The Stat3 pathway in self-renewal pluripotency and the germline

The goal of our research is to understand how certain cells have the ability to make all other types of cell. This property is call pluripotency. Pluripotency normally exists only in cells of the very early embryo. In each generation pluripotency is transiently recreated from the germ cells after fertilisation. It is possible to capture the pluripotent state in the laboratory in the form of stem cells derived either from early embryos or from germ cells. Recently a remarkable discovery has been made that allows scientists to convert normal adult cells back to a pluripotent state by genetic manipulation. A common feature of all truly pluripotent stem cells appears to be regulation by a specific signalling pathway called JAK/Stat. This makes the stem cells responsive to their environment. Scientists maintain pluripotent stem cells by activating this pathway with appropriate signals. The specific purpose of the research proposed here is to identify what activated JAK/Stat does inside pluripotent stem cells to control other genes. We will use technologies based on genome sequencing to identify all genes that are directly controlled by Stat3. We will then study the most interesting of these genes in detail to understand how they contribute to pluripotency. The experiments will primarily be performed in cultured mouse stem cells and developing mouse embryos but we anticipate that the results will be transferable to human. Ultimately we hope to understand in complete detail how pluripotency is created and maintained. This should bring close the use of pluripotent stem cells as a resource for drug testing and for cell therapies.

The broad goals of this project are, firstly to elucidate the molecular mechanism(s) by which activated Stat3 promotes self-renewal and counteracts Smad and Erk (MAPK) differentiation signalling, and secondly to define the function of Stat3 and its key targets in the mammalian epiblast and germline, and during induced reprogramming. Our primary experimental approach to the first goal is to identify direct targets of Stat3 by comprehensive genome-wide analyses based on high throughput sequence analyses of chromatin immunoprecipated DNA (ChIP-seq) and reverse transcribed RNA (RNA-seq). We will exploit a unique reagents developed in the Smith laboratory, namely Stat3 null embryonic stem cells and defined culture conditions, and for the data analyses will use computational tools developed by Bertone. In collaboration with Dr Kathryn Lilley at the Cambridge Centre for Proteomics we will also investigate the protein interactions of Stat3 to elucidate how it interacts with other transcriptional regulators including members of the canonical core pluripotency circuit. The second goal entails precise genetic manipulation of Stat3 itself and identified targets of interest both in ES cells in culture and in pluriptotent and germline cells in vivo. These studies will involve both loss of function by conditional deletion and gain of function by inducible expression. In tandem we will dissect the contribution of Stat3 and its targets to induced reprogramming of somatic cells to authentic pluripotent status (iPS cells).