Basic Biology I
All of the diverse cells in a human body contain the same genetic information, and originally arose from a single cell, a fertilized egg. Embryogenesis is a result of cell division followed by differential gene expression, to selectively activate only the genes needed for development of each specialized cell type. By understanding the multiple gene activities required to either maintain stem cell pluripotency or effect cell specific differentiation, it should be possible to define conditions under which undifferentiated stem cells may be grown in large volume in culture, or induced to become mature cell types of therapeutic interest. The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew or exit the pluripotent state and begin to differentiate. In preliminary studies, our laboratory has identified two protein complexes, SCC-A and SCC-B, required for the activity of genes needed for stem cell self-renewal. Here we propose to characterize the component proteins of these complexes. SCCs are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. We have also identified another protein, TAF3, that may play a central role in influencing whether stem cells self-renew or differentiate into various cell types. Understanding TAF3 function may provide a strategy to better control stem cell differentiation, a necessary step toward tissue replacement therapy. A third focus of the proposal is to understand the impact of eliminating specific stem cell gene regulatory proteins via intracellular protein degradation pathways. Our preliminary data suggest that degradation of proteins crucial to controlling gene expression in pluripotent stem cells is an important regulated step in initiating differentiation. We propose biochemical and molecular biological studies to identify protein degradation pathways involved in maintaining these gene regulatory proteins, or marking them for destruction. Similar to the SCC complexes, these proteins could provide excellent targets for drug design, to increase or decrease the ability of stem cells to divide or differentiate.
Statement of Benefit to California:
The ultimate goal of these studies is the development of therapies for diseases that are fundamentally the result of inappropriate levels of gene expression, cell division and differentiation. The proposed experiments will demonstrate how gene activity is controlled, either to maintain a renewing population of stem cells, or to direct differentiation of specific mature cell types. Because all biological activities; cell division, differentiation and function, are the result of differential gene expression, an understanding of the gene regulatory networks controlling these processes will be crucial for drug development and testing. The proposed experiments will benefit the people of the State of California immediately by supporting the state economy. This project will directly employ three people, supporting two graduate students, and one highly trained Research Specialist. Additionally, it will support the purchase of reagents and services necessary to conduct the proposed research, indirectly contributing to the employment of many other individuals in the biotechnology, education and service industries. In the longer term, this research will benefit the people of California by helping our state to maintain its status as a major force in biomedical research. A thorough understanding of basic stem cell biology will be necessary to support the ultimate development of safe and effective therapeutic applications, and the research efforts toward this end will contribute to the continued success of the outstanding universities and robust biomedical research community that have made California a leader in the fields of biotechnology and medicine. The work described in this proposal will likely reveal gene activities that are essential for the establishment, survival and maintenance of stem cells and differentiated cell populations. This knowledge may potentially identify previously unknown drug targets that will allow the screening of novel classes of pharmeceuticals, or to allow stem cells to be grown in culture as large volumes of homogeneous cells, a necessary prelude to their use in stem cell therapy. Understanding the role of coordinated turnover of transcriptional regulators during stem cell differentiation may also represent a promising new strategy to predictably influence the pluripotent state and differentiation program in stem cells.
This purpose of this proposal is to identify and characterize co-factors that regulate transcription as undifferentiated cells self-renew or exit the pluripotent state as well as to define the proteasome degradation machinery involved in controlling the protein stability of these co-factors. Specifically, the key components of two SCC complexes, SCC-A and SCC-B, will be identified and the potential role of TAF3 in stem cell self-renewal will be investigated. Following identification, the investigator will further characterize these complexes and novel factors by analyzing the biochemical activities and ascertaining their molecular targets using in vitro binding assays. Finally, the cellular machinery regulating protein stability will be examined through identification and isolation of the enzymes that control the degradation of these transcription factors. The reviewers described this proposal as well written and comprised of clear aims and appreciated the molecular focus of the proposal. The potential impact of the proposal was considered significant as data would be generated that could contribute to understanding gene regulation in stem cells, a necessary step to control stem cell differentiation for cell replacement therapies. The innovation of the proposal lies primarily in its focus on the novel co-factors, SCC-A, SCC-B, and TAF3, proposed to regulate gene expression in stem cells. The reviewers expressed several concerns regarding the feasibility of research plan, despite its use of established approaches, and considered the preliminary data and detail provided in the research plan to be generally insufficient. For Aim 1, the preliminary data supporting the role of the SCC complexes in regulating gene expression in stem cells was strong, but the data was from an embryonic carcinoma cell line rather than from human embryonic stem cells (hESCs). No data was presented that the investigator could successfully scale-up hESCs to perform the proposed experiments. Additionally, all the experiments proposed in Aim 1 require that the genes responsible for producing the SCC complex have been identified, and it was not clear from the preliminary data that genes from either of the complexes had been identified. For Aim 2, the preliminary data supporting the role of TAF3 in maintaining pluripotency was weak since appropriate controls were not included, and, for Aim 3, no data was presented. The lack of detail in the proposal was particularly evident in Aims 2 and 3. The proposal did not describe how the large amounts of data generated in Aim 2 would be analyzed or validated nor did it describe how factors from the RNAi screen proposed in Aim 3 would be identified. The reviewers were troubled that the proposal did not discuss any pitfalls or alternative strategies and that the only one of the three specific aims would be conducted in human cells as specified by the RFA. The reviewers universally praised the investigator as a world-class expert in studying mechanisms regulating gene transcription and well on the way to having the same stature in the stem cell field. The environment and investigator were considered outstanding and to be the strength of this proposal. Overall, despite strength of the investigator and the potentially high impact of this proposal, the reviewers concerns regarding the preliminary data and feasibility of the experimental design significantly dampened enthusiasm for this proposal.