ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression

Abstract

Transcription factors typically regulate a large number of genes. Here we found that transcription factor ZNF410 binds and activates the expression of a single direct target gene, CHD4, to enforce the silencing of the fetal hemoglobin genes (HBG1 and HBG2) in adult erythroid cells. ZNF410 is a pentadactyl DNA binding protein that emerged from a DNA binding domain-focused CRISPR-Cas9 screen aimed at the identification of new regulators of fetal hemoglobin silencing. Depletion of ZNF410 specifically diminished CHD4 expression, leading to reactivation of the normally silent fetal globin genes in both human erythroid culture systems and a human-to-mouse xenotransplant model. Combining RNA-seq and ChIP-seq analyses revealed that CHD4 is the sole direct ZNF410 target gene in erythroid cells, which was further validated by rescue of fetal hemoglobin silencing and other transcriptional changes upon CHD4 restoration in ZNF410-deficient cells. ZNF410 ChIP-seq detected only eight high-confidence peaks with seven associated genes including CHD4. Most strikingly, the two most predominant peaks are located at the CHD4 locus, which contains two highly conserved, dense clusters of ZNF410 binding motifs. The two motif clusters appear to be unique in the human and mouse genomes. Moreover, among the seven ZNF410-bound genes, CHD4 was the only one whose expression was down-regulated upon ZNF410 depletion, indicating that CHD4 is the sole target of ZNF410. Electrophoretic mobility shift assays (EMSAs) showed that the zinc finger (ZF) domain of ZNF410 is necessary and sufficient for DNA binding. When overexpressed, the DNA binding profile of ZF domain alone is very similar to full length ZNF410. Indeed, forced expression of the ZF domain displaced endogenous ZNF410 at all binding sites, including the CHD4 locus. This reduced CHD4 expression to levels comparable to those in ZNF410 deficient cells (and activated the fetal globin genes) but had no effect on the other ZNF410 bound genes, again confirming target specificity. ZNF410 depletion or expression of the dominant negative acting ZF domain lowered CHD4 only by ~65%-70%, which is very well tolerated by erythroid cells, as determined by morphology, cell surface phenotyping, and gene expression profiling. This exposes the fetal globin genes as highly sensitive to CHD4 levels. Lastly, we solved the crystal structure of the ZF domain-DNA complex at 2.75Å resolution pinpointing the protein-DNA contacts and showing that each of the five ZFs make specific DNA contacts. In sum, to our knowledge, ZNF410 is the only transcription factor with just one direct functional target gene in erythroid cells. Given the strong impetus to reactivate fetal globin gene expression in patients with sickle cell disease and some forms of b-thalassemia, it might be possible to exploit the exceptionally high transcriptional selectivity of ZNF410 to raise fetal hemoglobin expression for the treatment of these hemoglobinopathies. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.