Understanding how enhancer chromatin transduces extracellular signalling during developmental transitions in human pluripotent cells

A cell's identity is defined by its genes. A cell located in the heart, for example, will express heart genes, but not genes important for liver or brain function. Conversely, liver cells express liver genes but not heart or lung genes. During embryonic development animals are formed by different groups of stem cells, which are able to give rise to the myriad of different cell types found in an adult organism. Cells "know" which cell types they should become because of signals they receive, usually sent by neighbouring cells so that cells in the right place at the right time take on the appropriate identity. Responding appropriately when the levels of these signals reach certain thresholds is crucial not only during development but also in maintaining a healthy state. When these responses fail the consequences include developmental disorders and cancer.
When stem cells undergo the process of differentiating into a new cell type, they need to turn on genes appropriate for the new cell type, but also turn off the genes that define a 'stem cell' identity and also keep off any genes associated with other cell types. This process is tightly regulated by a group of proteins called chromatin remodellers. DNA is not "naked" within cells but exists in the form of chromatin, i.e. it is wrapped around proteins in a way that keeps it compact and stable. Chromatin remodellers are able to change how tightly packed specific regulatory parts of the chromatin are. In this way chromatin remodellers are important for making some genes available to respond if the right signal is received by a cell, while other genes are kept silent.
We are interested in how the signals which enter a cell are interpreted to produce a change in gene expression. We recently discovered a previously undetected step in an otherwise well studied signalling pathway in mouse pluripotent cells. Signal activation in these cells results in a very fast event in which the chromatin of key regulatory sequences gets reset from a 'waiting' configuration to an 'active' configuration. We further showed that different chromatin remodelling proteins are required for different steps in this resetting event. We now wish to determine how human pluripotent cells respond to activation of these same signals. Our current data indicates that a resetting event occurs in human cells, but we've already detected species-specific differences. In the current study we will move beyond the scope of the mouse work to define how cells respond to changes levels of different signalling pathways, and to determine why some cells are responsive to signals while others are not. We will use what we learn to devise methods for controlling the responsiveness of cells to signals, something that would be of enormous benefit in regenerative medicine and in treatment of diseases where cells exert aberrant signalling responses, such as cancer. We are using cutting-edge methodologies analysing chromatin behaviour at the genome-wide level and at the single molecule level.
Overall, this proposal represents a comprehensive investigation of how early embryonic human cells are directed to form the myriad of cell types present in an adult organism, through which we will devise ways in which to control this process.

A cell's identity is defined by its gene expression patterns, and gene expression potential is encoded in chromatin. Extracellular signals drive changes gene expression and thereby cell state during normal development by instructing very precise changes in protein occupancy and chromatin structure at key regulatory elements: the enhancers. This often involves activation of a signal-responsive transcription factor which can bind specific DNA motifs within a regulatory element to influence activity. Yet not all enhancers are immediately signal responsive, even if they contain DNA binding motifs for signal responsive transcription factors. Only those enhancers exhibiting an appropriate chromatin configuration can immediately respond to receipt of signal, thereby initiating a chain of events to propagate that signal. We find that signal activation causes a global change in enhancer chromatin and transcription factor activity in mouse pluripotent cells, an event we call Enhancer Resetting. We further found different nucleosome remodellers are required for different steps in Enhancer Resetting, and for the cell to mount an appropriate transcriptional response. While Enhancer Resetting occurs in human and mouse naïve pluripotent cells, both the activity of chromatin remodellers and the action of signalling pathways can have different consequences in human vs mouse pluripotent cell fate decisions. We will determine how signal activation engages with chromatin remodellers to invoke a transcriptional response in human pluripotent cells, and how that response directs cell fate transitions. We will define how graded changes in signalling levels influence enhancer activity and determine why naïve pluripotent cells require a period of capacitation to become signal responsive. We will use the information gained to devise strategies for controlling the signal responsiveness of human pluripotent cells as they undergo developmentally relevant cell fate decisions.