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链非编码RNA（lncRNA）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，在哺乳动物细胞中鉴定的数千种转录物对应于lncRNA。类似于蛋白质编码RNA，lncRNA可以是组织特异性的，在发育中高度调节并在疾病中改变。lncRNA在表观遗传基因调控或转录后事件中的功能最近已被认识。大量lncRNA起源于编码转录因子和发育调节因子的基因启动子处的趋异转录。这些不同转录的lncRNA/mRNA对显示相似的表达模式。我们的目标是确定与肺发育基因分化转录的lncRNA的作用机制，并评估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Gata 6AS在ESCs分化过程中与Gata 6类似地上调，与组蛋白修饰蛋白共免疫沉淀，并且其下调降低了Gata 6 mRNA的表达。在支持性研究中，我们发现人NKX 2 - 1AS 1在原代肺上皮细胞中表达，但在间充质细胞中不表达，并且定位于肺癌细胞的核仁和细胞质中。NKX 2 - 1AS 1的下调不影响NKX 2 -1 mRNA表达，而是导致细胞增殖减少和细胞周期基因的下调。我们推测肺发育基因位点转录的lncRNA/mRNA对的协调作用调节肺上皮细胞分化。我们将在小鼠胚胎干细胞培养中分化为内胚层和肺/甲状腺祖细胞以及小鼠胚胎中检验这一假设。在目标1中，我们将分析这些lncRNA在培养的ESCs敲低和过表达实验中对肺细胞命运指定和分化的时间和效率的影响，并通过RNA测序鉴定胚胎小鼠肺中表达的其他lncRNA，但不表达。在甲状腺或肝原基中。在目标2中，我们将确定Gata 6AS，Nkx 2 -1AS和其他肺lncRNA的分子作用，通过下拉实验识别相互作用的蛋白质，然后进行质谱分析。在目标3中，我们将测试lncRNA与染色质重塑蛋白的相互作用，通过评估lncRNA表达水平的变化是否以顺式或反式调节下游基因，通过微阵列测量附近或远处基因的mRNA表达变化，相应蛋白质和lncRNA与染色质的结合以及特定位点组蛋白标记的改变。对于与参与翻译的蛋白质相互作用的lncRNA，我们将测试在相同或远距离基因座中与lncRNA共享互补区域的基因的蛋白质水平。我们将测试这些lncRNA在表达靶向lncRNA的shRNA的敲除小鼠中的作用，并评估对肺器官形成的影响。总的来说，这些研究将测试lncRNA/mRNA趋异对在肺特异性基因表达、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.