Statement of Benefit to California:
SYNOPSIS: The goal of this proposed research is to understand the transcriptional program of human embryonic stem cells (hESCs) that maintains self-renewal and pluripotency. The approach uses a combination of global analysis of gene expression by microarray hybridization, computational analysis to identify potential regulatory sequences from the expression patterns, and biochemical assays of transcription factor specificity and function. Aim 1 will determine whether one of the transcription factors identified by this approach (NF-Y) regulates self-renewal of hEScs, as proposed. Aim 2 will test whether depletion of NF-Y via RNA interference causes renewal defects, and over-expression of NF-Y enhances renewal for a panel of hESc lines with different self-renewal properties. The second Aim will use biochemical approaches to identify and characterize the transcriptional regulators that bind two novel DNA motifs that computational analyses predict are specific to transcriptional regulation in ES cells. SIGNIFICANCE AND INNOVATION: The proposal is an integrated approach using microarray analysis, novel bioinformatics, and effective genetic manipulations of ES cells to identify new ES cell transcriptional regulators. This is a highly innovative program with promise for practical information on the control of ESC self-renewal and maintenance of pluripotency. This strategy should be applicable to understanding other aspects of hESC biology relevant to ESC-based therapy, such as the identification of regulators for cell-type specific differentiation in vitro. The proposed research has the potential to make contributions to three areas of CIRM emphasis: as the basis for diagnostic tests to evaluate the quality of newly-derived hESCs; to monitor the optimization of culture conditions for expansion of pluripotent human stem cells and for directed differentiation; to decrease ESC tumorigenicity; and possibly, in the long term, to provide the information for formulating a set of factors capable of reprogramming somatic cells from patients toward the ESC fate. The goal of this proposal is the discovery and analysis of novel transcriptional regulators in hESCs using a combination of gene expression, computational, biochemical and molecular genetic strategies. The combined use of various strategies is innovative and likely to provide fundamental insight into new molecular pathways that maintain the stem cell state. STRENGTHS: The main strenghts are (1) the strong prior experience of PI in identifying and analyzing genes specifically expressed in various types of stem cells. (2) Preliminary results deploying the proposed strategy appear to be compelling. Two seemingly significant manuscripts have been submitted for publication. (3) Identification of a set of genes that may regulate pluripotency; includes genes that encode chromatin re-modeling and RNA processing factors. (4) Discovery of novel transcriptional cis-regulatory elements that promote gene expression in murine and human ESCs. One of these motifs is recognized by NF-Y and this transcription factor is important for self renewal of murine ESCs. The PI has the training, expertise and perspective to make substantial contributions. The experimental plans are detailed, insightful and incorporate novel approaches. Plenty of convincing preliminary results demonstrate that much of the required expertise is in hand. A particular strength is the proven effectiveness of a highly inventive approach to identify novel DNA motifs of regulatory regions that drive the expression of genes preferentially in undifferentiated ESCs. One of these motifs is the binding site for NF-Y, which the PI has shown to affect hESC replication in culture. Two novel motifs with unknown binding factors were shown to be sufficient to drive gene expression in mouse and human ESCs, with greater activity in self-renewing rather than differentiated ES cells. This initial success indicates strongly that these new motifs may be important regulators of the hESC program. The PI has assembled an outstanding group of researchers and collaborators to help ensure success of the proposed studies. Assistant Professor Michael McManus, also in the Diabetes Center, will lend his extensive expertise with lentiviral delivery of RNAi to ESCs. Associate Professor Hao Li will continue his effective 2-year collaboration for bioinformatics of microarray analysis, genome comparisons and enhancer predictions which has led to the identification of NF-Y and the two novel motifs. The Co-Director of the Human ES Cell Biology Center Renee Reij Pera will provide expertise for the growth and manipulation of hESCs. Professor Susan Fisher will assist in the proteomic identification of the DNA-binding factors. If necessary, the Peter Walter group will lend their expertise in yeast genetics and one-hybrid technology to identify transcription factors for the new motifs. Postdoc Marica Grskovic, with proven success with the biochemistry of RNA-binding proteins, will lead the search for the new transcription factors. This is an outstanding environment conducive to the meaningful application of discoveries concerning human stem cell biology, especially those with relevance to diabetes. WEAKNESSES: One reviewer suggested that new genes need to be integrated into emerging gene regulatory networks that are operative in ESCs. The reviewer felt that there could be better delineation of roles of genes in cell survival, self renewal and maintenance of pluripotency. Another reviewer pointed out that although the proposed work seems too ambitious for the two-year time period, the insightful, detailed experimental plans with excellent preliminary results and recruitment of committed collaborators indicates that meaningful progress will be made. Although RNAi technology now seems applicable to hESCs, it would be useful to know how off-target and nonspecific effects will be controlled for in these experiments. Identification of DNA-binding factors for the two novel motifs will be difficult, especially with the relatively small amounts of hESCs that will be available. Nonetheless, the recruitment of Dr. Fisher for proteomic analysis and the prior experience of the researcher dedicated to this aim substantially increases the likelihood of identifying binding factors. It will also be difficult to verify convincingly that the identified factors mediate the activity of the motif for ES cells in situ. DISCUSSION: This promising young PI (a "star" from the Melton group) has submitted an exceptional grant proposal that has great potential to uncover important new elements of stem cell biology. The strategy will facilitate the assembly of a large set of transcription factor networks for differentiation combined with real developmental and molecular biology using hESCs as a model. The approach is inventive and proven effective for identifying regulators of stemness, an issue that is central to stem cell biology. The PI is a clear leader in the field as the first author of a paper describing stemness footprints, and has a collaborative history with key (credible) collaborators. There were a few minor technical weaknesses, but this work has the most potential to discover new elements of stem cell biology. CIRM needs to see and fund more applications like this.