The human body is composed of thousands of cell types, which all came originally from embryonic stem cells. Although all these cell types have the same genetic blueprint, different genes are active in different cells in order to give each its distinctiveness. The process by which the genes remember whether they are in liver, brain, or skin cells is called “epigenetics.” A central problem in regenerative medicine is to understand the epigenetic program so that human embryonic stem cells can be efficiently turned into the cell types required for each specific patient. Conversely, by manipulating the epigenetic program, adult cells may be reprogrammed into primitive cells that can turn into other cell types to repair diseased or damaged tissues.
The goal of the proposed research is to better understand the epigenetic program in human embryonic stem cells and adult cells. We want to tap into the natural mechanisms by which the body normally “remembers” what kinds of cells reside in each tissue and apply them to regenerative therapies. Specifically, the research will study the roles of a newly discovered type of genes, termed “noncoding RNAs”, in stem cell epigenetics.
A better understanding of how cells remember their own fates can improve regenerative medicine in several ways. First, by appreciating the roles of noncoding RNAs in this process, specific noncoding RNAs can be used as markers to track and predict when cells are acquiring or forgetting specific cell fates. For instance, it may be possible to learn from the pattern of noncoding RNAs that an embryonic stem cell is ready to become brain cells, which can be used to treat a patient with stroke. Second, beyond tracking cell fate, noncoding RNAs may be used to directly manipulate stem or adult cell fates. By introducing noncoding RNAs from different cell types, embryonic stem cells or adult cells may be directly reprogrammed into the desired cell type. While these potential application are far in the future, we believe that better knowledge of this new level of gene regulation will one day lead to more facile and efficient manipulation of cell fates for regenerative medicine.
The proposed research can benefit the state of the California in three ways. First, the research will generate important knowledge on new ways to manipulate cell fate potentials of stem cells and mature adult cells. The focus of this research is to explore the genetic circuitry that locks cells into particular fates, whether it is to become skin, muscle, or brain. Better understanding of these circuitries could allow human embryonic stem cells to be directed to become particular tissues—and remember such instructions permanently. Alternatively, interference with these circuitries could allow adult cells to be reprogrammed into stem cells, where they can be used to generate damaged tissues. This information could speed the development of regenerative medicine in California, benefiting patients with currently untreatable diseases.
Second, the proposed research will generate new tools for stem cell research and regenerative medicine. As a direct result of this work, we will provide a complete genetic and epigenetic characterization of some of the first human embryonic stem cells created in California. This information will allow future investigators, physicians, and potential patients to better utilize them in research and therapy, or conversely appreciate potential limitations or risks associated with these embryonic stem cell lines. Moreover, we are likely to generate derivatives of these embryonic stem cell lines that have altered potential to become specific cell types. Such cell lines with properties of “directed differentiation” may be particularly useful for treatment of diseases where deficits of specific cell types are known.
Finally, the proposed research will train young scientists to become skilled in human stem cell research. Graduate Ph.D. students and postdoctoral fellows in this California-based institution will gain the hands-on experience and expertise of manipulating human stem cells and of reprogramming adult cells. The training and experience of these young scientists will prepare them to develop new regenerative therapies, launch new companies based on stem cells, or teach future students about regenerative medicine. Creating a cadre of well-trained individuals would be a vital step toward making California a central hub for regenerative medicine.
SYNOPSIS: Expression of lineage specific genes in human embryonic stem (ES) cells, and conversely maintenance of pluripotency, is controlled in part by histone modification, particularly H3K27 methylation. Recent data show that non-coding RNAs (ncRNAs) may target histone methyltransferases (and perhaps demethylases) to specific genes. The two best described examples are XIST on the X chromosome and HOTAIR at the HOXD locus. Both bind to the Polycomb Repressor Complex (PRC), which is a histone methyltransferase and repressor of gene expression. The general goal of this proposal is to explore the roles of ncRNAs in determining human ES cell pluripotency and cell fate determination. The Principal Investigator (PI), an Assistant Professor of Dermatology at Stanford, proposes to study the role of ncRNAs in epigenetic regulation, specifically H3K27 methylases and demethylases. The ncRNA called HOTAIR has been shown to bind polycomb PRC2 and guide PRC2 in trans to silence HOX genes. The PI proposes to identify ncRNAs complexed with H3K27 methylases and demethylases in hES cells and more differentiated cells, and then examine the genomic targets. In Aim 1 he will use a battery of antibodies to various PRC associated components to isolate complexes and then compare the ncRNAs that are obtained. He will do this with hES cells and ES cells differentiated to endoderm/ectoderm (~90% purity).Retrieved RNAs will be identified by use of high density tiling arrays (termed RIP-chip). In Aim 2 the functional significance of these ncRNAs will be studied by mapping the genome wide sites to which they bind, and by RNAi strategies designed to knockdown expression of both the ncRNAs and the chromatin modifying factors they interact with.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The control of epigenetic regulation in ES cells is important and relevant to pluripotency and reprogramming, and in controlling lineage specific gene expression. This proposal contains very well-designed studies and is innovative in terms of its technology and focus on ncRNAs. The PI has an outstanding track record and is a pioneer in this area, having just published in CELL the discovery of the only ncRNA other than XIST that may function by recruiting chromatin-modifying enzymes to control gene expression during development. The PI is highly experienced in chip-based technologies and has excellent collaborators; if these experiments are going to be successful he is the one to get them done. In the first aim, different subunits of two histone methyltransferase and two histone demethylase complexes will be examined for ncRNA binding in embryonic and somatic cell lines. This important aim addresses a significant question as it may identify novel ncRNAs and reveal their potential role in cellular differentiation. Aim 2, which proposes to characterize the genomic binding sites of newly identified ncRNA(s), will be critical for understanding their role in directing cell fate and controlling gene expression. Most of the tools and expertise are already in hand to perform the experiments proposed, and other technology is provided by collaborators.
