The human embryonic stem cells (hESC) have the remarkable potential to replicate themselves indefinitely and differentiate into virtually any cell type under appropriate environmental conditions. They accomplish this through regulating the production of a unique set of proteins in the cells, a process known as gene regulation. While the genes encoding these stem cell proteins have been largely identified over the years, the mechanisms of gene regulation are not yet understood. This gap in our knowledge has seriously limited our ability to manipulate hESC for therapeutic purposes.
In Eukaryotic cells, gene regulation depends on specific sequences in the DNA known as transcriptional regulatory elements. These regulatory DNA consists of promoters, enhancers, insulators and other regulatory sequences. As a key step towards understanding the gene regulatory mechanisms in hESC, we will produce a comprehensive map of promoters, enhancers and insulators in the hESC genome. We will use a newly developed, high throughput experimental strategy to identify these sequences that are engaged in gene activation in hESC. Our strategy involves identifying the DNA sequences that are associated with the specific transcription factors or chromatin modification signatures known to be present at each type of regulatory elements inside the hESC. We will use biochemical procedures to isolate these sequences from the cell and determine the resulting DNA in large scale with the use of DNA microarrays, containing of millions of DNA species that together represent the complete genomic makeup of the hESC. Completion of the proposed research is expected to improve our knowledge of the gene regulatory mechanisms in hESC, which in turn will facilitate the development of new strategies for stem cell based therapeutics.
Benefits to the State of California: Our research is aimed to provide a foundation for analysis of the mechanisms that control the production of stem cell proteins, which in turn would help us design new ways to manipulate the stem cells so that they can differentiate towards specified cell types. The knowledge base resulting from our research will directly support the effort by us and other California researchers to investigate the mechanisms of stem cell biology, and design new stem cell therapies.
SYNOPSIS: The experiments in this proposal will utilize ChIP on Chip and other techniques to provide a comprehensive map of promoters, enhancers and insulators in hESCs. Genome-wide localization studies will map the position of transcription factors, as well as different types of chromatin modifications within the genome of hESCs. This will results in a catalog of information with which to manipulate the fate choices of these cells.
SIGNIFICANCE AND INNOVATION: The innovation in this proposal lies in the application of state-of-the-art genomic technologies to hESCs. Similar approaches have already been reported in the literature using the hESC and murine ESC systems. Thus the innovation is not particularly high. However, these are exactly the kinds of approaches that need to be pursued to attain the goals of the overall proposal. The significance is quite high given the potential to generate a comprehensive catalog of chromosomal phenomena that could correlate with pluripotency. These studies are largely descriptive; however, they are necessary before embarking on detailed and comprehensive functional efforts.
The secondary reviewer noted that the major goal of this proposal is to identify transcriptionally active regions, and the significance is that it will identify the location of putative pre-initiation complexes, enhancers, and insulators throughout hESCs.
STRENGTHS: The major strength of this proposal is the excellent level of expertise in all of the technologies to be employed. The PI's laboratory was one of the developers of the ChIP on Chip technology. There is a high level of confidence that valuable and large amounts of information will be obtained. The 4 Aims will proceed from the identification of active promoters, transcriptional enhancers, to insulator elements in hESCs. The final Aim will begin testing some of the identified elements in functional assays. Of further merit are the computational tools that will be employed in the analysis of high volume datasets.
Clearly the strength of this proposal lies with the investigator, who was one of the founders of the ChIP-chip assay and has a strong track record of productivity and expertise in ChIP-chip. There is very little doubt that the proposed aims will be accomplished successfully. The outcome will provide new knowledge on the location of pre-initiation complexes, enhancers, and insulators in the genome of hESCs.
WEAKNESSES: One reviewer felt that the major weakness of this proposal is its scope. Clearly, it seems to be an effort that will take more than two years. In fact, each of the four Aims could easily be a separately funded efforts. Nevertheless, this is a superb investigator, and even if not all goals are achieved, there will certainly be a wealth of information generated. There is some concern that the PI does not propose to integrate the data to be obtained with recently published datasets also obtained in the hESC system.
To the other reviewer, the major weakness is that the data that will be acquired will be a catalog of sites in hESCs, however, this is unlikely to make an immediate contribution towards the understanding of hESC regulation. Ultimately this information will be important in providing a base of knowledge. But at this stage the reviewer did not see the immediate downstream utility for understanding hESC regulation. Moreover, it is not clear that the major goal of this proposal could not be attained using tiled expression arrays.
DISCUSSION: There was no further discussion beyond the reviewers' comments.