ULK1 Regulates the Proliferation and Differentiation of Erythroid Progenitors

Erythropoiesis depends on the proliferation and differentiation of erythroid progenitors. However, underlying mechanism of balance of these two processes in erythroid progenitors is largely unknown. Unc-51 like autophagy activating kinase 1 (ULK1) drives the maintenance and differentiation of stem cells, and ULK1 keeps low expression in erythroid progenitors and rapidly increases during differentiation. Therefore, we hypothesize that the proliferation of progenitor cells and their transition to differentiation is regulated by increasing expression of ULK1. To explore the function of ULK1 in erythroid progenitors, ULK1 was inhibited by inhibitor SBI-0206965 (SBI) or ULK1 shRNA at day 4 (D4) during UCB-derived erythroid differentiation in vitro. Quantifying treated progenitors by colony-forming assay and flow cytometry indicated ULK1 influenced erythroid differentiation at CFU-E cell stages. The results showed that the differentiation of CFU-E cells was depressed, but their proliferation was enhanced when ULK1 was inhibited. Meanwhile, opposite results were observed when over-expressing ULK1 in erythroid progenitors. Moreover, the number of mitochondria, cell cycle, and apoptosis were not impacted by inhibiting ULK1. Further, the proteomics and RNA-sequencing data showed that the expression of AHR signaling-related genes like CYP1B1, CYP1A1, and AHRR were up-regulating by ULK1 inhibition suggesting the activation of AHR. Both AHR antagonist StemReginin 1 and AHR shRNA rescued the effects of ULK1 repressed in erythroid progenitors. We also observed that the number of AHR in the nucleus was increased, though the total expression of AHR was slightly decreased due to ULK1 inhibition, indicating that ULK1 plays a role in regulating AHR activation. Mechanically, we demonstrated that ULK1 directly interacted with AHR in cytoplasm, and phosphorylates Ser-73 of AHR. Then phosphomimetic (Ser-73 –> Asp, AHRS73D) and loss-of-function (Ser-73 –> Ala, AHRS73A) mutants were generated respectively to study whether Ser-73 of AHR can impact differentiation erythroid progenitors. Compared to AHRWT, AHRS73D promoted the differentiation, while AHRS73A showed the opposite result. Meanwhile, we observed that more AHRS73D were located in the cytoplasm, while more AHRS73A were located in the nucleus. These results were consistent with the observations of ULK1. Together, our data indicate that during the differentiation of erythroid progenitors, increased expression of ULK1 inhibited AHR translocated to the nucleus by phosphorylating Ser-73. On the contrary, low expression of ULK1 benefits AHR translocated to the nucleus, which enhances the proliferation of erythroid progenitors.