The genetic information contained in all human cells is spatially organized in a very precise manner and further 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 and often changing their physical location within the nucleus (which contain our genes) of a cell. This allows gene A to be turned off and genes B and C to start working. Importantly, these “fences” and interactions with other components in the nucleus can control large numbers of genes that regulate critical cellular processes. We have defined the borders/fences of a genetic region (NANOG-STELLAR-GDF3) that is important to maintain human stem cells (hESCs) in their most plastic state that is, having the ability to become any other cell type. To understand how the fences are opened or closed, we identified a critical protein called CTCF that interacts with the fences and serves as a “latch” to keep the NANOG gene working or resting in hESCs or differentiated cells. We further demonstrated that CTCF is necessary for both NANOG gene activity and hESC pluripotency.
We now propose to examine the broader role of CTCF in maintaining the proper physical organization/positioning of pluripotent genes within the hESC nucleus. Although critical genes have been identified in stem cells, their spatial organization, which regulates their coordinated activity, has not been determined. We will evaluate the role of CTCF in nuclear positioning and chromosomal interactions of several pluripotency genes in hESCs and measure how this organization changes upon stem cell differentiation when pluripotent genes like NANOG are shut off and new genes are turned on to confer a specific cell fate. We will also examine the relationship between CTCF and a specific signaling pathway that is essential to maintain pluripotency in the activation of their common target genes. Importantly, we will compare CTCF-dependent gene organization in hESCs with that of human induced pluripotent stem cells (hiPSCs) to evaluate how closely a “reprogrammed” nucleus actually resembles that of a bona fide stem cell. This information will be valuable before taking hESCs or hiPSC-based therapies to the clinic. We hypothesize that modulating CTCF function will significantly impact gene positioning and may redirect gene expression programs to promote pluripotency or differentiation. Such targeted changes in nuclear organization represent a novel approach to regenerative therapies by potentially improving the efficiency and accuracy of inducing reprogrammed somatic cells.
Statement of Benefit to California:
It is widely believed that the manner in which genes are organized or positioned within cells determines whether they are active or inactive. Human embryonic stem cells (hESCs) maintain their genetic information (genome) in a very unique organization so that genes can always be activated, which enables hESCs to develop into a wide variety of tissues (pluripotency). Although critical genes have been identified in stem cells, their spatial organization and unique chromosomal interactions, which regulate their coordinated activity, have not been determined. Our research will produce a comprehensive analysis of gene organization in hESCs and determine how these structures change when hESCs lose pluripotency and become a specific cell type. Significantly, we will perform the same analyses in human induced pluripotent stem cells (hiPSCs) to compare how accurately gene organization and chromosomal interactions are recreated in adult cells that have been “reprogrammed” to possess stem cell-like properties.
Reprogrammed adult cells (hiPSCs) have potentially immense therapeutic value for regenerative medicine due to their capacity to repair or replace damaged tissues. Of fundamental importance to achieving the full promise of stem cell therapy is the ability to produce hiPSCs with high efficiency that are physiologically functional as stem-like cells without acquiring the chromosomal instability associated with tumorigenesis. Towards this goal, we will experimentally manipulate gene organization in adult cells to more closely resemble that found in hESCs. We expect this to facilitate activation of critical genes and potentially increase the efficiency and pluripotent competence of hiPSCs while preserving chromosomal integrity, making the cells safer for use in humans. Such targeted changes in gene organization represent a novel approach to generate therapeutically valuable reprogrammed human adult cells. Our studies will also provide insight into genetic elements ("fences" or boundaries/insulators)that are required to organize human genes and chromosomes into functional units. These genetic elements can, in turn, be inserted into recombinant DNA vectors to improve their effectiveness of delivering genes that reprogram adult cells into hiPSCs.
The project is concerned with understanding how structural changes in chromosomal domains in human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) contribute to alterations in gene expression that promote pluripotency or development. The proposal is focused on CTCF, a chromatin insulator protein that appears to organize the genome into functional transcriptional units within nuclei. The first specific aim is to investigate how CTCF influences nuclear positioning of pluripotency genes in pluripotent and differentiated cells. The second aim is to analyze the relationship between CTCF and its associated cofactors with specific structures in the cell nucleus. The third specific aim is to examine genome organization patterns in hiPSCs and investigate whether manipulation of CTCF complexes influences cellular reprogramming.
Significance and Innovation:
- The project’s focus on understanding genome organization in hESCs and hiPSCs may provide new insights into reprogramming and differentiation.
- The proposed experiments will test an interesting and novel hypothesis that changes in CTCF complex composition in different cell types mediates cell-specific changes in chromatin organization.
- Modulating CTCF function may provide an innovative and safer approach to regulating hESC and hiPSC behavior.
Feasibility and Experimental Design:
- The study is supported by strong, appropriate, and convincing preliminary data.
- All experiments appear well developed with precise methodology, outcome analysis and clearly stated alternative plans.
- Reviewers found the proposed studies to be mostly descriptive with minimal attention to molecular mechanisms.
- The experimental plan did not adequately consider dynamics of the differentiation process or propose to assess changes over a series of time points.
- Reviewers expressed concern about the third aim, which they found too preliminary and lacking in a convincing rationale for manipulation of CTCF. Reviewers were also not convinced that retroviral overexpression of CTCF, which is already ubiquitously expressed, would be informative. It was suggested that the investigators might consider instead performing knockdown studies of CTCF binding partners.
Principal Investigator (PI) and Research Team:
- The PI has considerable expertise in transcriptional regulation and chromatin remodeling with a highly productive track record.
- Other research team members have complementary expertise and appropriate experience.
Responsiveness to the RFA:
- The project is responsive in its focus on a basic cellular regulatory phenomenon in human stem cells.