Novel Mechanism in Self-Renewal/Differentiation of Human Embryonic Stem Cells

Year 2

During the reporting period, we have made significant research progress towards (1) the epigenetic control mediated by cyclin dependent kinase 2-associated protein 1 (CDK2AP1) through Nucleosome Remodeling and Deacetylation (NuRD) complex and its functional contribution to the stem cell pluripotency, (2) Role of Cdk2ap2, a sibling molecule of Cdk2ap1 in stem cell self-renewal, (3) the role of cell cycle regulatory CDK2AP1 in the regulation of OCT4 promoter, and (4) genome wide molecular signaling pathways affected by CDK2AP1 in ESCs. These are briefly summarized as follows. (1) CDK2AP1 is a novel regulatory factor in NuRD-mediated Wnt signaling in embryonic stem cells (Manuscript submitted). NuRD complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered as a key player in the formation of functional NuRD complex. We show that CDK2AP1 plays a role in self-renewal/differentiation of ESC by engaging MBD3 to the promoter region of Wnt signaling related genes. The occupancy of MBD3 on several Wnt signaling gene promoters was significantly lost in the absence of CDK2AP1, resulting in hyper-activation of Wnt signaling. We propose that the transcriptional modulation of Wnt pathway mediated by MBD3/NuRD requires the presence of essential auxiliary component, such as CDK2AP1, that may aid the association of the complex with the specific focal region of the target promoters. (2) Cdk2ap2 Is a Novel Regulator for Self-Renewal of Murine Embryonic Stem Cells (In Press. Stem Cells and Development). To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2tr/tr). The Cdk2ap2tr/tr mESCs, when grown in a complete growth medium, showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation. Cdk2ap2tr/tr mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis. (3) Epigenetic regulation of Oct4 by CDK2AP1 (Presented in the ISSCR 2012, Manuscript in preparation). Cell cycle regulators are gaining a more prominent role, these include molecules such as p27 that have been shown to play a role in cell cycle kinetics to maintain a pluripotent state and regulate specific genes involved in pluripotency. In our studies we have found that CDK2AP1 plays a key role in NuRD-mediated Oct3/4 silencing by epigenetically regulating the Oct4 promoter during differentiation of both mESC and hESC. Detailed analysis of the Oct4 promoter revealed an absence of DNA methylation at the proximal enhancer (PE) region in differentiated Cdk2ap1-/- mESC. In parallel, we have seen an increase in H3K9 acetylation at the same region in Cdk2ap1-/- mESC. We have found CDK2AP1 occupancy at the PE region in mESC as well as hESC embryoid bodies. Furthermore, in ESC we have observed interdependency in CDK2AP1 and MBD3 binding to the OCT4 promoter. In hESC nuclear translocation of CDK2AP1 upon differentiation was distinct from mESC. CDK2AP1 plays a significant role in stem cell differentiation by association with the NuRD complex on specific promoter regions, changing chromatin accessibility and leading to the silencing of the Oct4 promoter during differentiation in ESC. (4) Global epigenetic signatures and signaling pathways affected by CDK2AP1 in ESCs (Manuscript in preparation). To gain insight into the genome wide molecular effects of CDK2AP1 in ESC differentiation, we have performed systems biology analysis by combining DNA methylation array analysis and gene expression array analysis on both mESC and hESC samples. From DNA methylation (MeDIP-seq on hESC) and gene expression array analysis, we found that there were global methylomic and transcriptional changes due to the deletion of in ESCs. Detailed analyses revealed that the deletion of Cdk2ap1 led to site-specific methylation changes. We have constructed a complete meta-network map of mESCs built from 12 different mouse gene expression arrays to explain how nucleosome remodeling and differentiation are interconnected and linked. In addition, by using our Cdk2ap1 knockout mESC model, we have demonstrated that CDK2AP1 may affect the repertoire and function of miRNA involved in stem cell self-renewal/differentiation. This finding demonstrates that CDK2AP1, either through cell cycle regulatory mechanism or transcriptional control, may affect the function of miRNA in ESC.