ZNF410 Represses Fetal Globin By Devoted Control of CHD4/NuRD

Abstract

Several major effectors of adult-stage fetal globin silencing have been identified, including the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening of 1591 transcription factors, we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements, one at its promoter and the other at an enhancer 6 kb upstream, with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. Peaks at these clusters display up to 57- and 77-fold enrichment respectively for ZNF410 binding compared to controls. Footprint analysis shows that the ZNF410 motifs are protected from nuclease cleavage. No other bona fide peaks could be detected across the genome in HUDEP-2 cells or primary CD34+ HSPC-derived erythroid precursors. We demonstrate that the ZNF410-bound elements at CHD4 completely account for ZNF410's effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios (6 homozygous Zfp410Gt/Gt gene-trap allele mice out of 20 live births) with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. Engrafting human erythrocytes induce HbF from ~2.5% in controls to ~17% in ZNF410 edited recipients, by hemoglobin HPLC. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex. FIGURE LEGEND: ZNF410 chromatin occupancy is restricted to CHD4. (A) Dense mutagenesis of ZNF410 shows its five C2H2 zinc-finger domains (red rectangles) are especially critical for HbF repression. (B) Genome-wide ZNF410 binding was identified by CUT&RUN in HUDEP-2 cells. Enrichment for ZNF410 binding was concentrated at just two sites in the genome - at the promoter and at an enhancer ~6 kb upstream of CHD4. (C) Genome-wide ZNF410 motif occurrences (JASPAR motif shown in center) were mapped using a 3 kb sliding window. Clusters of ZNF410 motifs were found only at three locations in the genome, including the CHD4 promoter and CHD4 -6 kb enhancer. (D) CHD4 locus showing ZNF410 binding (red peaks) at the CHD4 promoter and CHD4 -6 kb enhancer regions with anti-HA antibody recognizing epitope-tagged ZNF410 and anti-ZNF410 antibody recognizing endogenous ZNF410 in both HUDEP-2 cells and CD34+ HSPC derived erythroid precursors. CUT&RUN footprints demonstrate ZNF410 motif protection, ATAC-seq chromatin accessibility and phyloP DNA sequence conservation. Disclosures Bauer: Pfizer: Consultancy; Syros Pharmaceuticals: Consultancy; Fulcrum Therapeutics: Consultancy; Sanofi: Research Funding.