Generation of induced pluripotent stem cells (iPSCs) from somatic cells through cellular reprogramming offers tremendous potential for therapeutics, the study of disease states, and elucidation of developmental processes. Central to the process of generating a pluripotent cell from a somatic cell is an energy-dependent epigenetic reconfiguration event that must occur to produce iPSCs with characteristics similar to embryonic stem cells. However, the identity of nuclear factors that activate the metabolic programs linked to pluripotency and their contribution to epigenetic reprogramming remains largely unclear. Our lab is known for its discovery of the family of nuclear hormone receptors (NHRs) that use hormones to control genes and thereby regulate embryonic development, physiology and metabolism. Utilizing our specialized NHR knowledge and tools we identified a sub-population of cells that arise early in reprogramming that transiently express the NHR Estrogen Related Receptor alpha (ERRα). These rare cells provide the principal reservoir from which iPSC cells are produced.
Utilizing this newly identified cell population, we will determine the metabolic pathways during cellular reprogramming that reestablish ES cell-like chromatin patterns. Understanding the mechanism of epigenetic resetting could be exploited to deal with adult diseases such as cancer or even in ‘rejuvenating’ aged cells.
Our focus is on nuclear hormone receptors (NRs) that use hormones to control genes that regulate development, growth, and physiology. This has brought in more than $100M in private and federal funding to this lab over the last 30 years and has led to employment of 150+ people, publication of over 350 papers, and founding of three biotech companies that in aggregate raised more than $1B in research and development support. Several FDA-approved drugs for cancer, diabetes, osteoporosis, and leukopenia were developed with this technology. We found that a unique subset of 38 NRs are expressed in adipose-derived human induced pluripotent stem cells (hiPSCs), but little is known of how they control stem cell renewal and differentiation. Potential use of the extensive family of hormonal ligands to control iPSC generation, maintenance, and cell fate has profound implications for regenerative medicine. We wish to use our expertise to understand how NRs can be exploited to accelerate the use of iPSCs in regenerative medicine. Our proposed study should be beneficial to the State of California and its citizens in several ways: 1) by maintaining a unique training environment for students, postdocs and physicians; 2) discovering how to more efficiently generate and use human iPSCs; 3) deciphering the molecular genetic logic of nuclear reprogramming; 4) determining how a pharmacopeia of hormones and drugs can be brought to bear on directing stem cell renewal, differentiation, and therapy.
The major goal of this study is to determine the role of a nuclear hormone receptor, the estrogen-related receptor alpha (ERRα) in mediating the metabolic and epigenetic changes required during early reprogramming of human cells. Our recent findings have shown that in mice, its homologue ERRγ, is essential in mediating reprogramming. Gene expression analysis performed in human fibroblast cells indicated that in human cells, it is ERRα instead of ERRγ that is critical for initiation of reprogramming. Thus we proposed to specifically isolate the ERR-transiently expressing (tERRα) cells in early reprogramming and examine their properties in detail.
The first step towards achieving our goals is to overcome the difficulty in identifying and isolating sufficient quantities of tERRα cells for our proposed experiments. To do this, we first designed and tested various ways to isolate these cells using different reporter systems. We identified sequences in the human ERRα promoter region that allowed us to detect ERRα activity in vivo. After optimization of our reporter system, we confirmed that only a small percentage of reprogramming cells (in this case human fibroblasts) exhibit high ERRα expression. This small subpopulation of cells exhibits a detectably higher level of ERRα and its downstream targets, many of which are key metabolic enzymes, than the remaining population.
This demonstrates that we have optimized an efficient reprogramming system that will allow large-scale isolation of tERRα cells for our proposed genome-wide studies.
Our next steps will be to focus on the characterization of these tERRα cells. We will perform time-course RNA-Seq on tERRα cells and their counterparts, to determine the gene expression signatures of these cells. We will also examine whether these cells have a higher reprogramming efficiency compared to the rest of the cells. Furthermore, we will determine the epigenomic landscape of these cells by performing chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq) on isolated tERRα cells and compare this to the rest of the reprogramming population. This should provide valuable insights into the role of ERRα in regulation of metabolic and epigenetic changes required for reprogramming of human cells.
The major goal of this study is to determine the role of ERRα in mediating the metabolic switch and epigenetic changes during early reprogramming of human cells. In the human fibroblast, it is ERRα instead of ERRγ that is critical for initiation of reprogramming. Thus we proposed to specifically isolate the ERRα-transiently expressing (tERRα) cells in early reprogramming and examine their properties in detail.
In order to systematically characterize the tERRα cells, we designed and tested various ways to isolate these cells via different reporters. We found that the 1kb sequence immediately upstream of the human ERRα transcription start site is sufficient to recapitulate ERRα activity. As expected, only a small percentage of reprogramming cells (human IMR90 fibroblasts) exhibit high ERRα expression. These cells express ERRα and its downstream targets, many of which are key metabolic enzymes, at a higher level when compared to the ERRα low population. This demonstrates that we have optimized an efficient reprogramming system to allow large-scale isolation for genome-wide studies.
By using time-resolved RNA-Seq on tERRα cells and their counterparts, we successfully characterized the genome-wide transcriptomic dynamics in tERRα cells. Confirming our hypothesis, we found that these cells are undergoing a dramatic mesenchymal-to-epithelial transition, and exhibit a unique metabolic profile. Furthermore, we have charted the promoter and enhancer landscape by investigating the dynamic changes in H3K4me2 (a key transition histone mark) in tERRα cells compared to control cells. We identified significant changes in H3K4me2 levels at genes involved in development, including several key reprogramming genes, suggesting that the tERRα cells are undergoing extensive epigenetic reprogramming. Motif analysis reveals enrichment of optimal binding sites for ERRα, as well as other pluripotency factors, at up-regulated H3K4me2 peaks, suggesting the direct role of ERRα in mediating the epigenetic changes.
We will continue to investigate the dynamics of metabolite changes in tERRα cells. Additional genome-wide assays will also be performed to investigate other epigenetic markers changes in tERRα cells. This should provide valuable insights into the role of ERRα in regulation of metabolic and epigenetic changes required for reprogramming of