Transcriptional elongation and long noncoding RNA

转录延伸和长非编码RNA

• 批准号: 9226044
• 负责人: Nobuaki Kikyo
• 金额: $ 18.52万
• 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 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9226044
• 关键词: Alpha Cell; Antisense RNA; Basic Science; Binding; Binding Sites; Biological Assay; Cell Differentiation process; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA biosynthesis; Data; Development; Disease model; Down-Regulation; Electrophoretic Mobility Shift Assay; 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; Hybrids; Luciferases; Maintenance; Mediating; Medical; Messenger RNA; Metabolism; MicroRNAs; Modification; Molecular; Mus; Names; Nucleosomes; Oligonucleotides; Play; Pluripotent Stem Cells; Positive Transcriptional Elongation Factor B; Preclinical Drug Evaluation; Proteins; Publishing; RNA; RNA Binding; RNA analysis; Recruitment Activity; Regenerative Medicine; Regulation; Reporting; Ribosomal RNA; Role; Techniques; Testing; Transcription Elongation; Transcriptional Regulation; Transfer RNA; Transplantation; Untranslated RNA; 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（由应用程序提供）：多能干细胞，由胚胎干细胞（ESC）表示和诱导的多能干细胞（IPSC），可以区分体内的所有类型的细胞。这些细胞在基础科学中很重要，作为细胞分化和去分化的模型以及医疗应用，包括药物筛查疾病建模和移植。多能主要由三个主转录因子-OCT4，SOX2和Nanog控制。通过研究其相互作用蛋白，miRNA和表观遗传修饰的研究，通过三个因素进行了转录调节。然而，尽管已经报道了几种多能特异性的LNCRNA，但长期非编码RNA（LNCRNA）参与多能量的调节仍然难以捉摸。 lncRNA定义为超过200个不是mRNA，rRNA或tRNA的RNA。在人类基因组中发现了9000多个LNCRNA，它们几乎参与了RNA代谢和基因调节的各个方面。在当前的项目中，通过小鼠ESC的染色质免疫沉淀与OCT4和SOX2结合的LNCRNA。其中一个称为HAT1-AS是一种新型的反义RNA，该RNA在组蛋白乙酰转移酶1（HAT1）基因上编码。 HAT1和HAT1-AS在ESC中高度表达，并在分化过程中被下调。此外，HAT1-是HAT1的转录所必需的。此外，反映了与Oct4和Sox2蛋白的相互作用，HAT1-AS对于Oct4和Sox2的靶基因（例如其自己的基因）的转录很重要。 HAT1-AS与阳性转录伸长因子B（P-TEFB）复合物结合，这是转录伸长的中心调节剂。基于这些初步研究，假设HAT1-AS广泛分布在基因组中，作为Oct4和Sox2的辅助因子，可以根据P-TEFB募集到其靶基因并促进转录延长。该假设将以以下三个目标进行审查。在AIM 1中，将在寡核苷酸杂交，RNA-SEQ和GENE敲低的ESC中鉴定出HAT1-AS的全基因组靶基因。 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.