Workstream 1: Use of integration approaches to uncover genotype–phenotype interactions I. How genetic variation between cells impacts on their phenotypic behaviour in culture. This project was carried out in the framework of the Human Induced Pluripotent Stem Cells Initiative (HipSci). We integrated high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying dimensionality reduction approaches we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions, identifying genes associated with outlier cell behaviour. This study, published in Cell Report (Vigilante et al), established a strategy that we applied to other projects – see Mitchell et al – and provided the starting point for the MRC program grant detailed below. II. Using iPSC variation to define HIV-1 regulatory networks In this project, in collaboration with Prof Michael Malim and Dr Davide Danovi, we use the HipSci dataset to uncover the link between HIV-1 infectivity (phenotype) and genotype to identify regulatory factors and understand HIV-1 regulatory networks on a genome-wide level. This project was awarded a 5-years MRC program grant III. Integration of genetic and phenotypic heterogeneity within a mouse model of oral squamous cell carcinoma This study, in collaboration with Prof Fiona Watt and published as co-senior author (Sequeira et al), provided a foundation for exploring human oral squamous cell carcinoma, evolution, heterogeneity and progression. In particular, we have performed genomic analysis of mouse tongue lesions caused by 4-nitroquinoline N-oxide and studied the mutational signatures in relation to the lesions grade and phenotype. We can now build on these studies to improve diagnosis and treatment of human OSCC. IV. Naïve vs Primed states: transcription and post-transcriptional differences between clones This research, in collaboration with Dr. Danovi, is carried out as PhD project in my lab. We use 6 cell lines from the HipSci resource that we believe might have clones in two different states: naïve and primed. If our hypothesis is validated, this would represent the first time that not-lab induced human naïve stem cells can be investigated. While the experiments are ongoing to validate our premises, in parallel we are studying the impact of alternative splicing on the regulatory function of all human transcription factors in the different stem cell states. This will also result in an incredible useful resource: a census of all the alternative splicing scenarios and their functional impact in the transcriptional network.