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RS1-00195-1: Regulation of Specific Chromosomal Boundary Elements by CTCF Protein Complexes in Human Embryonic Stem Cells

Recommendation: Recommended if funds available
Scientific Score: 88

First Year Funds Requested: $358,670.00
Total Funds Requested: $678,788.00

Public Abstract (provided by applicant)

The genetic information contained in all human cells is arranged into distinct territories or “neighborhoods” with barriers or “fences” that protect the action in one neighborhood from spilling over into an adjacent region. In this way, one gene (A) can be working while its neighboring genes (B and C) are resting. As physiological conditions change in the body, appropriate signals are transmitted to cells that instruct genes to alter their genetic “programming” by opening or closing the fences. This allows gene A to be turned off and genes B and C to start working. Importantly, these “fences” can control large numbers of genes that regulate critical cellular processes. For example, a well-known fence borders a chromosomal region containing genes that encode oxygen-carrying hemoglobin. By opening or closing this fence, hemoglobin synthesis, and our oxygen carrying ability, can be turned on or off. Many, as yet, unidentified fences are likely to exist in our genetic material. This proposal is designed to find the fence(s) that border certain genes (Nanog-Stellar-GDF3) that are important to maintain stem cells in their most plastic state that is, having the ability to become any other cell type. Once we identify the borders/fences of this chromosomal region, we plan to investigate how they are themselves switched on or off. This switch is very likely to depend upon specific proteins that interact with the fences or borders and serve as “latches” to keep the gates open or closed and the Nanog gene working or resting. Information about the exact proteins or “latches” that control the Nanog neighborhood will enable us to begin to devise strategies, through genetics or pharmacological means, to open or close this particular fence at will and regulate the activity of the Nanog gene. The ability to maintain an active Nanog gene may facilitate stem cell self-renewal or reprogram adult somatic cells to progenitors that are more easily directed to another cell type. By contrast, the capacity to turn off the Nanog gene may be important for the treatment of stem cells that have acquired tumorigenic potential through persistent Nanog expression and inappropriate self-renewal. In the larger scope, information from this proposal may serve as a platform by which unique proteins that control other fences can be identified. Pharmacological manipulation of these unique proteins may selectively control the activity of chromosomal neighborhoods that specify distinct cell fates.

Statement of Benefit to California (provided by applicant)

All of our genetic information that regulates the proper function of our tissues and our overall health is arranged in large territories or “neighborhoods” that can be turned on or off with a genetic switch called a boundary. This acts like a fence to separate the influence of one neighborhood which may be working (active) from an adjacent one which may be resting (inactive). Organ function or tissue “identity” is conferred by the exact combination of our 35,000 genes that are working or resting. Diseased organs or tissues, including cancers, are characterized by having the wrong combination of genes that are inappropriately active or inactive. By being able to control the activity of chromosomal regions (“territories”) through switches or boundaries , we hope to devise new ways to more easily turn on and off many genes that determine tissue identity and proper organ function. This may lead to new therapeutic strategies to repair existing diseased tissues or replace them with new cells.

Review

SYNOPSIS OF PROPOSAL: The Principal Investigator (PI) proposes to analyze chromosomal boundaries in hESC before and after differentiation with a focus on a regulator with well-characterized features and functions. The PI proposes to perform protein localization studies using genes that are necessary for pluripotency. Component analysis of relevant complexes will also be carried out.

INNOVATION AND SIGNIFICANCE: Little information is available regarding the structure of chromatin in undifferentiated hESC and their differentiated progeny. Recently, several hallmark studies have clearly demonstrated that the chromatin structure and organization are critical mediators of ESC fate regulation. Further analyses of epigenetic and chromatin-mediated regulation in ESC are of the highest importance, and are likely to shed important light on how these cells may be controlled towards useful clinical ends. The proposed focus on genes critical for pluripotency is very appropriate, and will likely yield valuable information. The technical aspects of the proposed studies are not particularly innovative or original. However, they are entirely appropriate to address the questions asked.

The hypothesis may prove to be wrong given no preliminary data for this pilot study. Nevertheless, the experiment has a lot of merit. A fall-back option for defining the chromosomal boundaries important for differentiation of hESC has been proposed in the event that the primary approach fail.

STRENGTHS OF THE PROPOSAL: The proposed experiments are very solid, and based on a considerable amount of expertise. The proposed experiments utilize state-of-the-art technology and non-federally approved hESCs. Further experiments will use more directed hESC differentiated progeny. Subsequent studies will ask if the individual members of the chromosomal boundary element complexes are functionally required for binding. These experiments are particularly valuable given the possibility that it may be possible to modulate chromosomal functions in vivo. In summary, this is an outstanding set of proposed experiments that will surely provide invaluable information.

The strength of the proposal is the outstanding track record of the investigator in chromatin biology. The hypothesis is novel and potentially exciting if the data prove the hypothesis correct. The experimental plan is well-controlled, and adequately provides fallback approaches to demonstrate feasibility of the overall plan. This group has the skills to successfully take this project to completion and identify complexes that are altered by cellular differentiation.

WEAKNESSES OF THE PROPOSAL: Some concern resides in the PI’s lack of experience in working with hESC; although he/she has considerable experience with their murine counterparts. There are appropriate collaborative arrangements to alleviate this concern.

A weakness of this proposal is that Aim two is dependent on Aim one. The novelty of Aim one drives the overall enthusiasm for this project. The expertise of the group mitigates some of the concern that a differentiation specific boundary element can be identified.

DISCUSSION: The basis for the involvement of the regulator was a concern. There is no prior evidence for its involvement but this a novel and interesting hypothesis. The productivity of the PI was also brought up as a concern. There is only one publication since 2003 from the primary lab, but a number of good collaborative publications that are relevant and in good journals. Experience in biochemical purification was discussed as an asset for execution of Aim 1.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None