The life of every human starts with a fertilized egg. This single cell starts to divide and, in a truly amazing process, gives rise to a developed human being. Although each cell of a developed organism is a progeny of this single zygote, and shares the same genetic information with every other cell, cells differentiate to specialized forms such as skin, muscle or nervous cells. Thus, new information emerges during development, and is inherited in a fashion that does not involve changes in DNA sequence. This fascinating process is called epigenesis. Epigenetic changes underlie not only normal, but also pathological development. Abnormal epigenesis contributes to human pathology, such as aging, cancer, degenerative diseases, developmental defects and mental retardation. Embryonic stem cells (ESCs) share with the early embryo the potential to produce every type of cell in the human body. This rare biological property is known as pluripotency. Pluripotency is a unique epigenetic state, in that ESCs can self-renew, while retaining the potential for multilineage differentiation. The research proposed here aims at elucidation of the precise molecular nature of pluripotency. In the last decade evidence emerged that a substantial portion of epigenetic information is transmitted in a form of chemical modifications of histones and DNA, in particular histone methylation. The physiological template of our genome, called chromatin, is composed of DNA wrapped around histone proteins. Methylation marks are written and erased from histones by specific enzymatic activities and they are read by the specialized proteins to activate or silence gene expression. Here we propose to elucidate which writers, readers and erasers of histone methylation are required for maintenance of the unique epigenetic state of pluripotency. Building on this initial knowledge we will perform a series of biochemical experiments to understand the network of protein-protein and protein-DNA interactions involved in the epigenetic regulation of pluripotency. We are hoping that our studies will significantly advance our understanding of the unique properties of ESCs and bring us closer to the development of efficient technologies to direct the differentiation of stem cells into therapeutically useful tissues. Even more exciting is possibility that uncovered epigenetic regulators of pluripotency could be used to reset a patient’s differentiated cells to the pluripotent state, thus removing the current bottlenecks in stem cell derivation and requirement for human oocytes, and sidestepping the problems of tissue rejection. Last, but not least, understanding the mechanisms of epigenetic plasticity of human ESCs will contribute to the basic knowledge of human development. Basic knowledge has proven itself time and again to be the raw fabric of innovation and progress in medicine. Thus, in the long run our research may help the humankind in ways we are not yet able to predict.
Statement of Benefit to California:
We believe the proposed research will benefit people of California in the following ways. Direct benefits: It will increase experience and knowledge of human embryonic stem cells among residents of California. This project involves cooperation between two laboratories with complementary expertise. This interaction will facilitate skill exchange and staff training in cutting edge techniques of stem cell biology and epigenetics. It will result in development of new approaches – the proposed project involves technological approaches that to our knowledge have not been used before in studies of human stem cells, certainly not in the particular combination we propose. It will generate new reagents to study genetics and epigenetics of human stem cells, which will help position us and other Californian scientists at the forefront of embryonic stem cell research. Indirect benefits: We will contribute to basic knowledge of human development. Basic knowledge have proven itself time and again to be a raw fabric of innovation and progress in medicine. It is highly likely that discoveries resulting from proposed studies will identify molecules whose manipulation will contribute to current efforts to bring stem cell into realm of therapeutics, particularly in the area of directed differentiation and epigenetic reprogramming
SYNOPSIS: This proposal will address the roles of chromatin modifying and other epigenetically important gene-products in hES cells. The studies are largely molecular and biochemical in nature. The first Aim will perform a small scale RNAi screen focusing on a panel of candidate molecules (chromatin modifying and other epigenetic gene-products). The PI has constructed an Oct-4-driven GFP reporter hES cell line to use in the screen. The second Aim is focused on functional studies to evaluate the roles of candidates idntified in Aim 1. This will involve over-expression experiments and pull-downs to identify interacting complexes. The third Aim proposes to generate conditional mutations in hES cells of the identified gene-products using homologous recombination. SIGNIFICANCE AND INNOVATION: Epigenetic gene regulation (i.e. chromatin modification) plays a clear role in cell differentation and development. ESC differentiation may be significantly under the control of epigenetic marks. At least a good case is made to believe so. Wysocka is a new and credible expert in this area. The proposed work should lead to new insights into mechanisms by which ESC pluropotency is regulated. This proposal is innovative because it takes a head-on approach to tackling some significant and difficulty problems including reporter assay set up, siRNA delivery and target silencing. The role of epigenetics in hESC reprogramming is very much in its infancy, perhaps in large part due to the limitations noted above. This proposal has the potential to make a major advance in the field. STRENGTHS: The strength of this proposal is its logical experimental design, suggesting that at least some of the Aims will be accomplished. The expertise in the 2 collaborating laboratories is very complementary, and the experimental design suggests strong interactions. The experiments are very well-designed, and thoughtfully presented. There is a high probability that valuable information will be obtained. A second strength of the proposal is the investigator, who has an outstanding track record and is uniquely qualified to carry out the proposed experiemnts. A third strength is the significance of this work. If successful it will have a large impact on ESC research. Scientifically, the strength of the proposal is aim 1: the use of siRNA to identify chromatin regulation of ESC pluropotency. WEAKNESSES: There are 2 minor weaknesses in this proposal. First, the combined studies are very ambitious, and clearly will require more than 2 years for completion. However, as the project progresses valuable information will be accumulated in a continuous manner. Second, there is no discussion how the PI could take advantage of already published studies describing epigenetic phenomena and regulation in hES cells. There is similarly no discussion of a plan to integrate the data to emerge from this proposal with existing datasets. Such integration is essential given the issues of experimental variability among different independent efforts. DISCUSSION: This is a highly innovative and significant proposal given that little is known about chromatin modifiers and pluripotency. The individual parts in the head-on approach are not innovative, but the combination of the reporter and RNAi screen set up is. The proposal is logical, and the expertise of the PI and the collaborators is a strength. The experiments are well-designed and have a high-probablility of success. The PI needs to take advantage of current data in the work, especially since this a an ambitious proposal that will clearly take more than two years as proposed. Nonetheless, this significant work will have an impact on hESC biology.