Characterization of factors involved in the regulation of chromatin structure and human stem cell pluripotency
Unlike the majority of cells in the human body, stem cells can give rise to a wide variety of different cell types. This unique property – pluripotency – is the foundation of virtually all proposed uses of stem cells in regenerative medicine. Understanding the mechanisms that regulate stem cell pluripotency will facilitate the development of new approaches for isolating and culturing human stem cells in the laboratory and manipulating their development in the human body. Accordingly, the molecular mechanisms underlying the control of stem cell pluripotency and differentiation are currently a topic of intense interest. Members of the Polycomb group of regulatory proteins play important roles in the maintenance of stem cell pluripotency by repressing genes involved in cellular differentiation. A major question in the field of stem cell biology concerns how Polycomb repression is “switched off” to permit the differentiation of stem cells into specialized cell types. Although the molecular nature of this switch is unknown, genetic studies in simple model organisms have suggested that the human CHD7 protein plays a critical role in this process by counteracting Polycomb repression. The goal of our research is to test this hypothesis and determine the role of CHD7 in the control of stem cell pluripotency and differentiation. The proposed research may lead to novel approaches for the development of diagnostics, tools and therapies. If our hypothesis about the role of CHD7 in human stem cell development is correct, drugs that interfere with CHD7 function could be used to maintain or regulate stem cell pluripotency, either in culture or in patients. Such drugs might also prove extremely useful for creating new stem cell lines for use in regenerative medicine. An added benefit of our research is that it may suggest new treatments for CHARGE syndrome, a serious development that affects one in eight thousand births.
Statement of Benefit to California:
Pluripotency – the ability to differentiate into a seemingly unlimited variety of cell types – is a distinguishing feature of stem cells and the foundation of their potential uses in regenerative medicine. Recent studies have suggested that the human CHD7 protein may function as a genetic switch that triggers the differentiation of stem cells into specialized cell types. The information gained from the proposed research on CHD7 may facilitate the creation of new stem cell lines as well as new approaches for regulating stem cell pluripotency and differentiation in both the laboratory and the human body. In addition, the proposed research may permit the development of more effective treatments for CHARGE syndrome, a serious developmental disorder resulting from mutations in CHD7 that affects one in 8,000 births.
The long-term goal of this proposed research is to understand the role of Trithorax group chromatin regulators in the control of stem cell pluripotency and early differentiation. Specifically, three aims will test whether the chromatin-remodeling factor CHD7/Kismet promotes stem cell differentiation of cultured hES cells by counteracting the repression mediated by Polycomb complexes. The first Aim will determine whether changes in the level of CHD7 correlates with induction of hESC differentiation. Aim 2 will test whether forced expression of CHD7 can initiate the expression of differentiation markers. Aim 3 will use loss-of-function experiments to determine whether reduced expression of CHD7 in hESCs can prevent the induction of differentiation markers while maintaining the expression of genes responsible for stem cell propagation and pluripotency. The results are expected to determine whether CHD7 is an important player and to identify potential target genes. SIGNIFICANCE AND INNOVATION: Considerable recent evidence indicates that heritable changes in chromatin structure control the choice between maintenance of stem cell identity (including the ability to self-renew while maintaining pluripotency) and the initiation of differentiation. An understanding of the molecular and genetic mechanism of this critical decision will be essential for designing effective in vitro protocols for expanding human stem cell populations and switching their phenotype toward differentiated functions useful for a wide variety of therapeutic applications. CDH7/Kismet is a chromatin activator that is likely involved in the switch from stem cell renewal to programming differentiation, and so a test of its role and an analysis of its mechanism of action is highly relevant to the goals of stem cell technology. The innovation in this proposal lies in the insight into CDH7 function and the novelty of using such a regulator to control differentiation of cultured human ES cells. STRENGTHS: The proposed research address a central issue of stem cell biology: the mechanism controlling the transition between stem cell renewal and cell-fate decisions. The experimental plan uses standard approaches to ask fundamental questions regarding the role of CHD7, a potentially important regulator of stem cell identity. The association of heterozygosity at the CHD7 locus with human developmental abnormalities shows that CHD7 plays an important role in human development. This application illustrates the direct value of cross-over from discoveries in Drosophila development to human stem cell biology and the opportunity to bring the special insights of a young and accomplished investigator to this field. WEAKNESSES: Although CHD7 is a promising potential regulator of stem cell identity, a project focused on this one candidate may not turn out to be productive. As yet there is no compelling evidence that CHD7 is important in stem cells, rather than later developmental decisions. Some of the properties of Kismet/CHD7 suggest that it may direct the consequences of the decision to switch from renewal to differentiation rather than participate in the decision. Preliminary information implicating CHD7 in the decision itself would be helpful. Documentation of the necessary expertise within the nascent stem cell facility to grow and differentiate human ES cells and to transduce them with recombinant lentivirus would strengthen this application. DISCUSSION: Would recommend analysis of potential targets by Chip-on-chip and study of histone modifications at selected genes (eg nanog, oct 4, gata6, etc)