DDB1 and CUL4 Associated Factor 7 (DCAF7) Is Essential for Hematopoiesis

Abstract

Mutations in DDB1 And CUL4 Associated Factor 7 (DCAF7) have been detected in a subset of patients with Myeloid Leukemia in Down syndrome. DCAF7 is a highly conserved WD40 domain protein with 100% homology among vertebrates and is a prominent binding partner of DYRK1A, a kinase located on chromosome 21. Analysis of the DepMap database revealed that hematopoietic cell lines are highly dependent on DCAF7 compared to cell lines from other tumors. However, the function of DCAF7 during normal hematopoiesis is currently unknown. Thus, we aimed to study its function in hematopoietic cell lines and in a conditional knockout mouse model. We first deleted DCAF7 from a panel of hematopoietic cell lines by CRISPR/Cas9 editing and observed its absence led to cell cycle arrest and decreased proliferation. Rescue experiments in which a CRISPR resistant DCAF7 allele was expressed in the targeted clones restored proliferation and cell cycling. A comparison of RNA-seq data between parental HEL cells and those without DCAF7 showed decreased expression of genes involved in differentiation, TNF signaling and the IFN pathway, indicating that DCAF7 is important for cell differentiation. Next, we created a Dcaf7 conditionally targeted mouse strain (Dcaf7F/F Mx-1 Cre) and assessed its role in hematopoiesis in vivo. Following Cre induction by pIpC, we observed a decrease in white blood cells, hemoglobin and platelet counts in KO mice. Analysis of the bone marrow showed an accumulation of LT-HSCs and multipotent progenitors. In addition, we observed a defect in the generation of B-cells, reminiscent of the phenotype observed upon Dyr1ka deletion. We then tested the recovery of Dcaf7 KO hematopoietic cells after different types of stress, which included ex-vivo culture, transplantation, and myelosuppression. Dcaf7 deficient HSCs showed defective expansion in PVA-containing media and poor differentiation in single cell colony assays in methylcellulose. In non-competitive transplants, KO cells reconstituted hematopoiesis although the number of mature myeloid and lymphoid cells was dramatically decreased. In competitive transplants, Dcaf7 KO cells were outcompeted by wild-type cells. Lastly, upon myelosuppression with 5-FU, Dcaf7 deficient mice were not able to recover and died acutely. Overall, these results indicate that DCAF7 plays a critical role in HSCs during homeostasis and regeneration. To uncover the mechanisms by which DCAF7 contributes to hematopoiesis, we performed IP-MS using tagged DCAF7 as bait in a hematopoietic cell line. In addition to DYRK1A, we found that DCAF7 interacts with multiple components of the polycomb repressive complex1 (PRC1), specifically the non-canonical PRC1.5 complex, which has been previously implicated in gene activation and transcription. We confirmed that DCAF7 engages in the PRC1.5 complex by glycerol gradient centrifugation. Upon DCAF7 absence the size the PRC1 complex shifts towards a lower molecular weight, indicating that DCAF7 is a core subunit of the complex. We then performed RNASeq analysis in sorted LT-HSCs (Lin-cKit+Sca1+CD48-CD150+) and found that 70% of the differentially expressed genes were downregulated in KO cells. These downregulated genes were associated with diminished activation of GATA1, GATA2, RUNX1 and SP1 target genes, in agreement with the functional role of the PRC1.5 complex in gene activation and cell differentiation. Together our results reveal that DCAF7 regulates hematopoiesis by participating in transcriptional regulatory complexes, and we predict that DCAF7 mutations contribute to leukemia by cooperating with DYRK1A and altering the activity of the PRC1 complex.