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Investigating the role of DPPA2 and DPPA4 in the Epigenetic Control of Lineage Programs in Human Embryonic Stem Cells 10.17863/CAM.90427 Malcolm, Andrew 0000000162407701 Rugg-Gunn, Peter The precise co-ordination of cell fate specification during human early development is a vital yet poorly understood process. To navigate the dynamic transcriptional and epigenetic changes associated with germ layer allocation, pluripotent cells maintain developmentally important genes and their regulatory regions in a poised but repressed chromatin state. The poised state has been proposed to allow for precise and coordinated activation or complete repression of gene expression depending on the instructive signals from the external and intrinsic environment. Poised promoters and enhancers adopt multivalent histone modification states, comprised of both active and repressive modifications, such as H3K27me3, H3K4me3 and H3K4me1. Combining opposing modifications may help to maintain robustness of genes to low levels of signal, whilst retaining the capacity to respond upon the appropriate level. Despite their importance in controlling cell fate decisions, our understanding of the mechanisms by which poised states are established and maintained in human cells is currently lacking. Here, I discover a role for the transcription factors DPPA2 and DPPA4 in maintaining poised chromatin in human pluripotent stem cells (hPSCs). I found that DPPA2/4 bind to the majority of CpG islands, poised promoters and a large subset of poised enhancers. CRISPR-Cas9 mediated knockout of DPPA2/4 in primed hPSCs led to changes in the expression of developmentally critical genes, particularly those associated with signalling. Primed hPSCs lacking DPPA2/4 exhibit increased spontaneous differentiation even in self-renewing conditions, display altered cell fate commitment III during differentiation and show axial patterning defects upon human gastruloid formation. Epigenomic profiling following the loss of DPPA2/4 revealed a marked depletion of H3K27me3 at a subset of DPPA2/4-target regions, predominantly near to poised chromatin regions. Regions losing H3K27me3 were typically highly accessible and had high levels of H3K4me3 in both wild-type and DPPA2/4 DKO hPSCs. The depletion of H3K27me3 upon DPPA2/4 loss leaves these regions in a more active conformation, potentially driving changes in the expression of these genes. These data reveal new roles for DPPA2/4 in safeguarding the robustness of poised chromatin states and in regulating genes that are important for cell fate specification. Understanding further how these transcription factors and chromatin states jointly contribute to gene expression dynamics is critical to uncover the principles of developmental gene regulation and to improve generation of specialised cell types from human pluripotent cells. University of Cambridge 2022-03-31 Thesis Doctoral Doctor of Philosophy (PhD) eng Wellcome Trust Cambridge Stem Cell Institute Four Year Studentship in Stem Cell Biology https://www.repository.cam.ac.uk/handle/1810/343016 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1b630cb1-283b-46e5-8f21-13cdca206739/download 62152fa7693520589c7100ce068a9185 https://www.rioxx.net/licenses/all-rights-reserved/ Epigenetics Human Pluripotent Stem Cells Lineage commitment Polycomb Human Embryonic Stem Cells