This is certainly an emerging area of great interest, and one minor criticism might be that the study of ncRNAs is in its infancy with respect to PRC2 function; even in the case of HOTAIR, the mechanism of action is still uncertain, and it is somewhat unclear how far the PI will be able to take these studies. There is a diversity of mechanisms for recruiting chromatin modifying enzymes to gene promoters, such as via their interactions with other transcriptional regulatory proteins, and it may be too soon to tell how important ncRNAs will be relative to these other pathways. It could be argued that a more cautious approach would be to conduct pilot studies of the type described here, but on a smaller scale, in order to first demonstrate the possible validity of this model to gene regulation in ES cells. Going “all in” with a large scale, comprehensive attack is bold, but possibly premature given how little of the underlying biology is understood. Will these ncRNAs prove to be critical? Are 20 ncRNAs sufficient to get major insights? Are hESCs the best test system for study of ncRNAs? Would the mouse be more tractable at this time?
It might have been good to propose some experiments on the existing ncRNAs (e.g. HOTAIR) in case no other ncRNAs are identified, in which case both aims would not be very informative. However, if the PI’s prior experience with the HOX locus is any guide it is almost certain that many hundreds of ncRNAs will be identified by the approaches outlined in this proposal. In fact, little is said about how he would sort through this potentially vast array of ncRNAs for those 20 that will be studied at a functional level in Aim 2. Two other minor concerns were noted. First, as noted by the applicant, Aim 1 depends on having highly specific antibodies for immunoprecipitating components of the histone methylase and demethylase complexes. One reviewer who looked at the references provided in conjunction with the Table listing the ten target proteins and antibodies found that only two of the ten antibodies are described, and there are no specific details about how these apparently unpublished antibodies will be quality-controlled. These reagents are essential for the proposed experiments, and it is difficult to evaluate their feasibility without additional information about their utility and specificity. Second, little detail is given on the RNAi methodology of depleting ncRNAs in human cells and on the feasibility of the RAG-Chip approach. Preliminary data on the identification of the Hox cluster with HOTAIR as a bait for RAG-Chip would have strengthened the proposal.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI is a physician-scientist who obtained his MD and PhD degrees from Harvard and MIT in 2000, having done his graduate work with David Baltimore. His postdoctoral studies were performed with Pat Brown at Stanford. At the conclusion of his postdoctoral fellowship, he was appointed an Assistant Professor in the Department of Dermatology at Stanford, and in 2005 he became a member of the Stanford Comprehensive Cancer Center. His CV is excellent - he is very productive, has numerous first and now senior author papers in the top journals, and he has been a Damon Runyon Scholar and now an ACS Research Scholar. His current support is very extensive, with two RO1 grants, a KO8 and many more. His lab is tackling diverse problems in gene regulation, and is very well funded to do that. In addition, he is an active dermatologist. There is some question as to whether another large scale project would be appropriate at this stage, and the comments above regarding a pilot scale approach might also be considered in this context. The PI has outlined an excellent plan for his career, the environment is excellent, and he has excellent collaborators and mentors. This investigator may well become a leader in the field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institutional commitment is superb. They are providing excellent support to the PI, and have a superb track record with its trainees. While no specific comments are made in the letter are made about ongoing support, the PI’s lab space is very adequate, his interactions with colleagues outstanding, and the shared resources are outstanding.
DISCUSSION: This was considered an excellent, innovative, and nicely written proposal from a PI with excellent training and an excellent team of collaborators. The only real issue is how many ncRNAs will he find? Is human the right system, or should he look in the mouse first? These are only minor concerns as this PI, a physician-scientist who studied with David Baltimore and Pat Brown, is the type of investigator that we want to fund.