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

与肺发育基因不同的长非编码RNA转录物的分子和生物学功能

基本信息

批准号：
9135496
负责人：
Maria Isabel Ramirez
金额：
$ 41.27万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9135496
关键词：
Affect Binding Biological Process Cell Cycle Regulation Cell Differentiation process Cell Lineage Cell Nucleolus Cell Proliferation Cells Chromatin Code Cytoplasm Development Developmental Gene Disease Distant Down-Regulation Embryo Endoderm Endoderm Cell Epigenetic Process Epithelial Epithelial Cells Epithelium Event Fluorescent in Situ Hybridization Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Genome Goals Growth Health Histones Human Immunofluorescence Immunologic Liver Location Lung Lung diseases Malignant Epithelial Cell Mammalian Cell Mass Spectrum Analysis Measures Mediating Mesenchymal Messenger RNA Mining Molecular Mus Nucleotides Organ Organogenesis Pattern Proteins Publishing RNA RNA Sequence Analysis Regulation Role Scientist Small Interfering RNA Specificity Stem cells Technology Testing Thyroid Gland Thyroid Primordium Time Tissues Transcript Transgenic Mice Translating Translations Untranslated RNA Work cdc Genes cell fate specification chromatin remodeling differential expression embryonic stem cell histone modification human embryonic stem cell in vivo knock-down liver primordium lung Carcinoma lung development mRNA Expression mouse development mouse model next generation novel overexpression progenitor promoter research study stem cell differentiation transcription factor transcriptome sequencing

项目摘要

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.

描述（由申请人提供）：该提案的总体目标是研究长非编码 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出数千个转录本。与 lncRNA 类似，lncRNA 具有组织特异性，在发育过程中受到高度调控，并且在表观遗传基因调控或转录后事件中的功能发生改变。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，这些不同转录的 lncRNA/mRNA 对显示出相似的表达模式。基因，并评估其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 ESC 分化过程中与 Gata6 类似地被上调。与组蛋白修饰蛋白共免疫沉淀，其下调会降低 Gata6 mRNA 的表达。在支持研究中，我们表明人 NKX2-1AS1 在原代肺上皮细胞中表达类似 NKX2-1，但在间充质细胞中不表达。 NKX2-1AS1 的下调并不影响肺癌细胞的核仁和细胞质的 NKX2-1 mRNA 表达，而是导致细胞增殖减少和下调。我们研究从肺发育基因位点转录的lncRNA/mRNA对的协调作用来调节肺上皮细胞分化，我们将在培养物中分化为内胚层和肺/甲状腺祖细胞的小鼠ESC中以及在小鼠胚胎中检验这一假设。在目标 1 中，我们将在培养的 ESC 的敲低和过表达实验中分析这些 lncRNA 对肺细胞命运规范和分化的时间和效率的影响，并通过对胚胎小鼠肺中表达的其他 lncRNA 进行 RNA 测序，但在甲状腺或肝脏原基中不表达。在目标 2 中，我们将通过随后的下拉实验识别相互作用的蛋白质，确定 Gata6AS、Nkx2-1AS 和其他肺 lncRNA 的分子作用。在目标 3 中，我们将通过评估 lncRNA 表达水平的变化是否调节下游基因来测试 lncRNA 与染色质重塑蛋白的相互作用。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基因座中组蛋白标记的变化，我们将测试蛋白质水平的顺式或反式。 o 与 lncRNA 在相同或遥远位点共享互补区域的基因我们将测试这些 lncRNA 在表达靶向 lncRNA 的 shRNA 的敲低小鼠中的作用，并评估对肺的影响。总的来说，这些研究将首先测试 lncRNA/mRNA 不同对在肺特异性基因表达、细胞分化和发育中的功能作用。