Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: The focus of this proposal is to explore the possible biological role of different HDACs and associated cofactors in pluripotency and self-renewal of hESCs. HDACs are histone deacetylases that are believed to play a role in chromatin and transcriptional regulation. The PI’s laboratory has been involved with the isolation and characterization of human HDACs and has collected 18 of them. This proposal has three specific aims. The first is to evaluate the contribution of HDACs to the differentiation of hESCs into various progenitors. The second is to identify gene targets of individual HDACs in hESCs using microarrays. The third specific aim will identify the cellular partners of individual HDACs in hESCs using immunoprecipitation and proteomics. SIGNIFICANCE AND INNOVATION: One of the main challenges to moving from the discovery of human embryonic stem cells into the development of therapeutic products is to understand how to control and modify the pathways that control both the pluripotent state and the various states of differentiation. Epigenetic modification via chromatin remodeling has been shown to be a very important way to modulate the state of differentiation. This investigator, who is an expert in the field of chromatin remodelling via histone deacetylases (HDAC), proposes a series of studies that will provide much needed information regarding the role of HDACs in maintaining the undifferentiated pluripotent state and their role in driving differentiation. He also proposes to go beyond the analysis of HDAC expression alone, to include studies of the additional transcription factors (positive and negative) that are recruited due to the chromatin changes, and finally, to correlate expression of specific HDACs (either in a positive way or via knockdown strategy using shRNA) with in vivo differentiation into teratocarcinomas. These studies are not innovative per se, in that they are using reagents and methods that have been applied to many other cell types, but they are innovative in the application of these analyses to hESCs. The results of the studies could provide important information about the pathways and key switches in these pathways that would drive differentiation or maintain pluripotency. This type of information will aid in the ultimate development of specific differentiated cell products derived from hESCs. STRENGTHS: The PI is an expert in the study of HDACs and therefore, has the reagents and assays readily available to perform the proposed studies: quantitative RT-PCR, specific antibodies for Western blot analysis, and shRNAs that work to specifically knockdown expression of certain HDACs. Further, their preliminary studies in murine ESC have shown that driving differentiation down the neuronal pathway has resulted in selected loss of expression of certain HDACs, but not all, underscoring the basis of their hypothesis. The use of feeder-free culture conditions allow for a more precise measurement of the influence of the HDACs in the hESCs without the risk of contamination from the feeder cells. The Gladstone Institute has available a core program that supports the use of embronic stem cells by providing methods and reagents to the other investigators at the Institute. In addition, the Gladstone has a core Genomics facility that has experience using the ChIP on CHIP assay that is proposed by the investigators to map the gene targets of HDAC expression. The proposal has linked the mechanistic studies of looking at HDAC expression and the interaction of HDACs with transcription factors and genomic targets with functional assays to understand the phenotypic impact on differentiation when certain HDACs are either down-regulated by shRNAs or constitutively expressed. These types of functional outputs add an important dimension to the proposed studies. In summary, the proposal is well thought out and very feasible, based on the combination of the experience of the PI with the great support core services available through the Gladstone Institute. WEAKNESSES: In addition to the use of the in vivo differentiation assay (essentially, a gross assay for differentiation, by looking at teratoma formation in vivo), it might be easier to measure the impact of HDACs on differentiation if the investigators chose an in vitro pathway of differentiation to study -- much as they have done for their studies on murine ESCs (forcing them to differentiate into neuronal cells). A second concern is that there is a lot of weight put on the first aim. This problem might not be the most pressing issue that needs to be addressed in the first round of seed funds for hESC studies. DISCUSSION: This work is significant in that it deals with chromatin remodeling in gene regulation. The design of the experiments is fine, but tremendous weight is placed in the results of Aim 1. The main criticism is that the experiments rely on the HDAC association assumptions from the preliminary studies in mESC. Reviewers would also like to see more relevant in-vivo assays than teratoma formation.