DCAF13 Is Essential for Hematopoietic Stem Cell Function By Stabilization of Ribosome Biogenesis and P53 Pathway

The substrate-recognizing ubiquitin E3 ligase plays an important role in the regulation of cell fate decisions in hematopoietic stem cells (HSCs). The DCAF protein family are a group of approximately 60 ubiquitin E3 ligases that function as the substrate recognition protein of CUL4-DDB1 (CRL4) complex. Analysis of BloodSpot datasets revealed that DCAF13 is highly expressed in HSCs and progenitor cells compared to mature cells. However, the function and molecular mechanisms of DCAF13 in hematopoiesis, particularly in HSCs remain largely unexplored.

In this study, we generated Vav-Cre;DCAF13^fl/fl mice by crossing Vav-Cre;DCAF13^fl/+ with DCAF13^fl/fl mice. Surprisingly, no Vav-Cre;DCAF13^fl/fl newborn pups were obtained, suggesting that DCAF13 deficiency leads to embryonic lethality. Examination of the fetal liver at E12.5-E14.5 revealed the presence of Vav-Cre;DCAF13^fl/fl embryos with a number consistent with expected Mendelian inheritance. However, these embryos had significantly smaller and paler fetal livers compared to DCAF13^fl/fl embryos. Further analysis of the fetal liver of E12.5-E14.5 showed a significant depletion of HSPCs in DCAF13 knockout mice.

To investigate the role of DCAF13 in adult hematopoiesis, we used inducible Mx1-Cre and Poly (I:C) to knockout DCAF13. Similarly, Mx1-Cre;DCAF13^fl/fl mice died of bone marrow (BM) failure within 30 days after Poly (I:C) injections due to depletion of HSPCs. DCAF13 knockout led to severe cytopenia and reduced bone marrow hematopoietic cell proliferation. Functional studies demonstrated that BM cells from Mx1-Cre;DCAF13^fl/fl mice displayed a reduced CFU capacity and reduced competitive hematopoietic repopulation ability over serial transplantation when compared to BM cells from DCAF13^fl/fl controls. These findings underscore the essential role of DCAF13 in the development and maintenance of both embryonic and adult HSCs.

To dissect the underlying mechanism, we performed single-cell transcriptome analysis at E14.5. Lin^- cells were grouped into 15 clusters, with 8 subsets, including HSCs, significantly decreased in Vav-Cre;DCAF13^fl/fl fetal liver. The ratio of MEP and pro-E was increased. HSC and MEP subsets revealed disrupted cell cycles and enriched apoptosis mediated by P53 signaling pathway in Vav-Cre;DCAF13^fl/fl embryos. We also detected a decreased cell cycle and increased Annexin V⁺ cells by flow cytometry in DCAF13 KO cells. Notably, the down-regulated genes were enriched in ribosome biogenesis in Vav-Cre;DCAF13^fl/fl HSC and MEP cluster, polysome profiling revealed decreased 40S and 80S complexes in DCAF13 KO cells. Using O-propargyl-puromycin (OPP)-incorporation assay, we found that the protein synthesis of lin^- cells was significantly decreased in Mx1-Cre;DCAF13^fl/fl mice. These results demonstrated that DCAF13 could impair the ribosome biogenesis and protein translation of HSCs. Single-cell profiling also showed up-regulated P53 signaling pathway genes. Increased expression of P53-target genes, with unchanged P53 mRNA levels, suggests P53 activation via post-translational modifications.

In summary, our study provides novel insights into the critical role of DCAF13 in regulating HSC development and function through ribosome biogenesis and the P53 signaling pathway. These findings could potentially provide us novel therapeutic strategies for BM failure disorders and clinical application of HSCs.

No relevant conflicts of interest to declare.