Biochemical Basis and Regulatory Circuitries for Pluripotency of Embryonic Stem Cells

   Embryonic stem (ES) cells serve as a potentially inexhaustible source for tissue replacement in regenerative medicine due to their capability of unlimited self-renewal and multi-lineage differentiation. Vital cellular functions of ES cells require the coordinated action of a large number of proteins that assemble into an array of multi-protein complexes of distinct composition and structure (protein-protein interactions). In addition, physical interactions between regulatory pluripotency transcription factors and their target genes (protein-DNA interactions) provide insights into differential gene expression dictating the pluripotency program. Analysis of protein complexes encompassing intricate protein-protein and regulatory protein-DNA interactions is key to understanding stem cell pluripotency.

    Recently, we tested the utility of in vivo biotinylation of transcription factors in mouse ES cells, and have established an in vivo biotinylation system for BirA-mediated specific biotinylation of critical pluripotency factors in mouse ES cells. We developed and optimized an approach for affinity purification of pluripotency protein complexes involving streptavidin capture of biotinylated proteins (dubbed bioSAIP) and demonstrated the feasibility of in vivo biotinylation for mapping global/chromosomal targets of many different transcription factors (dubbed bioChIP-chip) (see Figure 1). Utilizing the technologies we developed, we have constructed a protein interaction network surrounding the pluripotency factor Nanog in mouse ES cells (Wang et al., Nature 2006) and mapped an extended transcriptional network for pluripotency of mouse ES cells (Kim et al. Cell 2008). The network is highly enriched for factors known to be critical in ES cell biology and appears to function as a module for pluripotency. Pluripotency is maintained by many transcription factors that form a highly interconnected protein interaction network including the two homeobox proteins Nanog and Oct4, and a battery of associated proteins of known and unknown functions linking to multiple co-repressor pathways (Figure 2).

    Further dissection of the pluripotency network in human ES cells (and induced pluripotent stem cells) and understanding molecular function of the novel factors should illuminate fundamental properties of stem cells and the process of cellular reprogramming, and ultimately lead to precise manipulation and realization of the full clinical therapeutic benefits of these unique cells. Therefore, my lab will be focusing on the following three research areas:

1. 1.Defining protein-protein and protein-DNA interaction networks for pluripotency of human ES cells (and human iPS cells);
   

2. 2.Dissecting molecular action of novel pluripotency factors on stem cell self-renewal and pluripotency;
   

3. 3.Elucidating functional significance of novel pluripotency factors in early development and somatic cell reprogramming.
   

Figure 1. Strategies for mapping protein-protein and protein-DNA interactions in mouse ES cells. The ES cells expressing BirA alone (as control) and BirA plus biotinylated transcription factors (bioTF) can be used for isolation of protein complexes using streptavidin (SA) immunoprecipitation (IP) coupled with LC-MS/MS (dubbed bioSAIP-MS) and construction of a protein-protein interaction network; meanwhile, the same ES cells can be subjected to in vivo biotinylation-mediated chromatin immunoprecipitation and microarray (dubbed bioChIP-chip) to identify protein-DNA interactions and construct a transcriptional regulatory network.

Figure 2. A protein interaction network in mouse ES cells. Proteins with red labels are tagged baits for affinity purification. Green and red lines indicate confirmed interactions by coimmunoprecipitation or published data. Dotted lines indicate potential association. Green circles indicate proteins whose knockout results in defects in proliferation and/or survival of the inner cell mass or other aspects of early development; Blue circles indicate proteins whose reduction by RNAi (or shRNA) results in defects in self-renewal and/or differentiation of ES cells; Yellow circles are proteins whose knockout results in later developmental defects; White circles denote proteins for which no loss-of-function data are available. Also indicated within the network are three major chromatin modifying complexes whose components are marked with black stars (Polycomb repression complex 1), red stars (NuRD complex) and a blue star (SWI/SNF complex), respectively.

© 2013 The Mount Sinai Hospital