Basic Biology I
Embryonic stem (ES) cells are able to give rise to all the cell types in an organism, a unique property called pluripotency. After ES cells differentiate into adult tissues, they lose the ability to respond to many environmental cues and can not differentiate into other cell types. This lack of plasticity is due to the permanent silencing of critical genes, a fact that in the past has been considered to be irreversible. However, recent achievements show that is possible to reprogram adult somatic cells to pluripotency by the overexpression of critical transcription factors. These induced pluripotent cells have the same properties as embryonic stem cells regarding self-renewal and pluripotency, and are thus of great importance for regenerative medicine. The process of somatic cell reprogramming is very inefficient because it entails the reactivation of the endogenous pluripotency genes that are permanently silenced in somatic cells and refractory to stimulation. The molecular basis of this silencing is found in the structural organization of DNA around histone proteins, called chromatin. Both DNA and histones can be modified by the action of enzymes and as a result, the chromatin structure changes to facilitate or block the expression of genes. These modifications are inherited from cell to cell in a process that is known as epigenetic memory. Our main goal is to gain insight into the epigenetic mechanisms that mediate the permanent silencing of pluripotency genes in human somatic cells and that contribute to the establishment of the repressive chromatin environment of these genes during the differentiation of human ES cells. We will address critical questions aimed at the identification of critical chromatin related factors that play a role in this process and to understand how these factors mediate their function. We anticipate that the knowledge generated from our proposal will shed light on the epigenetic barriers that need to be overcome during somatic cell reprogramming and inspire approaches to make this process more efficient. Ultimately, these new approaches could facilitate the use of induced pluripotent cells for clinical applications. Moreover, understanding the mechanisms of gene silencing will provide basic knowledge about the mechanisms that govern cell plasticity and will also help to understand pathological processes like cancer, in which the epigenetic deregulation of gene silencing plays an important role.
Statement of Benefit to California:
Embryonic stem (ES) cells have the ability to differentiate into a variety of cell types, tissues and organs, which opens the door to the possibility of tissue engineering, replacement, and cell transplant therapies to cure diseases ranging from Parkinson’s, Alzheimer’s, diabetes, blood disorders and a host of other debilitating disorders. Rarely, there comes along a new technology that has the potential to make such a major impact on human health. Recently, researchers have discovered methods to reprogram adult fibroblasts and skin cells back into a cell type referred to as an induced pluripotent stem cell, which appears to be indistinguishable from a pluripotent ES cell. This is accomplished without the need for embryo destruction and offers great potential to alleviate the problems of immune rejection in cell or tissue transplantation by allowing a patient’s own cells to be reprogrammed, expanded and then used in therapeutic applications. In this study we propose to gain insight into the basic mechanisms of gene silencing in the context of somatic cell reprogramming to pluripotency. Our findings could be used as a basis to enhance new strategies that render the process of reprogramming more efficient. These strategies might favor the generation of improved pluripotent cells that could later be used for therapeutic purposes, or as disease models by the pharmaceutical industry in order to test new drugs. We anticipate that our work will generate important contributions to understanding the basic mechanisms of gene silencing and epigenetics. This knowledge can be of great importance to uncovering the molecular mechanisms governing pluripotency, cell plasticity and differentiation. Moreover, epigenetic abnormalities have been found to be causative factors in cancer, genetic disorders and pediatric syndromes, as well as contributing factors in autoimmune diseases and aging. We feel confident that the proposal presented here will significantly contribute to understanding diseases characterized by epigenetic deregulation. Thus, our research will benefit the State of California by generating basic knowledge that will clearly reinforce the scientific leadership of California and that in the long term will inspire strategies applicable to therapeutic approaches that will directly benefit the people of California.
This proposal addresses how epigenetic mechanisms mediate silencing of critical pluripotency genes in human somatic cells and influence cellular reprogramming, with the hope that barriers to full reprogramming can be identified and overcome using insights gained from this study. The first aim centers on identifying chromatin factors enriched in somatic cells by comparative gene expression analysis based on microarrays, assessing the recruitment of those factors to silenced pluripotency genes in somatic cells by chromatin immunoprecipitation, and investigating their functional involvement in reprogramming by conducting gain and loss of function experiments. In the second aim, the principal investigator (PI) proposes to analyze the role of the chromatin factors identified in Aim 1 in silencing of pluripotency genes during human embryonic stem cell (hESC) differentiation. Although reviewers felt that this proposal addresses a major unsolved problem, they expressed serious doubt regarding its potential impact on the field of stem cell biology and regenerative medicine. This assessment was based on a central concern that the proposal lacks innovation, as it is focused on already well-characterized transcriptional and chromatin regulators. Furthermore, the approaches described are routine, and the depth and scope of the experimental plan is limited and builds only incrementally from the preliminary data. Reviewers pointed out that epigenetic analysis during hESC differentiation has been pursued by others in a highly systematic manner, and thus it is unclear what the proposed analysis would add to this field. The approaches presented for each aim are technically sound and doable, but they were not considered to be state-of-the-art. Reviewers were confident that the experimental plan is feasible. However, they were concerned that completion of Aim 2 depends too heavily on the success of Aim 1, and that the generation of antibody reagents required for chromatin immunoprecipitation experiments was not sufficiently supported by preliminary data or experimental detail. Reviewers criticized the PI's claim that the identification of several regulatory factors described under preliminary results provides proof-of-principle for the proposed screening methods, since functional significance of those factors has not yet been established, and was not even proposed to be validated. Instead, the proposal relies entirely on the identification of additional factors. Finally, reviewers objected to the underlying notion that mechanisms that lead to the release of repression of pluripotent genes during reprogramming of somatic cells are the same as those that lead to restoration of repression during differentiation of hESC. This assumption fails to consider that cellular context may demand use of subtly different mechanisms that achieve the same repressed state. The PI is a well-established scientist who has published substantially in the field of developmental biology and more recently in the pluripotent stem cell field. He/she is well qualified to conduct the research described in this proposal. The research environment is outstanding. In summary, the proposal addresses how epigenetic mechanisms mediate silencing of pluripotent genes in human somatic cells, a subject that remains an unsolved problem. However, reviewer enthusiasm was weakened by the lack of an overall focused working hypothesis and the heavy dependence of the project on an unfocused, global screening approach. The lack of innovative approaches in a rapidly changing field led reviewers to believe that the proposed research would not contribute to substantial advancement of the field.