Yi Zhang - US grants

Understanding mechanism and improving efficiency of somatic cell nuclear transfer (SCNT) Abstract Understanding the mechanism of cell fate reprogramming is important for both basic biology and regenerative medicine. Of the currently available reprogramming techniques, somatic cell nuclear transfer (SCNT) is the only one that allows efficient and rapid reprogramming of terminally differentiated cells to the totipotent zygote-like state. Totipotency is the ability of a cell to give rise to an organism and its placental tissues. However, despite more than 50 years of effort since the first successful cloning by SCNT, very little progress has been made in understanding how SCNT reprogramming is achieved. Although ectopic expression of certain pluripotency transcription factors (TFs) can reprogram somatic cells into induced pluripotent stem cells (iPSCs), these cells are not totipotent. Moreover, accumulating evidence suggest that SCNT-mediated reprogramming is mechanistically different from that of transcription factor-based iPSC reprograming. Since maintaining undifferentiated stem cells in a lineage-unrestricted naïve state is important for therapeutic purposes, understanding how differentiated somatic cells are reprogrammed into a totipotent state is of both biological and clinical importance. During SCNT-mediated reprogramming, donor cell genomes turn off their cell-type specific transcription programs and adopt a new gene expression profile that mimics that of totipotent zygotes. Our preliminary studies indicate that transcriptional reprogramming of donor cells is accomplished within 12 hours following SCNT, indicating that maternal factors present in oocytes can reset the chromatin state of somatic cells quickly upon nuclear transfer. Building upon this intriguing observation, as well as our recently developed techniques in analyzing chromatin accessibility of mouse zygotes and performing maternal factor depletion, we propose to understand the mechanism of SCNT reprogramming and improve SCNT efficiency with the following specific Aims: 1) Identifying and testing TFs and chromatin remodeling factors required for SCNT reprogramming; 2) Overcoming SCNT embryo developmental defects to increase animal term rate. Completion of the proposed study will not only identify oocyte factors important for SCNT-mediated reprogramming, but also improve the SCNT efficiency to achieve maximum term rate. These achievements will have far-reaching implications in the fields of development, stem cell, germ cell, chromatin biology, and regenerative medicine.

Role of DNA methylation in cocaine addiction Abstract Drug addiction is a chronic, relapsing brain disorder characterized by compulsive drug seeking and use despite harmful consequences. It is an urgent social and health problem contributing to more than 90,000 deaths and incurs a yearly cost of over $700 billion in the United States (see NIDA website). It is believed that long-term maladaptive changes in the mesolimbic dopamine reward system play a central role in the development of addictive disorders. However, the underlying molecular mechanism remains largely unknown. The long-lasting effect of drugs on animal behavior and the risk of relapse in human addicts indicate that some stable changes in the brain reward system induced by drugs of abuse mediate these long-term behavioral adaptions. Accordingly, accumulating evidence suggests that drug-induced epigenetic changes, particularly DNA methylation and histone modifications changes, play important roles in drug addiction. However, due to technical difficulties in dealing with the cellular heterogeneity of the mammalian brain, most of the molecular studies performed so far used mixed cell populations, making interpretation of the available data difficult. Consequently, limited progress has been made in understanding the molecular basis of drug addiction. To overcome the issue of cell heterogeneity and to advance our understanding of the epigenetic mechanisms underlying drug addiction, we propose to comprehensively analyze the role of DNA methylation in drug addiction in a neuron subtype- and projection-specific manner using a clinically relevant intravenous cocaine self-administration (IVSA) model. Specifically, we seek to elucidate how DNA methylation in ventral tegmental area (VTA) dopaminergic neurons regulates cocaine reinforcement. To achieve this goal, we have established the following specific aims: 1) Profile transcriptome and methylome of VTA dopaminergic (DA) neurons using a mouse cocaine IVSA model; 2) Functional analysis of key genes regulating DNA methylation in VTA DA neurons; 3) Understand the role of DNA methylation in cocaine reinforcement in projection-specific VTA DA neurons. Completion of the proposed study will not only advance our understanding of how DNA methylation contributes to drug addiction, but also reveal novel therapeutic targets for treating this disorder. Importantly, our study provides a novel, broadly applicable strategy for understanding epigenetic regulation in a neuron subtype- and projection-specific manner.