Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for β-hemoglobinopathy therapeutic intervention. The NuRD chromatin complex participates in γ-globin repression. Here we use pooled CRISPR screening to comprehensively disrupt NuRD protein coding sequences in human adult erythroid precursors. We find essential for fetal hemoglobin (HbF) control a nonredundant subcomplex of NuRD protein family paralogs, whose composition we corroborate by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identifies key protein interfaces where in-frame alleles result in loss-of-function due to destabilization or altered function of subunits. We ascertain mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrate that sequestering CHD4 from NuRD phenocopies these mutations. This work indicates a generalizable approach to discover protein complex features amenable to rational biochemical targeting.