Investigating the role of retinoblastoma-binding protein 9 in human pluripotent stem cells and embryonic development

Abstract

Many intricate networks are required to work together to regulate two defining characteristics of human pluripotent stem cells (hPSCs): the capacity to self-renew, and the potential to produce (through differentiation processes) any cell type of the body. The discovery of PSCs has provided an invaluable tool for investigating fundamental aspects of developmental biology, drug discovery, disease modelling, and tissue-replacement therapies. Master regulators of pluripotency have been identified in PSCs including OCT4, NANOG, and SOX2. However, the molecular events that drive self-renewal and differentiation currently are not completely understood. Previous results from our group identified the retinoblastoma (RB)-binding protein 9 (RBBP9) as a novel pluripotency regulator. Small interfering RNA (siRNA)-mediated loss of RBBP9 protein in hPSCs decreased expression of pluripotency and cell cycle genes, and increased expression of neurogenesis genes. RBBP9 is reported to have two potential mechanisms of action: the ability to i) bind RB protein and influence the RB/E2F pathway, and ii) serine hydrolase (SH) activity. To investigate the relative contribution of these two activities to hPSC maintenance and embryonic development in vitro and in vivo, we compared the responses of hPSCs and zebrafish treated with a recently identified selective chemical inhibitor of RBBP9 SH activity (ML114), to those treated with either RBBP9 siRNA or rbbp9 morpholino. Data presented in this thesis show the requirement of RBBP9, including RBBP9 SH activity, in hPSC maintenance in vitro, and possibly its requirement in early embryogenesis in vivo. In Chapter 2, selective loss of RBBP9 SH activity was investigated in hPSCs via ML114. This treatment resulted in the decoupling of hPSC self-renewal from differentiation, as seen by a significant reduction in hPSC proliferation without any observable decrease in pluripotency marker expression or morphological change. Chapter 3 then investigated effectors which could potentially be responsible for this unusual decoupling effect. Promoter analyses identified the Nuclear transcription factor Y subunit A (NFYA) as a highly-ranked candidate effector of RBBP9 SH activity through analyses of gene expression changes arising from ML114 treatment of hPSCs. The up-regulation of NFYA was hypothesised to mediate the changes in cell proliferation seen whilst maintaining pluripotency in hPSCs. Chapter 4 began to explore the impacts of RBBP9 and its activities in vivo. Rbbp9 is expressed in a large range of zebrafish tissues, and the data presented here is consistent with the idea that Rbbp9 and its activities are required for zebrafish embryogenesis. However, the complexity of the observed phenotypes suggests that toxicity might be an alternate explanation of the data. Further investigation into the role of RBBP9 activities during hPSC generation, maintenance and differentiation, as well as the early stages of embryonic development, is required to better understand the mechanisms behind developmental abnormalities resulting from Rbbp9 losses. A better understanding of RBBP9 activities and its role throughout embryonic development in vitro and in vivo will help us better understand the molecular networks required to: maintain hPSCs; generate hPSCs via somatic reprograming; and also drive or facilitate normal embryogenesis and cancer progression.

Date of Award	2016
Original language	English