. The transcription factors OCT4 and SOX2 are critical regulators of stem cell maintenance and pluripotency, but how they regulate gene expressions is poorly understood. This knowledge gap prevents us from understanding how OCT4 and SOX2 control pluripotency. The long-term goal of this project is to understand transcriptional mechanisms governing stem cell self-renewal and differentiation. Recruitment of transcriptional cofactors by activators is a key step in gene activation. However, the identity and function of these cofactors for OCT4 and SOX2 are not known. We developed a fully reconstituted in vitro transcription assay to detect and isolate novel cofactors for OCT4 and SOX2. One of these cofactors, the dyskerin ribonucleoprotein (RNP) complex, is mutated in the inherited stem cell disorder dyskeratosis congenita (DC), and is the focus of this application. The overall objective of this proposal is to understand how stem cell-specific transcription and pluripotency are regulated by the dyskerin RNP. The central hypothesis is that dyskerin RNP is an essential component of OCT4/SOX2 transcriptional machinery and a novel regulator of pluripotency and stem cell maintenance. The rationale for this work is that deciphering how the dyskerin RNP regulates pluripotency gene transcription will yield a new understanding of how transcriptional cofactors control stem cell pluripotency. The central hypothesis will be tested by pursuing three Specific Aims: (1) Identify transcriptional mechanisms by which the OCT4/SOX2/dyskerin ribonucleoprotein complex regulates stem cell maintenance and pluripotency, (2) Identify dyskerin-associated non-coding RNAs and the mechanism by which they activate transcription with OCT4 and SOX2, and (3) Determine how pathogenic mutations in dyskerin compromise RNA binding and transcriptional co-activation. Under the first aim, genome-wide analyses will be used to define transcriptional targets, gene expression programs, and stem cell differentiation pathways controlled by the dyskerin RNP. Under the second aim, by identifying RNAs associated with the dyskerin complex, fundamental insight into the mechanisms by which non-coding RNAs regulate cofactor activity will be investigated. Under the third aim, patient-derived induced pluripotent stem cells (iPSCs) will be used to identify changes in the RNA composition and transcriptional activity due to pathogenic mutations in the dyskerin gene (DKC1), and establish mechanistic connection between DKC1 mutations and a compromised pluripotency gene network that we found in DKC1-mutant iPSCs. The proposed research is innovative, because it employs in vitro reconstitution and transcription assay to uncover novel transcriptional mechanims in human pluripotent stem cells. This contribution is significant, because it is expected to yield a foundational understanding of how pluripotency is controlled by OCT4 and SOX2, thereby enhancing our ability to control stem cell fate for therapies and the treatment of diseases such as DC.
The ability of embryonic stem cells to propagate indefinitely and differentiate to generate essentially all cell types in the body is controlled by two essential transcription factors, OCT4 and SOX2. The overall objective of this project is to understand how OCT4 and SOX2 activate transcription in embryonic stem cells. The knowledge gained in these studies will enhance our ability to control human embryonic stem cell pluripotency for regenerative therapies.
