Transcriptional elongation and long noncoding RNA

转录延伸和长非编码RNA

• 批准号: 9111196
• 负责人: Nobuaki Kikyo
• 金额: $ 22.24万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9111196
• 关键词: Antisense RNA; Basic Science; Binding; Binding Sites; Biological Assay; Cell Differentiation process; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA biosynthesis; Data; Development; Disease model; Down-Regulation; Electrophoretic Mobility Shift Assay; Elongation Factor; Encyclopedia of DNA Elements; Enhancers; Epigenetic Process; Fibroblasts; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; HAT1 gene; Histones; Homologous Gene; Human; Human Cloning; Human Genome; Luciferases; Maintenance; Mediating; Medical; Messenger RNA; Metabolism; MicroRNAs; Modeling; Modification; Molecular; Mus; Names; Nucleosomes; Oligonucleotides; Play; Pluripotent Stem Cells; Preclinical Drug Evaluation; Proteins; Publishing; RNA; RNA Binding; RNA analysis; Regenerative Medicine; Regulation; Reporting; Ribosomal RNA; Role; Techniques; Testing; Transcription Elongation; Transcriptional Regulation; Transfer RNA; Transplantation; Untranslated RNA; Up-Regulation; base; cell dedifferentiation; cell type; chromatin immunoprecipitation; cofactor; differential expression; embryonic stem cell; genome-wide; induced pluripotent stem cell; innovation; knock-down; novel; novel strategies; overexpression; pluripotency; public health relevance; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): Pluripotent stem cells, represented by embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all types of the cells in the body. These cells are important in basic science as a model for cell differentiation and dedifferentiation as well as for medical applications, including drug screening disease modeling, and transplantation. Pluripotency is primarily controlled by the three master transcription factors-Oct4, Sox2, and Nanog. Transcriptional regulation by the three factors has been extensively characterized through the studies of their interacting proteins, miRNAs, and epigenetic modifications. However, the involvement of long noncoding RNAs (lncRNAs) in the regulation of pluripotency remains elusive although several pluripotency-specific lncRNAs have been reported. LncRNA is defined as RNA longer than 200 bases that is not mRNA, rRNA, or tRNA. More than 9000 lncRNAs have been discovered in the human genome and they are involved in almost all aspects of RNA metabolism and gene regulation. In the current project, lncRNAs that are bound to Oct4 and Sox2 are identified by chromatin immunoprecipitation from mouse ESCs. One of them called Hat1-AS is a novel antisense RNA encoded at the histone acetyltransferase 1 (Hat1) gene. Hat1 and Hat1-AS are highly expressed in ESCs and downregulated during differentiation. In addition, Hat1-AS is necessary for the transcription of Hat1. Furthermore, reflecting the interaction with the Oct4 and Sox2 proteins, Hat1-AS is important for the transcription of the target genes of Oct4 and Sox2, such as their own genes. Hat1- AS binds to the positive transcription elongation factor b (P-TEFb) complex, the central regulator for transcriptional elongation. Based on these preliminary studies, it was hypothesized that Hat1-AS is widely distributed in the genome as a cofactor of Oct4 and Sox2, regulating the recruitment of P-TEFb to their target genes and promoting transcriptional elongation. This hypothesis will be examined with the following three aims. In Aim 1, genome-wide target genes of Hat1-AS will be identified in ESCs with oligonucleotide hybridization, RNA-seq, and gene knockdown. The roles of Hat1-AS in the formation of iPSCs will be studied by up- and downregulation of Hat1-AS in fibroblasts in Aim 2. The regulation of the expression of Hat1-AS by Oct4 and Sox2 proteins will be investigated in Aim 3. Together, these aims are expected to unravel a novel lncRNA- mediated regulation of pluripotency by Oct4 and Sox2.

 描述（申请人提供）：多能干细胞，以胚胎干细胞（ESC）和诱导多能干细胞（iPSC）为代表，可以分化为体内所有类型的细胞。这些细胞作为细胞分化和去分化的模型，在基础科学以及医学应用（包括药物筛选、疾病模型和移植）中具有重要意义。多能性主要由三个主要转录因子——Oct4、Sox2 和 Nanog 控制。通过对这三个因子相互作用的蛋白质、miRNA 和表观遗传修饰的研究，对它们的转录调控进行了广泛的表征。然而，尽管已经报道了几种多能性特异性lncRNA，但长非编码RNA（lncRNA）在多能性调节中的参与仍然难以捉摸。 LncRNA 被定义为长度超过 200 个碱基的 RNA，不是 mRNA、rRNA 或 tRNA。人类基因组中已发现超过9000个lncRNA，它们几乎涉及RNA代谢和基因调控的所有方面。在当前的项目中，通过小鼠 ESC 的染色质免疫沉淀来鉴定与 Oct4 和 Sox2 结合的 lncRNA。其中一个称为 Hat1-AS 是一种由组蛋白乙酰转移酶 1 (Hat1) 基因编码的新型反义 RNA。 Hat1 和 Hat1-AS 在 ESC 中高表达，并在分化过程中下调。此外，Hat1-AS对于Hat1的转录是必需的。此外，Hat1-AS 反映了与 Oct4 和 Sox2 蛋白的相互作用，对于 Oct4 和 Sox2 的靶基因（例如它们自己的基因）的转录很重要。 Hat1-AS 与正转录延伸因子 b (P-TEFb) 复合物结合，该复合物是转录延伸的中央调节因子。基于这些初步研究，推测Hat1-AS作为Oct4和Sox2的辅因子广泛分布在基因组中，调节P-TEFb向其靶基因的募集并促进转录延伸。我们将通过以下三个目标来检验这一假设。在目标 1 中，将通过寡核苷酸杂交、RNA-seq 和基因敲低在 ESC 中鉴定 Hat1-AS 的全基因组靶基因。在目标 2 中，将通过成纤维细胞中 Hat1-AS 的上调和下调来研究 Hat1-AS 在 iPSC 形成中的作用。在目标 3 中，将研究 Oct4 和 Sox2 蛋白对 Hat1-AS 表达的调节。总而言之，这些目标预计将揭示 Oct4 和 Sox2 介导的新型 lncRNA 介导的多能性调节。