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Ddx41 function in erythropoiesis
Our model is that loss of Ddx41 contributes to excessive DDR signaling and subsequent cell cycle arrest in erythro- cytes, leading to anemia in ddx41 mutants. If correct, then inhibiting components of the DDR pathway would i) reverse cell cycle defects and ii) increase erythrocyte levels. In order to test this model, we examined how the two pri- mary mediators of DDR, Ataxia-telengiectasia-mutated (ATM) and Ataxia-telengiectasia and Rad3-related (ATR), affected erythrocytic cell cycle kinetics in ddx41 mutants. We assessed cell cycle status of 30 hpf ddx41-mutant gata1:dsred+ erythroid progenitors in embryos treated with DMSO vehicle control, the ATM inhibitor KU60019, or the ATR inhibitor AZ20. There was a significant increase of gata1:dsred+ ddx41-mutant cells in S phase when treated with either ATM or ATR inhibitors as compared to DMSO vehicle control (Figures 5A and B). Additionally, pharmaco- logical inhibition of ATM or ATR increased erythropoietic output in ddx41 mutants, as measured by quantification of gata1:dsred+ erythrocyte numbers per embryo using flow cytometry (Figures 5C to F). Although there was a trend towards an increase in erythrocyte numbers in control sib- lings treated with ATM or ATR inhibitors these changes were not statistically significant. Taken together, these data indicate that DDR signaling triggers a G0/G1 cell cycle arrest in ddx41-mutant erythrocytes that results in a reduc- tion of erythroid progenitor cell number.
Finally, we wanted to assess if increasing the number of erythroid progenitors via ATM or ATR inhibition would increase the number of oxygenated erythrocytes in ddx41 mutants. Surprisingly, we only observed a significant increase in o-dianisidine-positive erythrocytes in ddx41 mutants treated with ATM inhibitor, but not ATR inhibitor (Figures 6A and B). These data indicate that Ddx41 regula- tion of ATM might have a broader impact on erythropoiesis than ATR signaling.
Discussion
Although DDX41 mutations are found in numerous human hematologic diseases, its function in hematopoiesis is unknown. Our work is the first to estab- lish Ddx41 as a critical mediator of erythropoiesis with ddx41 loss suppressing the expansion and maturation of erythrocytes. We showed a profound effect on the expres- sion of cell cycle and DNA damage-associated genes in ddx41 mutant erythroid progenitors consistent with the observed cell cycle arrest. The DNA damage response is elevated in ddx41 mutant cells and triggers an ATM and ATR-triggered cell cycle arrest. Inhibition of ATM and ATR partially suppressed anemia in ddx41 mutants. These findings establish Ddx41 as a positive regulator of erythro- poiesis in part by preventing genomic stress and promot- ing proper erythroid progenitor expansion.
Patients with germline mutations in DDX41 do not develop hematologic symptoms until later in life,1 yet zebrafish ddx41 mutants show anemia within 40 hpf. We posit that the difference has to do with the extent of Ddx41 deficiency. Zebrafish homozygous mutants have maternally deposited Ddx41 that is naturally depleted over the first few days of life. When the levels drop below a certain threshold, the mutants die, demonstrating it is an essential factor. In contrast, zebrafish ddx41 heterozygous animals are phenotypically indistinguishable from wild- type animals during embryogenesis and in adulthood, sug-
gesting a 50% decrease of Ddx41 alone is insufficient to alter hematopoiesis. This is in agreement with the clinical observation that patients with germline DDX41 muta- tions who develop hematologic malignancies often acquire somatic missense mutations in the second allele that are thought to diminish DDX41 ATPase activity.1 Combined, the data indicate that when DDX41 levels decrease to less than 50%, this leads to hematologic defects, but when critically too low, it leads to lethality.
DDX41 was previously identified as a mediator of genomic stability in a cell line-based genome-wide siRNA screen.11 However, a role for DDX41 in genomic integrity as well as the downstream consequences of its loss were never demonstrated in vivo. Our current work revealed that Ddx41 regulates genomic integrity in vivo, and that loss of ddx41 leads to both cell cycle arrest and apoptosis in erythrocytes that contributes to anemia in ddx41 mutants. We established that ATM and ATR signaling contribute to these attributes, but only ATM inhibition significantly increased o-dianisidine-positive erythrocytes in ddx41 mutants. The differential impact on oxygenated erythrocyte output by inhibition of ATM and ATR might indicate that Ddx41-regulated ATM signaling is more crit- ical for proper erythropoiesis. ATM has an additional role in apoptosis, especially during development that might explain some of the phenotypic differences when compar- ing ATM and ATR inhibition effects on erythropoiesis. However, it should be noted that although the ATM and ATR kinases respond uniquely, there exists an extensive ‘cross-talk’ between them, which can make determining which precise pathway is involved in a phenotype confus- ing.32 Further dissection of the role of DDX41 in ATM and ATR pathway regulation will need to be investigated.
Splicing mutations are commonly found in hematologic malignancies.33,34 DDX41 interacts with multiple compo- nents of the spliceosome.1 Our work aligns with prior studies showing DDX41 insufficiency associates with numerous deleterious splicing outcomes. If and how these splicing events contribute to hematopoietic pathogenesis is unclear. We showed that components related to cell cycle and DNA repair are commonly mis-spliced in ddx41 mutants. Therefore, it is possible that loss of ddx41 may be mediating cell cycle arrest and activation of DDR via mis-splicing of crucial regulators of these pathways. The contribution of DDR pathway component mis-splicing in human cytopenias remains to be addressed.
In addition to the effect on cell cycle, we delineated maturation defects in ddx41 erythrocytes marked by aber- rant globin expression and a megaloblastoid-like morphol- ogy of mutant erythrocytes. Although we could not per- form a complete analysis of definitive erythropoiesis as ddx41 mutants die before EMP-derived or HSC-derived erythrocytes fully mature, the diminished expression of the larval βe2 globin suggests a decrease in EMP-derived definitive erythrocytes. This finding combined with the elevated cmyb and gata1 levels in EMP cells suggests that this defect could be caused in part by maturation defects. As the treatment with the ATM inhibitor KU60019 only partially increased hemoglobinized erythrocytes in ddx41 mutants, it suggests that deregulation of another pathway underlies additional maturational defects in ddx41 mutants.
In summary, our study unveils a critical role for Ddx41 as a key gatekeeper to maintain cell cycle progression, a necessary component for erythrocytic development. We
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