Deubiquitinase USP7 Regulates Erythroid Development Via Deubiquinating GATA1

Abstract

Erythropoiesis is the process which produces mature red blood cells from haematopoietic stem cell. Ubiquitination/deubiquititation plays important roles in many cellular processes. Previous studies have documented the roles of ubiquitination and several ubiquitinases in erythropoiesis. In contrast, the roles of deubiquitiation/deubiquitinases in erythropoiesis remain unexplored. In this study, we found that the expression of deubiquitinase USP7 was significantly upregulated during erythropoiesis. We then employed shRNA mediated knockdown approach to explore the role USP7, we found that USP7 knockdown significantly delayed terminal erythroid differentiation, inhibited the hemoglobin expression and cell proliferation, and induced apoptosis. To explore the underlying mechanisms for the observed phenotypic changes, we compared the expression of several key erythroid transcription factors including GATA1, KLF1, LSD1, SOX1 and IKAROS between control and USP7-knockdown cells by western blot analysis. We found that only the protein level of GATA1 but not other transcription factors was significantly decreased following USP7 knockdown. Moreover, GATA1 mRNA level was not affected by USP7 knockdown. The effect of USP7 on the stability of GATA1 protein was further demonstrated by the findings that inhibition of USP7 by USP7 inhibitors also resulted in decreased GATA1 protein levels and that knockdown/inhibition of USP7 shorten the GATA1 half-life and increased the ubiquitination of GATA1. Furthermore, we performed Co-IP, pull down and in vitro ubiquitization experiment which documented that USP7 directly binds to and deubiquinates GATA1. Our findings demonstrate that GATA1 is the substrate of USP7 and that USP7 stabilizes GATA1 by deubiquinating GATA1. To examine whether the impaired erythropoiesis following USP7 knockdown is due to decreased GATA1, we ectopically expressed GATA1 in USP7 knockdown cells and showed that the delayed differentiation was rescued by GATA1 overexpression. To demonstrate the role of USP7 in vivo, we established a stress erythropoiesis mouse model by phlebotomy and then treated the mice by intraperitoneal injection of USP7 inhibitor. We found USP7 inhibitor delayed the recovery of anemia. Collectively, our findings not only identified previously unknown roles of USP7 in erythropoiesis but also identified a novel regulatory mechanism for GATA1 expression.AcknowledgmentsThis work was supported in part by grants 81270576, 81470362 and 81530005 from Natural Science Foundation of China, by NIH grants DK100810 and DK26263, and by postgraduate innovation project of Central South University of China 2016zzts165.The authors declare no conflict of interest. DisclosuresNo relevant conflicts of interest to declare.