One of the major gaps in embryonic stem (ES) cell biology is our incomplete understanding of the mechanisms that enable the ES cells to differentiate into virtually all cell types in the body. While much progress has been made recently in determining key DNA sequences involved in ES cell differentiation, it is still unclear how such sequences work together to regulate ES cell differentiation. Based on existing evidence, we hypothesize that the way chromosomes are folded in 3-dimensional space would play a critical role in this process, but our current knowledge of chromosome folding principle is rather poor. Here, we propose to use a powerful DNA sequencing technique to analyze chromosome folding in the human ES cells before and after differentiation into four different cell types. We will carry out multiple analyses to study the role of chromatin architecture in gene regulation and ES cell differentiation. We anticipate that the proposed experiments will result in much improved understanding of the program controlling ES cell differentiation, which will translate into better means to manipulate the ES cells and development of better ES cell based therapeutics.
Statement of Benefit to California:
The proposed research will significantly improve our understanding of the mechanisms by which human ES cells differentiate along specific cell lineages. Such knowledge will facilitate the development of new methods for differentiating the ES cells and design new stem cell therapies.
The applicant proposes to perform a comprehensive investigation of chromosome folding in human embryonic stem cells (hESC) and four early embryonic lineages differentiated from them. This will provide insight into the mechanisms by which enhancers, i.e. short sequence elements in the genome that regulate expression of individual genes, exert their long distance effects. The proposed experiments include a genome-wide characterization of 3-dimensional (3D) chromosome organization, followed by an analysis of changes in chromosome organization and gene expression as a consequence of mutating certain genome boundary regions and binding sites for regulatory factors.
Significance and Innovation
- The significance of the proposed research lies in the genome-wide elucidation of 3D chromosome folding in hESCs and derivatives. However, the descriptive nature of this effort lowers enthusiasm.
- The proposed approaches for gaining mechanistic insight into the function of identified genome boundary regions are interesting, but the rationale and design of these experiments are not well focused on questions relating to stem cell biology.
- Innovative aspects of this proposal include the use of deep sequencing and informatics.
- The project is designed to fit into an ongoing large-scale epigenomics effort by the applicant. Reviewers viewed this both as a drawback in that the proposed studies lacked innovation, and also as a potential strength, since large data sets generated with other funds can be leveraged to support the analyses in the proposed work.
- The proposed studies are limited to the use of a single hESC line. It is unclear if the findings can be extrapolated to other heterogeneous hESC lines; the inclusion of additional lines would have improved the significance of the data generated from this study.
Feasibility and Research Design
- The experimental design is logical and well planned, the proposal is well written.
- Feasibility for the most part is convincingly supported by preliminary data, especially for the 3D chromosome analyses and other genome-wide assays. However, the proposed targeted mutations are difficult to achieve in hESC and no preliminary studies are presented to demonstrate their feasibility.
- Some of the proposed targeted mutations involve deletion of relatively large genomic regions, raising the concern that still unknown smaller regulatory elements might be present in those boundary regions, and upon concomitant deletion may confound interpretation of resulting phenotypes.
Principal Investigator (PI) and Research Team
- The PI is very successful and has an outstanding track record in the field of chromosome interactions, with relevant publications in excellent journals within recent years.
-The research team is excellent and includes a highly qualified collaborator.
Responsiveness to the RFA
- The application is somewhat responsive to the RFA, as it is focused on human cells, although its contributions to a mechanistic understanding of human stem cell biology are unclear.