A CRISPR screen for RBC regulators: Hemoglobin in red blood cells (RBCs) carries oxygen to the tissues. Sickle cell disease is an inherited condition that involves abnormal hemoglobin. Current treatments entail modulating the level of fetal hemoglobin expression. Grevet et al. performed a CRISPR-Cas9 screen for regulators of fetal hemoglobin in RBCs and identified heme-regulated eIF2α kinase (HRI). Depleting the kinase in RBCs led to an increase in fetal hemoglobin levels and reduced sickling of cultured human RBCs. Thus, HRI may be a therapeutic target for sickle cell disease and other hemoglobin disorders. Science , this issue p. 285

Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and β-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain–focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain–containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of β-hemoglobinopathies.

β‐thalassemia is a recessively inherited blood disorder affecting millions worldwide. Pharmacological induction of fetal hemoglobin (HbF) is an effective therapeutic strategy, yet existing DNA methyltransferase (DNMT) inhibitors, although effective HbF inducers, currently are not approved for β‐thalassemia treatment. Here, we report that DMT207, a novel non‐nucleoside DNMT1 inhibitor, robustly reactivates HbF in HUDEP‐2 cells and adult primary erythroblasts with minimal toxicity. In a mouse model of β‐thalassemia, DMT207 effectively elevates the levels of mouse fetal‐ and embryonic‐type hemoglobin, promotes the maturation of erythroid cells, and alleviates the splenomegaly. Further multi‐omics analyses expose γ‐globin as one of the most sensitive genes with promoter demethylation and transcriptional activation following DMT207 treatment. Mechanistically, DMT207 traps DNMT1 into a catalytically inactive conformation and concurrently enhances its interaction with UHRF1, which partially contributes to DNMT1 degradation. These findings highlight the therapeutic potential of DMT207 for β‐thalassemia and support its further preclinical development.

The clustering of multiple transcription factor binding sites (TFBSs) for the same TF has proved to be a pervasive feature of cis-regulatory elements in the eukaryotic genome. However, the contribution of binding sites within the homotypic clusters of TFBSs (HCTs) to TF binding and target gene expression remains to be understood. Here, we characterize the CHD4 enhancers that harbor unique functional ZNF410 HCTs genome wide. We uncover that ZNF410 controls chromatin accessibility and activity of the CHD4 enhancer regions. We demonstrate that ZNF410 binds to the HCTs in a collaborative fashion, further conferring transcriptional activation. In particular, three ZNF410 motifs (sub-HCTs) located at 3' end of the distal enhancer act as "switch motifs" to control chromatin accessibility and enhancer activity. Mechanistically, the SWI/SNF complex is selectively required to mediate cooperative ZNF410 binding for CHD4 expression. Together, our findings expose a complex functional hierarchy of homotypic clustered motifs, which cooperate to fine-tune target gene expression.

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.

SUMMARY: Transcription factors often regulate numerous target genes. However, ZNF410 controls only a single gene, CHD4, in human erythroid cells by its highly restricted chromatin occupancy to the CHD4 locus via two clusters of ZNF410 binding motifs. Here, we uncover that ZNF410 controls chromatin accessibility and activity of the two CHD4 enhancer regions. Combining CRISPR/Cas9 genomic deletion with CRISPRi approaches, we demonstrate that both enhancer regions additively contribute to CHD4 gene expression. Mutations of the di-adenine nucleotides within the ZNF410 binding motif fully disrupt ZNF410-DNA interaction. Luciferase assays, ChIP-seq, and ATAC-seq studies reveal that the homotypic clustered motifs within the CHD4 enhancers are recognized by ZNF410 in a collaborative fashion. Especially, the three ZNF410 motifs located in the 3’ end of the distal enhancer act as “switch motifs” to control the chromatin accessibility and activity of the whole distal enhancer region. Together, our findings expose a complex functional hierarchy of motifs, where clustered motifs bound by the same transcription factor cooperate to fine-tune the expression of target gene. Highlights: ZNF410 maintains the accessibility of the CHD4 enhancer regions The two enhancer elements additively modulate CHD4 gene expression The dinucleotide “AA” is the key bases within ZNF410 binding motif ZNF410 binds to homotypic clustered motifs in a cooperative manner