Transcription factor dynamics in control of pluripotent cell function and identity

Stem cells attract considerable attention because of their potential to repair damaged or exhausted tissue in patients. There are two properties that define stem cells and that account for their potential utility. First, stem cells can make identical copies of themselves and can do this indefinitely, a process we call self-renewal. Second, stem cells can change their properties and become a specialised type of cell that carries out a particular function in our bodies. Within an organism these two properties of self-renewal and specialisation must be balanced. If too many cells specialise, then the stem cell population may run out. If too many cells self-renew, there may be an insufficient supply of specialised cells to maintain an organisms functionality. How does an organism meet these demands upon its stem cell population? We believe we have uncovered an explanation and want to explore the detailed mechanisms behind our observations. We study a type of stem cell called an embryonic stem (or ES) cell. We study mouse ES cells because they are the most tractable experimentally.

We found that all cells in an ES cell population are not the same; some have a greater likelihood of specialising and others a greater likelihood of self-renewing. Unexpectedly we found that these two states could interconvert and the cells that were more likely to specialise could still self-renew and move back into a more niave state. Crucially we were able to determine that these two states could be distinguished by the presence or absence of a particular gene regulator, Nanog.

In the proposed work, we will ask how Nanog interacts with other gene regulators throughout the genome to turn genes on or off. We will identify genes that carry out the functions of Nanog. A major part of our work will be to determine the mechanisms that switch the Nanog gene on and off and that are therefore central to the function of the stem cell. We will relate our studies to stem cells from more mature embryos and test the relevance of our findings to the intact embryo.

Our studies will deliver a deeper understanding of the switches controlling the behaviour of ES cells. Not only will this be important in learning how to optimally apply ES cells in potentially therapeutic situations but may provide fundamental insights applicable to all stem cells.

We have shown that undifferentiated ES cells fluctuate between states in which they do or do not express the transcription factor Nanog and that these states are differentially responsiveness to differentiation cues. This raises several questions that form the basis of this proposal. Our overarching aim is to examine the function of pluripotency transcription factors at genes that critically modulate self-renewal efficiency. This will allow us to determine how the pluripotent population is compartmentalised into cells that self-renew efficiently and cells that are responsive to differentiation cues. Such knowledge is important because safe application of stem cells for regenerative therapies demands strict control of stem cells, so that their behaviour can be predictably and uniformly directed. We start from the premise that the molecular machinery of distinct pluripotent cell compartments is controlled by transcription factors that direct expression of the appropriate gene set. We will analyse the interdependencies of chromatin binding of pluripotency transcription factors and will identify genes that distinguish between ES cells of high and low self-renewal efficiency. We will determine which of the differentially expressed genes is responsible for the differences in phenotype by modulating the expression of these target genes. We will determine whether enforced expression of pluripotency regulators that control such target genes can complement the self-renewal efficiency of the low-efficiency compartment. We will analyse transcriptional controls operating at the Nanog gene that may influence Nanog heterogeneity and will relate these to chromatin states in Nanog expressing and Nanog non-expressing cells. We will assess single cells to determine the co-ordination between Nanog and other fluctuating genes and will assess the effect of altering transcription factor stability on the dynamics of heterogeneity. We will also extend our studies to epiblast stem cells cultured in vitro to determine whether overlapping mechanisms govern the identity of cells which can readily and quickly be derived from ES cells and will examine hypotheses that arise in the course of the studies to determine the relevance of our findings to cells in the embryo. We anticipate that these studies will lead to a deeper understanding of the mechanisms governing pluripotent cell function and that such knowledge can be used to advance the safe application of pluripotent cells for regenerative medicine.