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:
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.
SYNOPSIS: The PI proposes to analyze chromosomal boundaries in hES cells before and after differentiation. Specifically, the potential role of the CCCTC binding factor (CTCF). This protein regulates chromosomal domains in the beta globin family locus, the c-myc locus, and in imprinted gene loci. The CTCF protein contains 11 zinc fingers, and likely represents a versatile DNA binding molecule with distinct, locus-specific binding properties. The PI proposes to perform ChiP on Chip studies to analyze the localization of CTCF, as well as histone modifications in a locus the contains Nanog, Stella, and GDF3 on chromosome 12. These genes are necessary for pluripotency. The goal is to identify the boundary elements that flank this locus. In the second Aim, Mass Spectrometry will be employed to identify the compositions of ctcf protein complexes at the boundaries of the Nanog locus. Composition will be determined before and after hES cell differentiation. SIGNIFICANCE AND INNOVATION: Little information is available regarding the structure of chromatin in undifferentiated hES cells and their differentiated progeny. Recently, several hallmark studies have clearly demonstrated that the chromatin structure and organization are critical mediators of ES cell fate regulation. Further analyses of epigenetic and chromatin-mediated regulation in ES cells are of the highest importance, and are likely to shed important light on how these cells may be controlled towards useful clinical ends. The experiments in this proposal focus on chromosomal domain boundaries, and how these are set by CTCF, a protein known to play such a role in a number of other systems. The focus on a genetic locus (12p13) containing three genes critical for pluripotency is very appropriate, and will likely yield valuable information. The PI will identify boundary elements or insulators that flank the Nanog locus. The hypothesis is that these elements are critical in keeping this locus on in hES cells, and possibly important in turning the locus off after differentiation. The technical aspects of the proposed studies are not particularly innovative or original. However, they are entirely appropriate to address the questions asked. This proposal seeks to define the boundary elements in chromatin that mediate nanog expression using chromatin immunoprecipitation and tiling arrays. The specific hypothesis being tested is that the boundary element CTCF may regulate boundary formation during differentiation. This hypothesis is innovative, however may prove to be wrong given no preliminary data for this pilot study. Nevertheless, the experiment is novel and has a lot of merit based on the notion that CTCF mediates DNA methylation dependent gene expressions changes, and nanog is known to be under epigenetic control. As a fall-back option for defining the nanog boundary during differentiation of hES cells, the investigator proposes to utilize histone modifications as an unbiased surrogate for open and closed chromatin. The second aim is dependent upon finding a boundary element in aim one. The investigator proposes to utilize this sequence to biochemically purify and characterize protein complexes bound to these target sites in undifferentiated and differentiated cells as a means to identify subunits that selectively regulate the nanog chromatin domain. By identifying such a factor, the group proposes they will seek to reprogram cells by selectively regulating nanog expression, rather than utilizing a broader approach such as CTCF knockdown that has confounding effects on cell growth. STRENGTHS: The proposed experiments are very solid, and based on a considerable amount of expertise. In the first Aim ChIP on Chip techniques will be used to identify CTCF binding regions on chromosome 12. Nimblegen arrays will be used. The entire human chromosome 12 is represented by two chips at a 100 bp resolution. These arrays are state-of-the-art for these kinds of studies. The PI will design a custom Nimblegen array the contains the 12p13 region aas well as flanking regions. ChIP on Chip will be performed using hES cells derived by Dr. Melton's laboratory, and on control fibroblast cells. The material immunoprecipitates with the CTCF antibody will be analysed in 2-color hybridiaztions by Nimblegen. The potential undifferentiated cell specific binding sites will be confirmed in EBs. Further experiments will use more directed hES cell differentiated progeny. There is a very high likelihood that numerous CTCF-binding sites will be identified around the Nanog locus. Further studies will ask if the binding sites constitute actual boundary or insulator regions. This will be accomplished by monitoring histone modifications that flank the CTCF binding sites using ChIP on Chip to monitor modifications that are indicative of active or repressed chromatin loci. Finally, transcription around the 12p13 region will be measured using Nimblegen and Affymatrix arrays. The former have 50 bp resolution and will be useful to detect non-coding transcripts. All of these studies are very well-described, and clearly will provide invaluable information. In the second Aim the PI will employ protein purification to identify the composition of CTCF containing complexes that bind to the boundary elements identified in the previous Aim. Immobilized regions of DNA will be used to capture complexes that contain CTCF. These DNAs will be larger than simply CTCF binding sitessince the goal is to identify the complex within the context of a functional boundary element. The proteins that are in the complex will be identified by Mass Spectrometry following purification by 2D-PAGE. The PI has ample experience in these methodologies, and potential problems and their solutions are well-described. Subsequent studies will ask if the individual members of the Nanog boundary element complexes are functionally required for binding. These experiments will utilize specific antibodies as well as RNAi reagents. The latter are particularly valuable given the possibility that it may be possible to moculate boundary 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 that CTCF may regulate the complex of pluripotency genes on chromosome 12 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. The second aim ties into the strength of the laboratory. Provided a sequence is identified that is meaningful through the results from aim one, this group has the skills to successfully take this project to completion and identify complexes that are altered by cellular differentiation. WEAKNESSES: Some concern resides in the PI's lack of experience in working with hES cells; although s/he 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 CTCF was a concern. There is no prior evidence for its involvement, but a number of CTCF binding sites have been mapped to clusters of genes under epigenetic control, which makes 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.