Miguel Ramalho-Santos, PhD
Pluripotent stem cells, such as Embryonic Stem (ES) cells, have the fascinating ability to give rise to any cell type of the body. Because of this property, pluripotent stem cells have allowed detailed studies of cellular differentiation and may open new avenues in Medicine, whereby cells are engineered to model or treat diseases. Understanding the regulation of pluripotency is a fundamental biological question with great potential for impact in other fields. We aim to understand the transcriptional regulation of pluripotency.
To begin to understand the transcriptional regulation of pluripotency, we identified and compared the global patterns of gene transcription of ES cells with those of adult stem cells, which are not pluripotent (Ramalho-Santos et al, Science, 2002) . This work provided an important resource for the stem cell community, contributed to the characterization of other stem cells, and lead to the identification of functional regulators of multiple stem cells. We have collaborations with other laboratories to define regulatory modules that are shared by different stem cells. This work also revealed that some genes are preferentially expressed in ES cells relative to other stem cells or differentiated cells, and we are studying these genes in innovative ways.
Transcriptional Regulation of ES cells
Knowledge on how the transcriptional program of ES cells is regulated derived mostly from the identification of the targets of transcription factors previously shown to be important for ES cells, using a technique called chromatin-immunoprecipitation (ChIP). This approach, going from transcription factors to targets, is limited because it requires prior knowledge of the transcription factors to be studied. We decided to reverse this approach, and proceed in an unbiased fashion from targets to transcription factors. We hypothesized that genes preferentially expressed in ES cells were controlled by the same transcription factors. It followed that a search of the non-protein-coding DNA sequences of these genes should allow the identification of the relevant genetic switches, or transcription factor binding sites (TFBS), that regulate their expression. In collaboration with Hao Li’s lab here at UCSF, potential TFBS shared among genes preferentially expressed in ES cells were identified computationally. My lab systematically demonstrated that these putative TFBS are highly active in undifferentiated ES cells, identified the transcription factor that binds to one of these sites and showed that it regulates pluripotency-associated genes and is required for ES cell proliferation (Grskovic et al, PLoS Genetics, 2007) . It should be noted that this innovative and systematic dissection of the regulation of a cohort of genes had not previously been implemented in mammalian genomes. We are currently using genetic and biochemical approaches to identify other transcription factors that bind to the motifs. Integration of the data is expected to reveal the transcriptional networks that underlie ES cell pluripotency.
We also developed very powerful genetic assays to dissect pluripotency. We implemented a quantitative RNA interference (RNAi) screen in ES cells. My lab has to date identified 7 putative transcriptional regulators of ES cells that had not previously been implicated in ES cell biology. For example, we discovered that a chromatin remodeling enzyme maintains an open, transcription factor-accessible chromatin state in ES cells and is required for pluripotency. We are characterizing in greater detail the role of these new regulators of ES cells.
Dissection of Cell Reprogramming
A fascinating aspect of pluripotency is that it can be “re-awakened” in differentiated cells by experimental manipulation. Reprogramming of cells to pluripotency allows an investigation of how the differentiated cell state is maintained, and how it can be subverted. Therefore, reprogramming may lead to new approaches in Regenerative Medicine, such as the generation of disease- or patient-specific pluripotent stem cells, and may provide insight into the process of cellular transformation in cancer. We have developed improved methods for reprogramming somatic cells to pluripotency, using fusion with ES cells or over-expression of transcription factors (Blelloch et al, Cell Stem Cell, 2007). My lab is using these assays to dissected the molecular mechanisms that underlie reprogramming. We are also interested in how the state of the differentiated cell affects its ability to be reprogrammed.
Significance of Pluripotency In Vivo
Pluripotent stem cells are cultured and predominantly studied in vitro. However, pluripotency must have evolved for specific functions in the context of the developing embryo, and therefore it is important to investigate the significance of pluripotency in vivo. To begin to do this, we improved methods for analyzing global gene expression patterns from low numbers of cells using microarrays, and recently carried out a comprehensive analysis of the transcriptional program of mouse pluripotent cells in vivo. Our analysis reveals that there is a remarkable global maintenance of the transcriptional profile of pluripotency in the embryonic germline, long after the embryo has began to differentiate all the major tissues. We are using innovative genetic approaches in the mouse embryo to investigate the role of regulators of pluripotency in the germline.