show Abstracthide AbstractCellular differentiation-trajectories require extensive alterations in chromatin structure and function, elicited by a diverse array of chromatin-factors (CFs). Using haematopoiesis as a model-system, we combined bulk ex vivo and single-cell in vivo CRISPR screens to comprehensively characterise the role of CF families in cell-fate decisions. We uncover marked lineage specificities for many CFs, dissecting specific CF-dependencies in vivo at single-cell resolution. This highlights functional diversity among related CFs (i.e. BAF-subcomplexes, MLL-methyltransferases), but also reveals functional redundancy among unrelated Repressive-complexes that restrain excessive myeloid differentiation. Epigenetic-profiling, molecularly explains CF lineage-dependencies, identifying differentiation stage-specific interactions between individual CFs and lineage-determining Transcription Factors (TFs). Intriguingly, non-canonical BAF remodeler disruption abrogates myeloid-TF function generating a pre-leukaemia phenotype. Screening CF functions in leukemia, we show that leukaemia cells abrogate normal CF function, restraining differentiation trajectories by engaging in novel CF-TF interactions that suggest potential therapeutic avenues. Together, our work greatly elucidates the role of CFs and their TF-partners across normal and malignant haematopoiesis. Overall design: Here we have analysed the binding patterns of the canoncal-BAF subunit Smarcb1 and the H3K4-methyl writers Kmt2d (MLL4) and Kmt2a (MLL1) in normal myelopoiesis and in a murine leukemic model driven by the Npm1c/Flt3-ITD mutations. For the normal hematopoiesis we have profiled early myeloid progenitors (GMPs) FACs-sorted from bone-marrow as Lin-, ckit+, sca1-, FcgR-III+, cD34-), in vivo FACs-sorted bone marrow monocytes (Lin-, CD11b) and exvivo derived monocytes (expanded from LSK prgenitors with Flt3-ITD, G-CSF, IL3 and GM-CSF). Leukemic cells were purified from primary tumours and expanded exvivo for few passages with SCF, IL3 and IL6. Freshly isolated cells were dual-crosslinked with 3 mM EGS+DSG+DMA for 20 minutes followed by 1% Formaldehyde for 5 minutes. Chromatin extracts were prepared by nuclei isolation with an hypotoniic buffer (25 mM Hepes 7.5 M, 10 mM NaCl, 0.2% Igepal + Protease Inhibtors), followed by sonication in 0.5% SDS, 5 mM EDTA. Chromatin extracts were quantified and diluted 5x to reach a concentration of 150 mM NaCl, 0.1% SDS, 1% Tx-100, 1 mM EDTA, 1X Prot Inh. Immunoprecpitation was carried out overnight (10-12 h) with the appropiate amount of the relevant antibody and the immuno-complexes were captured on the following day by incubation with ProtA + ProtG Magna-ChIP beads (Sigma). After that, DNA was purified with SPRI beads and libraries were prepared using the NEB Next Ultra II kit (NEB).