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Mechanisms of NCOA4-maintained murine erythropoiesis
mone-dependent transcriptional program that supports erythropoiesis. While our results do not exclude this pos- sibility, it is clear that the erythroid-specific function of Ncoa4 does not account for the entirety of the anemia effect in systemic Ncoa4 KO mice. These results will have to be reconciled with the clear role of Ncoa4 in mediating ferritinophagy7,8 as well as reports that Ncoa4 participates in DNA replication origin activation.46 Contrasting these results from the postnatal stage with those from adult mice, Ncoa4-deficient mice recover from the profound postnatal anemia either because they are no longer dependent on cell autonomous or non-autonomous Ncoa4 function or because compensatory mechanisms replace Ncoa4 function. For the first time, we are now able to temporally control Ncoa4 ablation. Our results show that Ncoa4 is important in the adult mouse given that an acute ablation of Ncoa4 leads to anemia. While we have not determined the cause of the acute anemia it is interesting to speculate based on results from Gao et al. that show a block in terminal differentiation of primary human ery- throblasts upon acute NCOA4 knockdown, which may render a subset of newly synthesized reticulocytes partic- ularly sensitive to turnover.
The role of Ncoa4 in erythroid differentiation has been studied intensively with mixed results. Our results with targeted constitutive ablation of Ncoa4 in the erythroid compartment showed no appreciable defect in differentia- tion of erythrocyte precursors derived from an adult mouse. Likewise, we saw no effect on erythroid differenti- ation after acute systemic Ncoa4 depletion. Our results could suggest a number of possibilities with respect to the role of Ncoa4 in erythroid differentiation: (i) erythroid intrinsic Ncoa4 function may not be important for in vivo differentiation; (ii) erythroid intrinsic Ncoa4 function in differentiation may be dispensable and compensated for by other intrinsic or extrinsic mechanisms; and/or (iii) ery- throid differentiation is dependent on Ncoa4 in postnatal mice but not in adult mice. Erythroid compartment-specif- ic Ncoa4 rescue in the setting of a Ncoa4 null mouse may answer the question of extrinsic effects on differentiation.
Overall, we have shown that Ncoa4 is important for
basal and stimulated erythropoiesis by regulating both sys- temic and RBC iron metabolism. Loss of Ncoa4 triggers significant compensatory mechanisms to mitigate baseline and induced anemias. In response to Ncoa4 depletion, the Hif-2a-Epo axis is upregulated to compensate for anemia. We note that with a concurrent iron deficiency, activation of Irp1 may repress Hif-2a translation; however, the net result in our system is induction of Hif-2a protein expres- sion. Activation of the Hif-2a-Epo pathway mediates a compensatory increase in Fpn expression, which increases iron availability from tissues and iron import from the diet. Furthermore, both the Epo system and extramedullary ery- thropoiesis are required for recovery from chemically- induced anemias in Ncoa4-null mice. As Ncoa4-depleted RBC maintain their ability to synthesize heme and hemo- globin, there must exist additional baseline or compensa- tory pathways to ensure continued iron delivery to the mitochondria. Our erythroid compartment-specific mouse model shows a cell autonomous role of Ncoa4 but also highlights the non-autonomous role of Ncoa4 in maintain- ing erythropoiesis. Future work will be directed at deter- mining the non-autonomous contribution of Ncoa4 to ery- thropoiesis, with the liver (hepatocytes) and macrophages being two likely cell types given their established roles in systemic iron homeostasis.
Acknowledgments
We thank Roderick Bronson in the Rodent Histopathology Core at the Dana-Farber/Harvard Cancer Center in Boston, MA for tissue processing and histopathological interpretation services. We thank Steven Gygi for use of CORE for mass spectrometry data analysis. This work was supported by a Burroughs Wellcome Fund Career Award for Medical Scientists, Brigham and Women’s Hospital MFCD Award, Sidney Kimmel Foundation Kimmel Scholar Program, and Dana-Farber Cancer Institute Claudia Adams Barr Program for Innovative Cancer Research Award to JDM, and NIH grant R01GM095567 to ACK and JWH WF and BM are supported by the First TEAM of the Foundation for Polish Science. Dana-Farber/Harvard Cancer Center is supported in part by a NCI Cancer Center Support Grant # NIH 5 P30 CA06516.
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