Specific Aims proposed in this application are designed to significantly advance our understanding in hESC biology towards (1) the epigenetic control of OCT3/4 and its functional contribution to the stem cell pluripotency, (2) the role of cell cycle regulatory mechanism in stem cell self-renewal/differentiation decision, and (3) the potential utilization of molecular regulatory mechanism for future regenerative therapeutics. There are three main specific aims proposed.
Specific Aim 1: Significance of CDK2AP1-mediated molecular regulation in hESC self-renewal/differentiation.
Specific Aim 2: Systems biology analysis to identify key molecular pathways in CDK2AP1-mediated hESC regulation.
Specific Aim 3: Identification of small molecule activator for OCT3/4.
These aims are based on our preliminary study using mouse embryonic stem cells.
The main goal of this proposal is to further translate what we have found with Cdk2ap1 knockout mESC model to human embryonic stem cell biology. Acknowledging the difference between mouse model and human model, we have performed the proposed study in parallel by using our established mESC model and also generating and testing hESC model.
For Specific Aim 1 in the Year 1, we have further detailed our knowledge on how CDK2AP1 epigenetically regulates Oct3/4 promoter at the molecular level. We have recently made a novel finding that CDK2AP1 may play a role in the regulation of Oct3/4 in the sequence specific manner along with NuRD complex. This has been also confirmed in hESC model. We have also found that CDK2AP1 may exert its effect through changes in cellular localization and conformation during the differentiation of hESC. We are in the process of further detailing this mechanism to link the cell cycle regulatory role of CDK2AP1 through CDK2/RB and the epigenetic role in stem cell differentiation. We have generated necessary hESC cell lines to further perform the proposed studies. At the end of the Year 2 as planned, we are expecting to submit a manuscript and related to the Aim 1. An NIH RO1 grant will be submitted for the next cycle related to our new findings.
For Specific Aim 2, we have performed and obtained significant amount of data that advance our understanding on the molecular role of CDK2AP1 in stem cell differentiation. We have newly found CDK2AP1 significantly alter DNA methylation of key signaling pathways, especially Wnt pathway that is known to have a significant impact on stem cell maintenance and differentiation. We have detailed molecular axis related to the epigenetic regulation of Wnt pathway by CDK2AP1 and a manuscript is in the preparation. Using the hESC cell lines we have generated, we will perform systems biology approach as proposed in the Year 2. This will enable us to generate significant amount of data related to the epigenetic role of CDK2AP1 in hESC differentiation.
For Specific Aim 3, we have generated hESC sublines with human OCT3/4 promoter-GFP and human CDK2AP1-GFP for chemical library screening. We will generate more deletion mutants of CDK2AP1 promoter-RFP lines and test them before we initiate the works proposed in the Aim 3. We will initiate the proposed Aim 3 near the end of the Year 2.