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

Area of Science:

• Molecular Biology
• Gene Regulation
• Chromatin Biology

Background:

• Developmental silencing of fetal globins is crucial for gene regulation and a target for treating β-hemoglobinopathy.
• The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a role in repressing fetal globin gene expression.

Purpose of the Study:

• To comprehensively identify NuRD complex components involved in fetal hemoglobin (HbF) repression using pooled CRISPR screening.
• To understand the functional interfaces within the NuRD complex and their role in HbF regulation.
• To explore therapeutic strategies targeting the NuRD complex for β-hemoglobinopathy.

Main Methods:

• Pooled CRISPR screening in human adult erythroid precursors to disrupt NuRD protein coding sequences.
• Affinity chromatography and proximity labeling mass spectrometry to determine NuRD complex composition.
• Functional analysis of mutations in CHD4 in erythroid precursors and transgenic mice.

Main Results:

• A non-redundant subcomplex of NuRD protein paralogs is essential for HbF control.
• Key protein interfaces were identified, with in-frame alleles causing loss-of-function via subunit destabilization or altered function.
• Mutations in CHD4 were found to dissociate its role in cell fitness from HbF repression.
• Sequestering CHD4 from NuRD mimicked the effects of these mutations.

Conclusions:

• The study elucidates critical NuRD complex features governing HbF repression.
• Targeting specific protein interfaces within the NuRD complex, particularly involving CHD4, presents a rational approach for biochemical intervention.
• This work provides a generalizable strategy for discovering druggable targets within protein complexes for therapeutic purposes.