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Interplay between the Trithorax Group protein Ash2L and the dynamic epigenome in the regulation of open chromatin, pluripotency, and cell fate commitment in embryonic stem cells

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07097
Funds requested: 
$1 178 370
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
With their remarkable potential to develop into virtually all cell types in the body, human embryonic stem cells (hESC) represent a powerful research tool to study fundamental mechanisms regulating development and disease. Notably, adult human cells can be induced to revert back to an immature stage of development, showing properties similar to hESC. Maintaining this pluripotent cell state requires precise control of gene regulation, processes that keep genes turned on or off. Chromatin, the intranuclear genetic blueprint made up of DNA and histone proteins, has emerged as one component which controls self-renewal and pluripotency potential of hESC. Essentially, chromatin determines how information stored in DNA is interpreted and influences cellular behavior through alterations in patterns of gene expression. Here, we propose to investigate the initial events during which chromatin-modifying enzymes and binding proteins interact with the genome. We want to know how the chromatin structures/domains that permit or impede factor binding are constructed both in hESC and maturing cells differentiating along specific lineages. Results from the proposed studies will advance our understanding of the unique properties of chromatin configurations in hESC and of mechanisms that control pluripotency and lineage specification. This will provide a foundation for the development of new strategies to manipulate and reprogram hESCs for regenerative medicine.
Statement of Benefit to California: 
Many California citizens suffering from heart disease, diabetes, osteoporosis, cancer, stroke, Alzheimer’s and Parkinson’s diseases, and other injuries could benefit from treatments using human stem cells. In order to maximize therapeutic potential, we need to better understand the basic molecular mechanisms that maintain the pluripotent state of human stem cells, enhance differentiation of stem cells along specific cell lineages, and allow reprogramming of specific adult cells. Our research will reveal important fundamental principles of gene regulation during early human development and further our understanding of the dynamic balance between cellular pluripotency and cell fate commitment. We aim to create both a solid foundation of knowledge and novel tools to develop new therapies based on directed, disease-specific manipulation of stem cell populations. This information could expedite the development of regenerative medicine in California, benefiting both young and elderly patients with currently incurable diseases. Other direct benefits of our work include the training of a new generation of scientists (graduate students and postdoctoral fellows) proficient in human stem cell biology as well as the potential creation and commercialization of intellectual property supporting local institutions, the community-at-large, and the biotechnology industry.
Review Summary: 
Human somatic cells can be induced to revert to an immature stage of pluripotency similar to hESC, which to retain, requires precise control of gene processes that keep genes tuned on or off. Chromatin, the intranuclear genetic blueprint comprised of DNA and histone proteins, is one component that can control self-renewal and pluripotency. Chromatin structure can affect access of regulatory transcription factors to specific regions in the DNA to positively or negatively control transcription of genes. The goal of this Track 1 (Fundamental Mechanisms) project is to investigate regulators of chromatin structure and how they affect reprogramming by manipulating a specific DNA-binding protein then quantifying the downstream changes and documenting the effects on chromosomal organization in hESC, human-induced pluripotent stem cells (iPSCs), and differentiated cells. Significance and Innovation - The regulation of chromatin change is a fundamental cell biology question. The studies should provide some insights into the epigenetic regulation of the global chromatin changes and thus would provide some significance. - The innovation is significantly diminished by a recent publication (Wan 2013) on a similar topic, and some of the proposed experiments have already been done in the published manuscript, which was not discussed in the proposal. - The proposed work is clearly interesting by the use of point mutations in specific DNA binding domains, but the overall application shows only modest innovation. Feasibility and Experimental Design - A significant weakness in the feasibility is that the proposal relies extensively on cells lines that have yet to be generated, and it is not clear why new iPSC lines will be derived instead of using existing lines. - For the unbiased analyses, it is not clear as to how the analyses will be performed. In addition, the term “differentiated” is used very generically to assume that all differentiated cells will have the same epigenetic markers, which may not be accurate. - It is unclear and not well justified how the proposed rodent experiments enhance the knowledge to be gained. The alternative plans would benefit from the use of more advanced and available techniques. - Most of the proposed experiments and key data generation are straightforward and feasible, but the multi-component approach would make it difficult to achieve these aims within the 3-year timeline. The preliminary data and the design of the genomics experiments are strong. Principal Investigator (PI) and Research Team - The PI has excellent training, a strong publication track record, and has included outstanding collaborators that should provide the necessary expertise to conduct the proposed research. However, no budget or letters of support from the collaborators are included. - There is a considerable amount of genomic work to be performed and data to be analyzed. It is not clear who is performing this work. - Additional expertise in pluripotent stem cells, especially in reprogramming and differentiation, would further strengthen the project. Responsiveness to the RFA - The proposal addresses a basic mechanistic question of the maintenance and induction of pluripotency by regulation of the chromatin three-dimensional structure in human pluripotent stem cells, which is responsive. - Some of the detailed work described in the rodent model does not comply with the goal of this RFA and is not responsive.
Conflicts: 
  • Todd C. McDevitt

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