Public Summary: We have performed additional analysis during the NCE period (Aug. 1, 2013 – Nov. 30, 2013) to continue and finish what has been attempted during the Yr 3 funding period. The following is the Summary of research findings during the NCE period.

To define molecular signatures that are regulated by CDK2AP1 either in association with MBD3 or independently of MBD3, we have taken advantage of our ESC model system. We have generated mESC lines from Cdk2ap1-/- cells by restoring either the wild type Cdk2ap1 or the MBD3 binding mutant form of Cdk2ap1. We then performed gene expression microarray analysis. During this NCE period, we have completed bioinformatics analysis of data as briefly presented in the progress report. From this approach, we were able to identify significant differences in molecular roles of CDK2AP1 and MBD3. Some of important pathways are regulated collaboratively by CDK2AP1 and MBD3. In addition we found some important cellular functions that are distinctively regulated by either CDK2AP1 or MBD3. This result demonstrates that CDK2AP1 may participate in the regulation of certain genes through interaction with MBD3/NuRD, while it maintains its own molecular role apart from NuRD complex. We will continue to further define the significance of these molecular roles in ESC biology and also their in vivo significance in development.

As a way to define molecular targets that are specifically associated with CDK2AP1/MBD3-NuRD in hESCs, we have performed the genome-wide ChIP-seq analysis with undifferentiated and differentiated hESCs. We have identified molecular marks that CDK2AP1, MBD3, Mi-2beta or active histone H3 associates and examined how those marks change during the differentiation of hESCs into embryoid bodies. This analysis enabled us to profile gene targets that are under the regulation by MBD3/NuRD during the differentiation of hESCs and also targets of CDK2AP1-mediated MBD3/NuRD control. Results from this analysis will set the basis for further analysis towards defining molecular mechanisms underlying the target selection or specificity of epigenetic regulation by MBD3/NuRD in hESCs. As an additional approach to define the role of CDK2AP1/MBD3/NuRD in neural differentiation of hESCs, we have established a neural induction model from hESCs and performed ChIP-seq and gene expression analysis. Currently we are in the process of analyzing the data and plan to continue beyond the funding period. Our ultimate goal of this approach is to define the role of CDK2AP1 in conferring molecular specificity in the spatial or temporal regulation by MBD3/NuRD in ESCs and during development.