Hematopoietic Stem Cell Function and Differentiation Is Regulated By DCAF7 through Polycomb Repressive Complex 1 (PRC1) Modulation

The regulation of hematopoietic stem cell (HSC) function has been extensively studied at genomic and transcriptional levels, but the roles of ubiquitin E3 ligase-associated factors in the hematopoietic system are not well understood. DCAFs (DDB1-CUL-Associated Factors) interact with Cullin-Ring Ligases to recruit proteins for ubiquitination, facilitate protein assemblies, redirect E3 ligase complexes, and recognize chromatin features. Our studies focus on elucidating the function of DCAF7, a poorly characterized protein and one of the few DCAFs (a family of more than 100 proteins) expressed in HSCs. Previous studies on DCAF7 were performed in cell lines, and its physiological role in vivo is unknown. DCAF7 has been shown to bind kinases and target substrates for degradation such as Menin and TFEB. Mutations in DCAF7 have been reported in myeloid leukemia in Down Syndrome and other hematological malignancies. Thus, we aimed to investigate DCAF7 function in hematopoiesis using a novel conditional knockout mouse line. Dcaf7-deficient mice (Dcaf7F/F Mx1-Cre+) showed an increased frequency of phenotypically defined long-term HSCs and multipotent progenitors, with a decrease in more committed progenitors. Similar results were observed in Dcaf7F/F Vav1-Cre+ mice. We assessed DCAF7 function in HSC regeneration upon myelotoxic stress by injecting mice with 5-fluorouracil (5-FU). KO mice showed delayed recovery and died earlier than WT mice upon serial injections. To investigate the effect of Dcaf7 loss on hematopoietic cell fitness and long-term self-renewal, we performed bone marrow transplants. In competitive transplants, KO cells (CD45.2) were outcompeted by CD45.1 cells with no differences in KO-derived lymphoid or myeloid cells. In non-competitive transplants, KO-recipient mice showed impaired hematopoietic reconstitution and hypocellular bone marrow, consistent with a bone marrow failure phenotype. Using single-cell RNA sequencing and single-cell time-lapse microscopy, we observed that loss of Dcaf7 accelerates HSC differentiation including their direct differentiation into megakaryocytes. KO cells show a dysregulated trajectory toward erythroid, basophil/mast cell, and dendritic progenitors, with reduced stemness in HSCs. Overall, these results indicate that lack of Dcaf7 induces HSC differentiation into the myeloid lineage, leading to stem cell exhaustion. Next, we investigated the molecular mechanism by which DCAF7 contributes to hematopoiesis. By performing IP-MS, we found that DCAF7 interacts with components of the E3 ligase-containing polycomb repressive complex 1.5 (PRC1.5), which is involved in gene activation unlike other PRC1 complexes. DCAF7 absence did not alter PRC1.5 protein levels. Glycerol-density sedimentation analysis showed that without DCAF7, all PRC1 subunits eluted in lower molecular-weight fractions compared to WT complexes, indicating DCAF7 promotes the assembly of large PRC1.5 complexes. Given that PRC1 binds chromatin and its configuration varies among cell types, we performed chromatin profiling in HSCs and progenitors. We analyzed the genome-wide distribution of RING1B (the core PRC1 component that ubiquitinates H2A), H2AK119ub, H3K27me3, and H3K4me3 using CUT&RUN. Our results revealed that the loss of DCAF7 leads to increased RING1B localization at transcriptionally active loci marked by H3K4me3. This increased binding was observed at genes involved in myeloid differentiation and was associated with increased mRNA expression in HSCs. Together, our study elucidates a new pathway for the regulation of PRC1 complex activity mediated by DCAF7 that controls HSC differentiation.