Transcriptional elongation and long noncoding RNA

 描述(申请人提供)：多能干细胞，以胚胎干细胞(ESCs)和诱导多能干细胞(IPSCs)为代表，可分化为体内所有类型的细胞。这些细胞在基础科学中作为细胞分化和去分化的模型以及医学应用，包括药物筛选、疾病建模和移植，都是重要的。多能性主要由三个主要转录因子-Oct4、Sox2和Nanog控制。通过对这三种因子相互作用的蛋白质、miRNAs和表观遗传修饰的研究，已经广泛地表征了这三种因子对转录的调控。然而，长非编码RNA(Long Non Coding RNAs，LncRNAs)在多能性调控中的作用仍不清楚，尽管已有几个多能性特异的LncRNAs被报道。LncRNA被定义为长度超过200个碱基的非信使核糖核酸、核糖核酸或核糖核酸。目前已在人类基因组中发现了9000多个lncRNAs，它们几乎参与了RNA代谢和基因调控的各个方面。在目前的项目中，结合Oct4和Sox2的lncRNAs是通过小鼠ESCs的染色质免疫沉淀来鉴定的。其中一个称为Hat1-AS，是一种新的在组蛋白乙酰转移酶1(Hat1)基因编码的反义RNA。Hat1和Hat1-AS在ESCs中高表达，在分化过程中表达下调。此外，Hat1-AS是Hat1转录所必需的。此外，Hat1-AS反映了与Oct4和Sox2蛋白的相互作用，对Oct4和Sox2的靶基因如自身基因的转录具有重要意义。Hat1-AS与正转录延伸因子b(P-TEFb)复合体结合，后者是转录延伸的中央调节因子。在这些初步研究的基础上，推测Hat1-AS作为Oct4和Sox2的辅助因子广泛分布于基因组中，调节P-TEFb向其靶基因的招募，并促进转录延长。这一假设将以以下三个目标进行检验。在目标1中，将通过寡核苷酸杂交、RNA-SEQ和基因敲除来鉴定胚胎干细胞中Hat1-AS的全基因组靶基因。在AIM 2中，将通过上调和下调成纤维细胞中Hat1-AS的表达来研究Hat1-AS在IPSCs形成中的作用。在AIM 3中，将研究Oct4和Sox2蛋白对Hat1-AS表达的调节。总之，这些目标有望揭示Oct4和Sox2对多能性的一种新的incRNA调节。

 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.