Basic Biology II
Life begins with single fertilized zygote that through the process of differentiation gives rise to every cell type in human body. Amazingly the embryonic stem cells and induced pluripotent cells retain that potential and thus provide exciting hope for therapeutic applications. In the process of differentiation cells “make a decision” and “learn” what their job in the body is and often have to remember that decision for a lifetime, while retaining the ability to interact with their environment. In our proposal aims at investigating the nature of this cellular memory and plasticity and at understanding the mechanism by which certain genes are turned on or turned off during differentiation. Scientific evidence suggests that an indexing system exists that is based on chemical modifications of proteins organizing DNA into chromatin to regulate gene expression. Some of these modifications act as signals marking particular genes to be active, while others mark inactive genes. This indexing system needs maintenance and there are specialized proteins, called chromatin modifying enzymes that maintain chromatin modifications, thus acting as a “memory”. However, chromatin modifying machines are also capable of switching one indexing state (e.g. active) to another (e.g. inactive) at a particular gene locus in response to a signaling event. We recently discovered a novel chromatin modifying complex, that may work in mediating such epigenetic switches during development and embryonic stem cell differentiation. We propose here to test the hypothesis that this complex enzyme is indeed a switch, to investigate the exact mechanism of the switch at the molecular level and determine which regions of genome are the targets for the switch complex at different times during differentiation. We anticipate that these studies will advance our understanding of basic biological processes underlying the developmental decisions of differentiating stem cells. Such advancements could contribute to improvements in stem cell derived therapies.
Statement of Benefit to California:
Since the nature of basic science is to tackle the “unknown unknowns” we can’t make specific promises for short-term health benefits to the residents of California. Nevertheless, research proposed here addresses fundamental questions regarding mechanisms of gene regulation in stem cells and during human development and thus, in the long-term, it is likely to have a high impact on development of cell replacement therapies, for harnessing the potential of personalized medicine and for identification of novel drug targets to combat cancer and age-related diseases. Other tangible and immediate benefits for the community include: - creations of at least 2 new jobs in a high skill sector - contribution to the training of new workforce in a set of unique skills in human stem cell technology - creation of new intellectual property that would benefit local institution and by extension local community. - boosting local economy since we buy our supplies from local vendors whenever possible.
EXECUTIVE SUMMARY The proposed research concerns epigenetic changes associated with cellular differentiation. The applicant proposes that during differentiation, changes in gene expression in response to transient signaling cues are directly and mechanistically associated with epigenetic switches, and that these epigenetic switches are coordinated so that positive and negative changes are appropriately synchronized. The applicant describes this process as a bistable dynamic system of epigenetic memory with switch-like behavior. The principal investigator (PI) has identified a “multi-protein complex” in mouse embryonic stem cells (mESC) that contains both chromatin modifying enzymes (epigenetic regulators) and co-activators of certain transcription factors (gene expression regulators), which appears to mediate such an epigenetic switch. In Aim 1, the PI seeks to identify mechanisms underlying the proposed epigenetic switch, by first generating genome-wide chromatin occupancy maps of “multi-protein complex” components and associated transcription factors in mESC and human embryonic stem cells (hESC) undergoing mesoderm induction. The PI then plans to test the functional significance of candidate target genes by employing genomic reporter constructs and knockdown of “multi-protein complex” components, structure-function analyses of “multi-protein complex” components, and quantitative studies of promoter occupancy and epigenetic status. The goal of Aim 2 is to investigate whether “multi-protein complex” behavior fulfills the theoretical requirements of a bistable epigenetic system by conducting a series of biochemical, structure-function, and knockdown studies using changes in epigenetic status at select genetic loci as a readout. This proposal addresses a significant issue in basic stem cell biology that could have a broad impact, beyond simply understanding the mesodermal phenotypes being specifically examined by the proposed studies. The major innovation comes from the question posed and the use of very sophisticated new methods in some of the proposed experiments. However, the justification for these complex methods is not well developed. Additionally, reviewers felt that the PI attempted to describe too many elements of the bistable dynamic system in parallel, thus confounding efforts to resolve underlying mechanisms. The project is based on interesting ideas, but the experimental goals were not well supported by preliminary evidence. For example, pertinent details regarding the initial biochemical purification of the “multi-protein complex” were not included, and the findings regarding promoter occupancy during early mesoderm induction require more consideration and analysis before the underlying hypothesis can be pursued further. The proposed studies were inadequately supported by the preliminary data, and much additional background work is required to build support for the complex hypothesis; the project is too conceptual and premature at this point. In general, some reviewers felt the experimental plan was relatively straightforward, whereas others felt that the experimental design was lacking important detail and controls, raising doubts that consistent and interpretable data will be obtained. For instance, the use of viral integrated reporters for purifying cell populations during differentiation requires better validation to demonstrate that the reporter faithfully identifies cell populations based on endogenous patterns of gene expression. Furthermore, some reviewers questioned whether the genome-wide mapping approach as proposed in Aim 1was inherently more suited to addressing the hypothesis than direct testing of defined mesodermal target genes. Overall, reviewers judged this proposal to be enormously overambitious, and they had serious concerns about the project’s feasibility. The applicant is a well-trained junior investigator but has no independent track record, no senior author publications, and no other pending proposals or active extramural support. Reviewers expressed concern that these factors, along with a rather weak letter of support from the Department Chair, do not support the independence of the investigator. The environment is outstanding and the collaborators are highly qualified, but they or members of their laboratories are noticeably absent from the budget, calling into question their commitment to the overall success of the project. In conclusion, reviewers thought the proposal addressed an interesting problem in stem cell biology but felt its complex hypothesis was not sufficiently supported by the preliminary data and its experimental design was excessively overambitious and not adequately developed. This, together with the inexperience of the PI, led the reviewers to doubt the likelihood for the project’s success.
- Ali Brivanlou