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.

PUBLIC HEALTH RELEVANCE: This study will uncover the regulation and functions of a novel antisense RNA of the Hat1 gene. Because the RNA regulates transcription of genes involved in pluripotency, this study will contribute to the development of regenerative medicine.