Basic Biology II
Embryonic stem cells (ESC) hold great promise for the treatment of debilitating diseases and for use as research tools to elucidate the molecular mechanisms underlying many diseases. Current knowledge is already quite advanced and has allowed researchers to begin making use of stem cells for clinical applications. However, the full clinical potential of ESC cannot be realized without a better understanding of their fundamental properties. Increased fundamental knowledge is necessary to help overcome technical limitations in our ability to differentiate ESC into clinically useful cell types and to enhance the efficiency and consistency with which differentiated cells obtained from patients in need of stem cell-based therapies can be reprogrammed to an ESC-like state. ESC lines are of great value because of their unique ability to continually divide on laboratory tissue-culture dishes while maintaining pluripotency, which is defined as a capacity to differentiate into almost any cell type. Pluripotency is known to be dependent on the expression of key genes whose protein products function by turning on many other genes that are needed to achieve the pluripotent state. Considerable attention has also been focused on genes encoding proteins that regulate the differentiation of ESC into specific cell lineages. These genes possess intriguing properties that keep them silent in ESC, but poised for activation when the ESC receive appropriate differentiation signals. The research proposed in this application focuses on a third class of genes that have received relatively little attention from researchers studying pluripotency: genes expressed only in differentiated cell types, which generally remain silent until long after the ESC have differentiated into a specific cell lineage. Although prior models suggested that the cascade of events leading to the activation of these typical tissue-specific genes does not begin until the ESC have differentiated, recent evidence from our laboratory and others supports a hypothesis in which the competence of these genes for expression in differentiated cells is dependent on specific marks established at their DNA regulatory regions in the pluripotent state. The proposed examination of this hypothesis will reveal whether the proper marking of tissue-specific genes in ESC is essential for their differentiation into clinical useful cell types and tissues and for the proper functioning of mature ESC-derived tissues.
Statement of Benefit to California:
The research proposed in this application will increase our knowledge of the fundamental properties of embryonic stem cells (ESC) that are important for their pluripotency, defined as their capacity to differentiate into almost all human cell types and tissues. ESC hold great promise for treating or leading to a better understanding of human diseases, which would greatly benefit the State of California and its citizens. However, despite this potential and the rapid progress that has been made toward its fulfillment, our incomplete knowledge of the properties of ESC critical for their pluripotency has limited their utility. Previous studies of the key determinants of pluripotency have focused on genes that are actively expressed in ESC or that play a role in the initial decision to differentiate into specific cell lineages. The research proposed in this application focuses on an emerging hypothesis that pluripotency also requires the active marking of typical tissue-specific genes, which generally remain silent until long after the ESC have begun to differentiate. The proper marking of these genes in ESC may be essential for their competence for activation in differentiated tissues. A rigorous evaluation of this hypothesis will contribute to efforts to identify the sources of variability in the differentiation potential of ESC lines and to develop improved methods for the reprogramming of differentiated cells to a pluripotent state.
EXECUTIVE SUMMARY The goal of this proposal is to investigate the functional role of specific enhancers, chromatin features at gene regulatory regions, associated with tissue-specific genes in pluripotent stem cells. In a recent publication, the applicant demonstrated that these enhancers are biochemically marked in mouse embryonic stem cells (mESCs) in advance of differentiation, and that this marking may play role in subsequent gene activation after the cells have become differentiated. The applicant proposes to extend these studies through two Specific Aims. First, the function of chromatin marks on two specific enhancers in mESCs will be analyzed by incorporating reporter genes, mutating relevant sequences and introducing chromatin motifs of interest. In Aim 2, the applicant proposes to examine the conservation of enhancer marks between mouse and human pluripotent cells and determine how variability in these features might contribute to differences in tissue-specific gene activation. Reviewers agreed that this proposal addresses an important, fundamental question in stem cell biology: how pluripotent cells suppress the expression of tissue-specific genes while maintaining their competence to be activated during and following differentiation. Insights into molecular mechanisms that mark tissue-specific genes could be used to enhance the efficiency of somatic cell reprogramming and shed light on the inherent variability in differentiation potential of different pluripotent stem cells lines. A key concern of reviewers was that although much of his work seeks to describe and address the consequences of pioneer factor binding, there was relatively little attempt to identify or study these factors directly. Additionally, it was unclear why the applicant chose the particular tissue-specific genes proposed for study and little justification for these choices was provided. Moreover, reviewers viewed the study as having only a limited impact, as the research concentrates on only a small set of regulatory elements. Finally, reviewers judged the proposal to be only marginally responsive to the RFA, since the bulk of the work focuses on mouse cells. The reviewers found the research plan to be sophisticated and clever but also overly ambitious and risky. They noted that the proposal contains little preliminary data; this is limited to one figure and a textual account of published results. This was viewed as a particular weakness. Reviewers also were concerned that the much of the analysis relies on ectopically introduced DNA, which may not accurately recapitulate the behavior of endogenous sequences and may enhance the risk of artifactual results. Additionally, reviewers were concerned that the dependence of certain experimental objectives on the success of other ones might lead to delays in the anticipated timelines. Reviewers found the research team to be well qualified to carry out the proposed studies. They noted that the PI has an excellent publication record and runs an active program on the molecular mechanisms of transcriptional control. They did feel that four full-time research personnel staffing the project are excessive and recommended elimination of one position. Overall, although reviewers appreciated that this proposal addresses an interesting biological problem, they questioned its responsiveness to the RFA and had serious reservations about its potential impact and feasibility.
- Ali Brivanlou