This research proposal integrates into our long-term goal to identify and characterize the specific molecular determinants (both genetic and epigenetic) that define the identity of human embryonic stem cells (hESCs). Recent discoveries in mouse embryonic stem cells (mESCs) have identified the protein MYC as a "master" regulatory molecule that maintains the ability of embryonic stem cells to divide indefinitely and to give rise to all tissues of the human body. The MYC protein (in conjunction with 3 other factors) when introduced into an adult tissue cell (for instance a skin cell that has limited life span) can "rejuvenate" the adult cell into a pluripotent stem cell that can divide indefinitely and give rise to all types of tissues in vitro. This is a major step toward being able to generate an unlimited supply of hESCs for regenerative medicine. Howeve MYC also has a "dark side". In our lab we have been working for several years at understanding how MYC immortalizes cells in the context of cancer. Indeed most human tumors contain large amounts of MYC and MYC stimulates tumor formation when aberrantly overexpressed. So MYC can have both beneficial (rejuvenation/immortalization) and detrimental (tumor formation) effects on cells. We do not understand yet how this balance is regulated. The current project aims at understanding (i) how MYC and one important cooperating partner of MYC called GCN5 regulate genes in hESCs, (ii) which genes are directly regulated, and (iii) how MYC functions might be modified in hESCs as compared to cancer cells and other adult body cells. This understanding will be critical to eventually develop safe methods to generate large amounts of hESCs for regenerative therapy from adult body cells by exploiting selectively the beneficial functions of MYC (or its partners such as GCN5) while avoiding its oncogenic activities (i.e., its "dark side").
Statement of Benefit to California:
The proposed research project will investigate at the molecular level the role of emerging"key" regulators of human embryonic stem cell identity and self-renewal. It is speculated that these factors control the self-renewal and pluripotency of human embryonic stem cells by modifying the structure of chromatin and the expression of specific genes in the nucleus of these cells. The network of genes regulated by these key factors in human embryonic stem cells will be identified by the proposed analyses.
These studies will contribute to a better understanding of the molecular genetic mechanisms and gene regulatory networks that identify specifically human embryonic stem cells and differentiate them from all other human cells. This fundamental undertanding of the molecular biology of human stem cells will be essential for the eventual rational design of methods to control the self-renewal and differentiation of these cells and, in the longer term, to exploit their therapeutic potential. As such the proposed research will contribute to the effort of the State of California and its many research institutions and dedicated individuals to accelerate discovery in the stem cell field in order to advance at a faster pace towards the goal of curing many degenerative diseases and tissue injuries that affect many, if not most, people at some point in life.
SYNOPSIS: This proposal will explore the molecular mechanisms that define pluripotency of human embryonic stem cells (hESCs). In particular, the applicant proposes that the MYC oncogene is a key regulator of hESC self-renewal and pluripotency and that its associated histone acetyltransferase (HAT) GCN5, have marked differences in gene network control in ESCs than in differentiated or cancer cells. The applicant will firstly analyze the expression and sub-cellular localization of MYC and associated proteins in hESCs before and after induction into embroid bodies, and possible post-translational modifications of these factors. Secondly, the applicant will identify the promoters that are bound to MYC and GCN5 by microarray analyses. These will be compared to MYC bound genes in cancer cells.
INNOVATION AND SIGNIFICANCE: The genes controlling self-renewal and pluripotency in hESCs are of great importance in the understanding of the molecular mechanisms of these processes. While it has been shown that c-MYC is a critical factor in self-renewal and pluripotency in mouse ESCs, it is clearly important to test whether this is also the case in hESCs as major differences are known to exist between mouse and human ESCs.
STRENGTHS: The strength of this proposal is that it might identify the statistical enrichment of MYC, GCN5, and its partners genome-wide in hESCs. Furthermore, the applicant has a strong academic background appropriate to the proposed studies. UC Riverside is developing strengths in hESC research and Dr. Sato will be an important collaborator.
WEAKNESSES: The primary weakness of the proposal is that aim 1 is not very substantial. GCN5 is ubiquitous and so is likely to function broadly in gene expression (i.e. at most genes) rather at selected genes involved in hESC regulation. Hence there is no evidence that GCN5 plays important roles in ESC pluripotency or differentiation. MYC has been implicated in ESC regulation, but it's not clear whether it is a major player. The basis for conducting expensive ChIP-chip on these factors is not established. Another weakness is that the specificity of the ChIP is not established. GCN5 ChIP-chip could generate a lot of noise. It is not clear how the PI plans to distinguish false positives from true positives. Most importantly, it is not clear how these results will tangibly produce a better understanding of hESC regulation.
DISCUSSION: There was no further discussion following reviewers' comments.