Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
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