Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: The goal of this project is to comprehensively characterize DNA methylation patterns at a large number of promoters, focused mostly on polycomb (PcG) target genes, to determine if stem cells are subject to aberrant irreversible gene silencing. The PI describes currently unpublished data to support the logic. PcG is known to target repressive marks (e.g. H3K27me3). The PI looked at 177 PcG target genes in primary colorectal tumors and found that 77 had cancer-associated hypermethylation marks compared to normal. Based on this result, they suggest the interesting hypothesis that repression of differentiation genes in cancer cells contributes to their stem cell properties such as unlimited proliferation. This supports the concept of the stem cell basis for tumors and suggests that it may be very important to monitor the epigenetic state of ES-derived cell populations prior to use in therapies. The goal here is to demonstrate feasibility of screening human embryonic stem cell (hESC) lines or derivatives. In aim1, the applicant will use a high throughput DNA methylation Illumina-based platform to screen a number of cell lines. He will attempt to analyze over 1000 PRC2 target promoters and classify hESC lines based on the epigenetic profile. Aim 2 will monitor epigenetic abnormalities of selected hESC lines under different culture and/or differentiation conditions. INNOVATION AND SIGNIFICANCE: The potential danger of introducing irreversibly self-renewing stem cells in hESC-based therapies is an important issue that is well recognized, and the development of assays to screen for this potential could be very valuable. This project uses innovative approaches, developed largely by the PI, to evaluate promoter methylation state, and although descriptive, the comparison of different lines will be interesting and provide valuable information. STRENGTHS: Strengths involve the application of high-throughput, sensitve technology to profile DNA methylation patterns in several embryonic stem cell lines. The technology will then be applied to monitor DNA methylation abnormalities that might occur under various culture and differentiation conditions in stem cells. While the details of the two approaches were not well explained in the brief application, they are bisulfite-based PCR protocols that have been used in previous publications. The big advantage of Aim 1 experiments is that the PI does not even need to culture cells, but only needs to obtain DNA samples for the analysis (although it will be important to know the state of the cells when harvested). Even in the absence of DNA methylation abnormalities, a strength of the proposal is that a large amount of valuable comparative data should be generated that may provide a unique database for the community and that could serve as a benchmark for stem cell quality. The proposal applies powerful technology that could help scrutinize stem cells and material derived from them before they are transferred to patients. The PI is a leader in the field of DNA methylation and cancer. The degree of confidence for successful execution of the experiments is therefore high. A backup plan for obtaining ESCs is in place in case ESC lines from the international stem cell consortium cannot be obtained. WEAKNESSES: The research plan could be much more focused and well described. While the rationale for aim 1 was well described, it is less clear how the experiments will proceed in aim 2. There is not a clear plan or set of priorities in terms of differentiation protocols. Will cell subsets be sorted for comparison? Overall, the research plan is presented without well-defined benchmarks or priorities. There is some concern that abnormal methylation patterns may not be found in hESC lines. If rare methylation marks are detected it is not clear how this could be used to determine meaning or relevance. What will determine success of the project? There is a strong hypothesis presented but beyond the admittedly interesting characterization, how will these experiments really test the hypothesis? While the expertise of the PI in this field is appreciated, there is some concern regarding available effort. Also. depite being supported by 3 RO1 grants over the past 4-5 years, the PI's published productivity has been modest. DISCUSSION: The applicant proposes to profile DNA methylation patterns of hESC lines with the Illumina DNA methylation analysis platform; this methylation technology is not real high throughput but is sensitive, it can detect 1 aberrantly methylated promoter in a background of 10000 cells. One reviewer questioned how would know in aim 2 whether epigenetic abnormalities detected under different differentiation and/or culture conditions were positive or negative.