Public Summary: During the reporting period of the Yr3 (Aug. 1, 2012- Jul. 31, 2013), we have made significant research progress towards (1) Epigenetic role of CDK2AP1 in ESC maintenance/differentiation by regulation of WNT pathway. This work was an extension of the previous year and completed for a publication. (2) We have performed bioinformatics analysis on our own datasets and publicly available datasets to identify novel molecular factors in self-renewal of hESCs. Findings from this study were submitted to Stem Cells and now in revision for publication. (3) We have performed detailed analysis to define molecular role of CDK2AP1 and MBD3 in mouse and human ESCs, which are briefly summarized as below.
(1) CDK2AP1 is a novel regulatory factor in NuRD-mediated Wnt signaling in embryonic stem cells (Published in JBC 87(49): 41103–41117): In continuation of work from the previous year, we have completed the study and successfully revised the manuscript for publication in JBC during the first quarter of this reporting period. Potential role of MBD3/NuRD in the repression of pluripotency genes in ESC has been demonstrated. However, it is still elusive on the detailed molecular mechanism, such as the necessity of any auxiliary factor priming the complex formation or conferring target specificity. Our study further elucidates the epigenetic role of CDK2AP1 in stem cell maintenance via association with MBD3/NuRD complex and demonstrates the importance of a complimentary molecule to MBD3/NuRD in the regulation of ESC. One area of interest is how NuRD recruitment occurs on the target gene promoters, whether it is focal, broadly distributed or dependent on the pattern of the gene. In this study, we found a novel regulatory factor within the NuRD complex, which may function as a guide in NuRD recruitment to specific WNT genes to regulate stem cell pluripotency.
(2) Discovery of novel molecular factors in self-renewal of hESCs (Manuscript in revision: Stem Cells). Our understanding of self-renewal and differentiation capacity of human embryonic stem cells (hESCs) remains elusive on the detailed molecular mechanisms. Molecular markers defining self-renewing pluripotent embryonic stem cells have been identified by relative comparisons between undifferentiated and differentiated cells. Most of previous analyses have been done under a specific differentiation condition that may present significantly different molecular changes over other experimental conditions. Therefore, it is currently unclear if there are true consensus markers that define undifferentiated hESCs. Our current study elucidates the global regulation of stem cell beyond the well-known stem cell factors by combining over 33 microarrays and the latest bioinformatic tools. We examined if there are a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions. By comprehensive genome-wide consensus microarray analyses, we have profiled gene expression signatures that are most significantly affected by differentiation in hESCs from our own microarray data sets as well as publically available microarrays. Our finding has unveiled the novel molecular markers that determine self-renewal and form intramodular hubs. Bioinformatics approach for the identification of new molecular markers defining undifferentiated hESCs, interacting partners and interconnectivity analyses may contribute to delineating molecular mechanisms of stem cell self-renewal/differentiation and can be a useful tool to identify molecular factors inducing stemness from different cell types.
(3) Identification of molecular targets that are specifically regulated by CDK2AP1/MBD3 in mESCs and hESCs: To examine the specific molecular effect of CDK2AP1/MBD3 interaction, we restored Cdk2ap1 wt or Mbd3 binding mutant in Ckd2ap1 ko mESCs and performed gene expression microarray analysis (Affymetrix GeneChip Mouse Genome 430 2.0 array). In addition, we have performed genome-wide ChIP-seq analysis to identify specific molecular targets in hESCs that are associated with CDK2AP1, MBD3, Mi-2beta or active histone H3. Analysis is being done to profile changes in molecular marks that are associated with these molecules during differentiation of hESCs. Results will be essential to defining molecular mechanisms in the regulation of hESC self-renewal/differentiation by MBD3/NuRD complex.