Despite the remarkable progress made in deciphering embryonic stem cell (ESC) self-renewal, the molecular circuitry that endows ESCs with the ability to form a broad range of differentiated derivatives remains poorly understood. The continued existence of this gap in knowledge limits our ability to efficiently and safely manipu- late pluripotent cells for theurapeutic and research purposes. Our long-term goal is to better understand epige- netic mechanisms responsible for the maintenance of the pluripotent state. By utilizing shRNA-based functional genomics approach developed in our previous studies, we identified novel chromatin-associated factor Dppa2 as critical for the maintenance of developmental potency in ESCs. Furthermore, we demonstrated that forced expression of Dppa2 facilitates acquisition of the pluripotent state during cellular reprogramming. The objective in this application is to determine how Dppa2 sets up and maintains the epigenetic landscape of the pluripotent state. The central hypothesis, formulated on the basis of our own preliminary data and work by others, is that Dppa2-associated protein complex maintains unique epigenetic signatures at the promoters of poised devel- opmental genes that ensure proper activation of these genes upon induction of differentiation and during re- programming. The rationale for the proposed research is that in-depth understanding of Dppa2 function has the potential to translate into novel strategies to enhance stem cell maintenance and derivation of the pluripo- tent cells. This hypothesis will be tested by pursuing three specific aims: 1) Identify epigenetic footprints at Dppa2-bound poised promoters in ESCs and EpiSCs. Determine how these footprints are altered in Dppa2- depleted cells;2) Determine the composition and function of the Dppa2 complex in ESCs;and 3) Determine the function of the Dppa2 complex during cellular reprogramming. Under the first aim, Dppa2 binding, histone modifications, patterns of DNA methylation and gene expression will be compared in wild-type and Dppa2- depleted ESCs and EpiSCs after which mechanistic models of chromatin regulation by the Dppa2 complex will be developed. Under the second aim, Dppa2-associated protein complex will be purified, protein identities de- termined by mass-spectrometry, and individual genes inactivated by shRNAs in order to determine the roles of these interacting partners in chromatin regulation at the Dppa2-bound genomic loci. Mechanism(s) of target recognition will also be investigated under this aim. Under the third aim, iPSCs will be generated from fibro- blasts with or without Dppa2 and their ability to form germ layer derivatives in vitro and in vivo will be analyzed. Molecular analyses will be performed in order to define the function of the Dppa2 complex during the repro- gramming process. The proposed research is significant, because it is expected to vertically advance and ex- pand understanding of developmental potency and its regulation at the chromatin level. Such knowledge has the potential to enhance stem cell maintenance and differentiation - critical steps for new and innovative ap- proaches to treatment of a variety of diseases.
The proposed research is relevant to public health because a thorough understanding of embryonic stem cell pluripotency is a crucial requirement for developing regenerative therapies to effectively treat human disease. Patients with neurodegenerative diseases such as Parkinson's and Alzheimer's diseases and spinal cord injuries could potentially benefit from this research. Thus, it is relevant to the part of NIH's mission that pertains to developing of fundamental knowledge that will help reduce the burdens of illness and disability.