The reprogramming of skin fibroblasts into pluripotent stem (iPS) cells represents a significant milestone towards the goal of developing stem cell therapies tailored to the patient. However, the reprogramming is slow and inefficient. A key step is the ability of four exogenously introduced transcription factors to turn on or activate the endogenous versions of their own genes, a process termed autoregulation. In fibroblasts, the genes for the endogenous transcription factor genes are turned off because they are assembled into a repressive structure termed silent chromatin. The repressive structure must be removed to activate the endogenous stem cell transcription factor genes. The efficiency by which the endogenous genes are activated is poorly understood. Dozens of different proteins assist the four transcription factors during the reprogramming process. However, little is known of how the dozens of proteins coordinate their actions to remove the silent chromatin structures and replace them with active chromatin. Our goal is to study this process and use the resulting knowledge to improve the efficiency of iPS cell formation.
This process can be studied by recreating it in a test tube or in vitro. This biochemical recreation involves three steps: 1. Generating highly pure versions of the four transcription factors. 2. Reproducing the silent chromatin environment of the gene. 3. And finally, identifying each of the steps involved in reactivating the endogenous genes. We have developed a technology that permits us to biochemically recreate silent chromatin on the transcription factor genes in vitro. We then attach the silent chromatin to magnetic beads, add protein mixtures from disrupted stem cells, and use magnets to capture beads with the attached proteins, which are in the process of converting silent chromatin to active chromatin. This “immobilized template capture” technique also allows us to delineate the steps involved in the reprogramming process. The proteins are identified by a state-of-the-art method known as multidimensional protein identification technology (MuDPIT). By combining the immobilized template and MuDPIT techniques, we will be able to provide detailed knowledge about how autoregulation is achieved. As such, we will be able to determine which specific steps are limiting in the conversion of fibroblast to iPS cells. Knowledge of the mechanism of stem cell gene regulation is essential for fully understanding stem cell self-renewal and the transition of fibroblasts to iPS cells. The information can be utilized to improve the efficiency of iPS cell formation.
Statement of Benefit to California:
California is investing 6 billion dollars in stem cell research with much of the principal being spent in the first 10-15 years on research and development. To date, much research has focused on the therapeutic potential of stem cells and understanding a few fundamental aspects of how stem cells self renew and differentiate. A major gap in our understanding lies in the most basic aspect of stem cell biology, the mechanics by which stem cell genes are regulated. To fully understand and to optimally manipulate the technology, a significant amount of basic knowledge must be obtained.
The lack of basic mechanistic knowledge is what initially limited gene therapy and immunotherapy from achieving clinical application. For example, in gene therapy, we knew how to design therapeutic genes but knew little about how to deliver and regulate them. The situation has improved considerably in recent years, driven not by new technologies per se, but due to an investment in understanding basic mechanisms. We are now starting the same process with stem cells. Our technology and knowledge base have improved considerably but there are large gaps. Few scientists doubt the potential. However, many overestimate our knowledge base and ability to achieve therapeutic goals. It is important that we determine at the most basic level how stem cell genes are regulated.
We know that four gene regulatory factors, when added to skin fibroblasts, can convert these into stem cells. However, we do not know precisely how they do this. To date, CIRM has invested heavily in labs trying to understand the functions of these four transcription factors in living cells. However, in the gene regulation field of biology, many of the major advances derived from biochemical or in vitro studies.
My laboratory works on the fundamental aspects of how genes are regulated. We have recreated key events in a test tube. We have developed new biochemical approaches for determining how stem cell genes are regulated and for understanding how the four transcription factors function when bound to chromatin. Our studies will provide insight into and understanding of the processes by which stem cells self renew and differentiate. As such, the basic knowledge will help further the transition of into stem cells into clinical applications.
This proposal is focused on the molecular events that occur at the promoters of key regulators of pluripotency during reprogramming of induced pluripotent stem cells (iPSCs). The general strategy is to reconstitute the minimal transcriptional machinery, called the “enhanceosome”, on DNA templates representing the promoters of Nanog, Oct4 and Sox2 and then use histone modifications to mimic active or repressive chromatin states. In Aim 1, the applicant proposes to characterize how recombinant Oct4, Sox 2, Klf4, Myc and Nanog proteins interact and assemble into functional enhanceosomes at these promoters. In Aim 2, the applicant will incubate human embryonic stem cell (hESC) extracts with this system and identify co-factors in hESCs required for transcription using multidimensional protein identification technology. Finally, in Aim 3, the applicant proposes to use the information derived from Aims 1 & 2 to improve iPSC derivation.
Reviewers expressed divergent opinions about the significance and potential impact of this proposal. One reviewer found it to be highly important, as little is known about the basic transcriptional mechanisms of reprogramming. This reviewer also found the proposal innovative because, although these technologies have been extensively applied to other transcription factors, the use of the approach in studying cellular reprogramming is novel. However, another reviewer raised significant concerns about the relevance of the “completely artificial” system described in the proposal. This reviewer asserted that minimally recapitulated chromatin structures using artificial DNA and recombinant proteins would be unlikely to yield physiologically relevant data. Furthermore, this reviewer questioned the novelty of the proposal, since most of the proteins that bind to these promoters are already known.
Reviewers agreed that the research plan is feasible but raised a number of issues with the experimental design. They found the preliminary data convincing, demonstrating the ability to produce functional recombinant proteins and induce chemical modification of histones. However, there were concerns about several key assumptions such as whether the modified histones would be appropriately spaced and whether higher order chromatin structure (which would not be recreated in the in vitro system) would be important. Reviewers also questioned the assumption that the pluripotency factors themselves recruit additional factors critical for reprogramming rather than acting by activating other genes that mediate reprogramming; the latter possibility would be missed by the proposed approach. There was also concern that the second specific aim was too open-ended and was not sufficiently supported by preliminary data.
Reviewers praised the applicant as an excellent investigator with a very productive track record and many years of experience studying transcription in vitro. They agreed that the applicant has assembled a strong research team, which is eminently qualified to carry out the proposed experiments.
Overall, reviewers appreciated the mechanistic focus of this proposal and the applicant’s expertise. However they raised a number of significant concerns about the research plan and the physiological relevance of its approach.
A motion was made to move this proposal into Tier 1 in order to add a very basic, discovery research proposal to CIRM’s portfolio. CIRM staff noted that there was a large standard deviation of reviewer scores for this application. The reviewers summarized their opinions about the strengths and weaknesses of the proposal. The motion failed.
The vote of the GWG on the motion to advance this proposal to Tier 1 was 8 For, 9 Against, and 1 Abstention. The minority who supported recommending the proposal for funding felt that the strengths far outweighed the proposal’s weaknesses. Their support for advancement of the proposal to Tier 1 was largely based on the following points:
The PI is an expert in the field and will bring extensive knowledge and experience in analysis of transcriptional complexes to stem cell biology
Proposed research comprises a novel, reductionist approach to study regulation of induced pluripotency
Project has potential to provide a promising discovery tool for the field