In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis.

Acknowledgements: We thank M. Wiederstein, University of Salzburg, for HPC support, K. Kania from the CRUK Genomics Core for assistance with single-cell methods and N. Cano and M. Dawes for superb administrative support. We thank D. Kent and A. Sebe-Pedrós for their constructive advice and discussions regarding the manuscript. The CRISPR sequencing-BFP backbone (plasmid no. 85707, Addgene) was produced by I. Amit, Weizmann Institute of Science. pMD2.G (plasmid no. 12259, Addgene) and psPAX2 (plasmid no. 12260, Addgene) was produced by D. Trono, École Polytechnique Fédérale de Lausanne. This project was funded by the ‘La Caixa’ Foundation (agreement no. LCF/PR/HR20/52400016, B.J.P.H., F.P.), Marie Skłodowska-Curie International Fellow (no. 886474, D.L.-A.), a European Hematology Association Junior Research Grant (D.L.-A.), Cancer Research UK Programme Grant (no. DRCRPG-Nov22/100014, B.J.P.H.), the European Research Council (no. 647685, B.J.P.H.), the Cancer Research UK Cambridge Major Centre (no. C49940/A25117, B.J.P.H.), a Kay Kendall Leukaemia Fund Junior Fellowship (no. KKL1440, N.N.), the National Institute for Health and Care Research (NIHR) Cambridge Biomedical Research Centre (no. BRC-1215-20014, B.J.P.H.), the UK Research and Innovation Medical Research Council (MRC) (no. MC_PC_17230, B.J.P.H.) supporting the Wellcome–MRC Cambridge Stem Cell Institute, Wellcome Trust (no. 203151/Z/16/Z, B.J.P.H.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.Funder: European Hematology Association (EHA); doi: https://doi.org/10.13039/100008594Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis