Higher-order chromatin structure and regulatory sequence variation in human induced pluripotent stem cell (iPSC) self-renewal and differentiation

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The human body contains over 200 different cell types which fulfil highly specialized functions in different organs such as the liver, brain, heart, and skin. Pluripotent stem cells (PSCs) are fascinating because they can give rise to each of these specialized cell types, in a process called cell differentiation. This unique property (pluripotency) makes PSCs not only immensely valuable to study development, but also endows them with great promise for medical applications. A discovery honoured with the 2012 Nobel prize found that the process of differentiating PSCs into specialized cells can be reversed: mature skin cells can be reprogrammed to become so-called induced PSCs (iPSCs). In the clinic, this reprogramming makes it possible to derive an entire palette of bespoke cells from a patient's skin cells (via iPSCs), for example to replace damaged cells or even entire organs. This is a key aim of regenerative medicine, and it has the potential to solve the problems of the shortage of organs available for donation, and of organ transplant rejection.All cells contain the same genes; what makes cell types different is which sets of genes are 'on' and 'off' in a particular cell type. This determines which proteins (the cell's workhorses) are present in a cell; only 'on' genes are expressed and translated into proteins (for example, in liver cells a different set of genes is 'on' than in PSCs). Interestingly, although genes control cell function, they make up only a small amount of the DNA in our cells (~2%). The vast majority of our DNA (~98%) is non-coding, i.e. does not carry the information to make proteins. Once thought to be 'junk' DNA without a function, we now know that this non-coding DNA contains a plethora of so-called regulatory elements. These regulatory elements function like molecular switches by controlling which genes are 'on' and when. Regulatory elements make physical contacts with their target genes to switch them on. This is achieved by DNA looping; the genome folds in three dimensions (3D) in a cell-type specific manner to bring the appropriate switches and genes together.We often talk of THE human genome, but this is misleading as there are billions of human genomes - we are all different genetically (apart from identical twins). The sequence differences in our genomes are largely confined to the non-coding genome, and can explain not only why we all look different, but also why the cells in our bodies function in a slightly different way, for example in the context of disease. In iPS cells, this naturally occurring sequence variation is thought to be the reason for functional heterogeneity: some iPS cell lines can be differentiated more efficiently into liver cells, whereas other iPS cell lines can be differentiated more efficiently into brain cells, and yet others show a generally poor differentiation efficiency towards all more specialised cell types. Understanding this phenomenon mechanistically is important for the clinic, as it will in the future help to tailor treatment options in regenerative medicine specifically to the requirements of individual patients; this is a key aim of personalised medicine.Because of their immense potential for basic research and regenerative medicine, it is crucial that we understand the molecular mechanisms that endow iPSCs with their unique properties. The aim of this project therefore is to i) map the 3D genome folding in iPSCs to uncover which regulatory elements they use, ii) elucidate how this repertoire of regulatory elements changes as iPSCs differentiate, and iii) identify the genetic variants in these regulatory elements that cause iPS cell lines to differentiate more/less efficiently into specialised cell types.This knowledge is also important in research outside of PSCs, as aberrant 3D genome folding can bring together the wrong pairs of regulatory elements and genes, which can lead to disease including developmental malformations and cancer.