Transcriptional elongation and long noncoding RNA

 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用于医学应用（包括药物筛选、疾病建模和移植）是重要的。多能性主要由三个主转录因子Oct 4、Sox 2和Nanog控制。这三个因子的转录调控已经通过对它们相互作用的蛋白质、miRNA和表观遗传修饰的研究得到了广泛的表征。然而，尽管已经报道了几种多能性特异性lncRNA，但长链非编码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。在人类基因组中发现了9000多个lncRNA，它们参与了RNA代谢和基因调控的几乎所有方面。在目前的项目中，通过染色质免疫沉淀从小鼠ESC中鉴定与Oct 4和Sox 2结合的lncRNA。其中Hat 1-AS是一种编码组蛋白乙酰转移酶1（Hat 1）基因的新型反义RNA。Hat 1和Hat 1-AS在ESCs中高表达，并在分化过程中下调。此外，Hat 1-AS对于Hat 1的转录是必需的。此外，反映与Oct 4和Sox 2蛋白的相互作用，Hat 1-AS对于Oct 4和Sox 2的靶基因（例如它们自身的基因）的转录是重要的。Hat 1- AS与正转录延伸因子B（P-TEF B）复合物结合，后者是转录延伸的中心调节因子。基于这些初步研究，推测Hat 1-AS作为Oct 4和Sox 2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。我们将从以下三个方面来检验这一假设。在目标1中，Hat 1-AS的全基因组靶基因将通过寡核苷酸杂交、RNA-seq和基因敲除在ESC中鉴定。Hat 1-AS在iPSC形成中的作用将通过在Aim 2中成纤维细胞中Hat 1-AS的上调和下调来研究。目的3将研究Oct 4和Sox 2蛋白对Hat 1-AS表达的调节。总之，这些目标有望揭示Oct 4和Sox 2对多能性的新型lncRNA介导的调节。

 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.