Basic Biology II
Our goal is to understand the processes that control how human embryonic stem cells differentiate to become various distinct cell types. The control of several important genes that determine this process relies on a process known as DNA methylation. DNA methylation involves the reversible "decoration" of our DNA at specific gene locations, resulting in the silencing of that gene. This is a key step in the commitment of these cells to particular differentiated endpoints. Understanding what controls the DNA methylation and silencing of key genes in human embryonic stem cell differentiation is the goal of this proposal. RNA can regulate how proteins function in the cell, and based on many examples involving DNA methylation, we will test if RNA controls when and where DNA methylation occurs during the differentiation of human embryonic stem cells. We propose to use methods relying on the use of human embryonic stem cells and purified enzymes that carry out the DNA methylation itself. The identity of RNA molecules that bind to these enzymes will be determined by two distinct methods, and the biological importance of these RNA molecules to embryonic stem cell differentiation will be determined. The mechanism of how these RNA molecules alter the function of the the DNA methyltransferase enzymes will be determined. The realization of our goals is anticipated to provide the basis for controlling the function of the critical DNA methyltransferases, which has already been demonstrated to be important in influencing the cellular differentiation process.
Statement of Benefit to California:
The ability to use human embryonic stem cells depends on being able to control their differentiation. Thus, while these cells have the potential to become a different cells within different human tissues, our current ability to direct such changes is limited. This proposal will benefit the State of California and its citizens by providing improved ways of achieving this goal, of being able to direct these cells to particular differentiated cell types.
EXECUTIVE SUMMARY The overall goal of this project is to understand how DNA methylation regulation occurs during human embryonic stem cell (hESC) differentiation. DNA methylation involves the reversible alteration of DNA at specific gene locations and plays a critical role in the regulation of several genes. The applicant proposes to test the hypothesis that gene-specific de novo DNA methylation is an RNA-driven process during hESC differentiation. In Aim 1, the Principal Investigator (PI) proposes to use immunoprecipitation to identify RNA sequences associated with two specific DNA methyltransferases (DMNTs) during hESC differentiation and to validate identified sequences. In Aim 2, the PI proposes a complementary approach to identify RNA sequences that bind to purified DMNTs. S/he proposes an in vitro selection followed by the subsequent bioinformatics and hESC expression analyses. Reviewers appreciated the proposal’s mechanistic emphasis and the novelty and innovation of the proposed hypothesis. They were, however, divided in their assessment of its potential impact. One reviewer noted that the mechanisms of gene-specific DNA methylation are not well understood and that these studies could contribute to our understanding of how to reliably control lineage specific differentiation. Another reviewer questioned the significance for stem cell biology, given that there is limited evidence to suggest that the proposal will uncover a new biochemical mechanism important for hESC function. Reviewers raised several issues when evaluating the proposal’s feasibility and experimental design. They commented that the preliminary data presented does not directly demonstrate feasibility of the approach. Specifically, the feasibility of using immunoprecipitation to characterize a single known RNA is missing thus the specificity of the immunoprecipitation technology for RNA isolation is not validated. Moreover, the proposed application of RNA sequencing for global discovery was not a logical extension in the experimental approach, as a design to eliminate false positive hits was lacking. Validation of the results of the second aim is dependent on the first aim being successful. Therefore the preliminary data on RNA selection by the in vitro approach in a different system does not provide sufficient feasibility data for the proposed Aim of identifying RNA that binds to DMNT and is relevant for function in hESC. Reviewers also found the proposal’s feasibility diminished by some gaps in the discussion of potential pitfalls and alternative approaches. One reviewer found this particularly important given that a part of the preliminary data appear to contradict, as noted by the PI, the central hypothesis. Reviewers noted that the PI has put together a team comprised of productive and innovative investigators who have strong expertise in enzymology and computational molecular genetics. They highlighted the PI’s effort commitment to the program and the excellent research environment. They appreciated that the co-investigator has been responsible for development of the novel technique for the in vitro selection approach. The reviewers, however, considered the lack of stem cell biology expertise on the team to be a significant weakness of the proposal. In summary, although the reviewers considered the proposal to be innovative and of potential significance, and the PI and co-investigator very strong; they had concerns about the project’s feasibility given some weaknesses in the preliminary data and the experimental design as well as the apparent lack of key personnel with stem cell biology expertise.