The human ES cells are euploid cells that can proliferate without limit and maintain the potential to differentiate into all cell types. Differentiation of human ES cells involves selective activation or silencing of genes, a process that involves not only combinatorial interactions between the cis-regulatory sequences and DNA binding transcription factors, but also post-translational histone modifications and other epigenetic mechanisms such as DNA methylation and non-coding RNAs. A number of transcription factors have been found to be essential for the ES cells to maintain their identity or differentiate along specific lineages. These regulators exert their effects through interacting with the promoters, enhancers or silencer elements to modulate the expression of target genes. Currently, the molecular details of how transcription factors modulate target gene expression upon binding to DNA and how they mediate ES cell differentiation are still unclear. The proposed project is aimed to determine the role that histone modifications play in this process. We will conduct experiments to identify the DNA binding proteins, chromatin modifying enzymes and chromatin binding proteins that are responsible for the specific histone modification profiles at regulatory DNA sequences , and investigate how these proteins activate target genes and contribute to the unique properties of human ES cells. Results from the proposed study will improve our understanding of the mechanisms that control pluripotency and lineage specification, and lay a foundation for development of better tools for manipulating and reprogramming human ES cells for regenerative medicine.
Our research will provide better understanding of the mechanisms by which human ES cells differentiate along specific cell lineages. Such knowledge will facilitate the development of new methods for manipulating the ES cells, and enable a mechanistic understanding of the reprogramming of somatic cells to the pluripotent state. These results will directly support the efforts by us and other California researchers to investigate the mechanisms of stem cell biology, and design new stem cell therapies.
The applicant proposes to delineate the mechanisms that establish, maintain and remove specific histone modifications at transcriptional enhancers and to understand the role of these histone modifications in controlling gene expression and differentiation of human embryonic stem (hES) cells. Based on the hypothesis that enhancer function requires binding of specific transcription factors that recruit histone modification enzymes, the principal investigator (PI) seeks to determine which transcription factors are involved in establishing a specific histone modification; to determine the chromatin modification enzymes required for establishing and removing that histone modification, and to determine the chromatin binding proteins that recognize that modification. Finally, the PI plans to determine the functional requirements of proteins identified in the proposed experiments in hES cell differentiation using a specific differentiation protocol.
Reviewers differed in their assessment of the potential significance of the proposed work. Some felt that it covers an exciting area of study, and information gained from the proposed experiments may have significance for attempts to direct differentiation of hES cells toward specific early precursor lineages. The fundamental assumptions underlying this proposal were judged to be reasonable and to have a lot of support in the current literature. Conversely, other reviewers questioned the assumption that histone modifications play an active role in establishing cellular identity, but conceded that this proposal, if successful, would provide a clarification for whether this is the case or not. They pointed out that the proposed approach will help identify large-scale genomic modifications, but specific control of unique gene elements appears to still reside in the transcription factors, not the histone modifications or the proteins responsible for those modifications.
The applicant presents a well-written, coherent and ambitious research plan, the proposed experiments are sophisticated, and the preliminary data support the feasibility of the research plan. However, the reviewers identified a limitation of the proposed studies in that the differentiation experiments proposed in aim 4 do not include selection for the rare subset of hES cells that are responding to the differentiation stimulus. Thus, the PI will examine very heterogeneous populations of cells, and the bulk population response may not reveal the subtle changes that are occurring in the subset of hES cells that are committing to the differentiation pathways. Furthermore, the PI limits his/her analyses to only one NIH-approved hES cell line. Different hES cell lines can respond in a variable manner to the same differentiation signals, and significant, useful information could be obtained by comparing and contrasting the epigenetic changes among various lines.
The applicant is a very accomplished mid-career scientist who was recently promoted to Associate Professor. S/he is extramurally funded, has an outstanding publication record, and is a renowned expert in applying epigenetic analyses to questions in the stem cell field. However, the career development plan and the mentoring plan contained only minimal information, one mentor is mentioned. As outlined in the institutional letter, the PI is a highly valued member of the home institution, s/he receives generous space and support, and all of the necessary state of the art equipment is readily accessible. The institution is small but growing, and is associated with a large university.
A motion was made to recommend that this application be moved from Tier 1 to Tier 2 – Recommended for Funding if Funds Available. The working group considered and discussed whether the applicant met the objective of this RFA, i.e. to promote the careers of promising junior investigators. Although the panelists did not question the qualifications of the PI, they agreed s/he is already an accomplished, established investigator, and therefore was not an appropriate target for this initiative. The panelists further raised the programmatic point that this proposal is fundable by the NIH. The motion to move this application to Tier 2 carried.