合以及特定位点的组蛋白标记的变化，来评估lncRNA的表达水平的变化是否调节顺式或反式的下游基因，从而测试lncRNAs与染色质重塑蛋白的相互作用。对于与翻译相关蛋白相互作用的lncRNAs，我们将测试与lncRNA在相同或远距离基因座上共享互补区域的基因的蛋白质水平。我们将测试这些lncRNAs在表达针对lncRNAs的shRNAs的敲除小鼠中的作用，并评估其对肺器官发生的影响。总而言之，这些研究将首次测试lncRNA/mRNA发散对在肺特定基因表达、细胞分化和发育中的功能作用。

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to investigate the molecular and functional roles of long non-coding RNAs (lncRNAs) in regulating lung cell differentiation and development. Recent studies revealed that thousands of transcripts identified in mammalian cells correspond to lncRNAs. Similarly to protein-coding RNAs, lncRNAs can be tissue specific, highly regulated in development and altered in disease. Function of lncRNA in epigenetic gene regulation or in posttranscriptional events has been recently recognized. A significant number of lncRNAs originate by divergent transcription at promoters of genes encoding transcription factors and developmental regulators. These divergently transcribed lncRNA/mRNA pairs display similar expression patterns. Our goal is to define the mechanisms of action of lncRNAs divergently transcribed with lung developmental genes, and to evaluate their function in lung cell differentiation and development. We have preliminary evidence that the mouse Gata6AS is up-regulated similarly to Gata6 during ESCs differentiation, is co-immunoprecipitated with histone modifying proteins, and its down-regulation reduces the expression of the Gata6 mRNA. In supporting studies, we showed that human NKX2-1AS1 is expressed like NKX2-1 in primary lung epithelial but not mesenchymal cells and is localized in nucleoli and cytoplasm of lung carcinoma cells. Down-regulation of NKX2-1AS1 does not affect NKX2-1 mRNA expression but rather results in reduced cell proliferation and down regulation of cell cycle genes. We hypothesize that coordinated actions of lncRNA/mRNA pairs transcribed from lung developmental gene loci regulate lung epithelial cell differentiation. We will test this hypothesis in mouse ESCs differentiating in culture into endoderm and lung/thyroid progenitors and in mouse embryos. In Aim 1, we will analyze the effect of these lncRNAs on timing and efficiency of lung cell fate specification and differentiation in knock-down and over-expression experiments in ESCs in culture, and identify by RNA-sequencing other lncRNAs expressed in embryonic mouse lung, but not in thyroid or liver primordia. In Aim 2 we will determine the molecular role of Gata6AS, Nkx2-1AS, and other lung lncRNAs, by identifying interacting proteins by pull-down experiments followed by mass spectroscopy analysis. In Aim 3, we will test lncRNAs interacting with chromatin remodeling proteins by evaluating whether changes in expression levels of the lncRNA regulate downstream genes in cis or in trans by measuring changes in mRNA expression of nearby or distant genes by microarrays, binding of the corresponding proteins and lncRNAs to chromatin and alterations in histone marks in specific loci. For lncRNAs that interact with proteins involved in translation we will test protein levels o genes sharing complementary regions with the lncRNA in the same or distant loci. We will test the role of these lncRNAs in knock-down mice expressing shRNAs targeting the lncRNAs and evaluating the effect on lung organogenesis. Collectively, these studies will test for the first tie the functional role of lncRNA/mRNA divergent pairs in lung specific gene expression, cell differentiation and development.