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L. Liang al.
In vitro deubiquitylation assays
In vitro ubiquitylation assays were performed as previously
described28 and additional details are provided in the Online Supplement.
Statistical analysis
All data are presented as mean ± standard deviation (SD), and the results were analyzed using the SPSS 18.0 software package. Significant differences between groups were determined using analysis of variance and the Tukey range test.
Results
Deficiency of USP7 impairs human terminal erythroid differentiation
To explore the roles of deubiquitylases during erythro- poiesis, we first analyzed the expression patterns of de- ubiquitylases in human erythroblasts at different stages of differentiation from our RNA-sequencing data.2 Figure 1A shows the expression patterns of USP family members and reveals that the expression levels of USP7 are signifi- cantly increased during erythropoiesis. Based on the pre- viously identified important role of USP7 in cell differenti- ation in other cellular systems,17-19 in the present study we focused our attention on the role of USP7 in erythroid dif- ferentiation. We confirmed the increased expression of USP7 during late stages of erythroid differentiation by both real-time PCR (Figure 1B) and by western blot analy- sis (Figure 1C). To examine the effect of USP7 on erythro- poiesis, we employed a shRNA-mediated knockdown approach in human CD34+ cells.27,29,30 As shown in Figure 1D, USP7 knockdown impaired the terminal erythroid dif- ferentiation as demonstrated by the decreased surface expression of the erythroid marker GPA, delayed loss of α4-integrin expression in association with decreased sur- face expression of band 3. There was also a marked decreased in the extent of enucleation. USP7 knockdown also inhibited the expression of hemoglobin (Figure 1E). The significant impairment of the growth of late-stage erythroblasts caused by USP7 knockdown was accompa- nied by increased apoptosis (Online Supplementary Figure S1A, B). Similar to USP7 knockdown, USP7-specific inhibitors P5091 and P2207731,32 also impaired human ter- minal erythroid differentiation, inhibited hemoglobin expression (Figure 1F, G) and cell proliferation (Online Supplementary Figure S1C, D). These results imply that USP7 plays an important role in human terminal erythroid differentiation.
USP7 regulates erythroid differentiation by modulating GATA1 protein levels
We subsequently explored the molecular mechanism(s) of the altered erythropoiesis due to USP7 deficiency. Given the fact that USP7 functions in the nucleus,33,34 we hypothesized that USP7 might affect erythropoiesis by regulating erythroid differentiation-related transcription factors. As shown in Figure 2A and Online Supplementary Figure S2, GATA1 was the transcription factor most signif- icantly decreased after knockdown of USP7, although KLF1 levels also decreased. Since KLF1 expression is regu- lated by GATA1,35 we suggest that the decreased expres- sion of KLF1 is a consequence of GATA1 downregulation. Interestingly, mRNA levels of GATA1 were not affected by USP7 knockdown on day 9 (Figure 2B), suggesting that
the observed decrease in GATA1 levels is at the post-tran- scriptional level. We noted decreased levels of GATA1 mRNA levels on days 11 and 13 following USP7 knock- down (Online Supplementary Figure S3A, B), likely due to the fact that GATA1 mediates its own regulation at the late stages of erythropoiesis.36,37 Similarly, inhibition of USP7 activity by the USP7-specific inhibitors P5091 and P22077 also resulted in significant decreases in GATA1 protein levels in a dose-dependent manner (Figure 2C, D), with no obvious effects on other transcription factors beside a slightly decreased expression of KLF1 (Online Supplementary Figure S4), implying that USP7-mediated regulation of GATA1 depends on the enzymatic activity of USP7. To further confirm that USP7 deficiency-induced defective erythropoiesis is due to downregulation of GATA1, we performed rescue experiments by ectopically expressing GATA1 in USP7 knockdown cells. Figure 2E shows that the delayed erythroid differentiation as well as impaired erythroblast enucleation could be rescued by restoring GATA1 levels. Furthermore, the expression of GATA1 target genes such as 4.1R and HBG were also res- cued (Figure 2F). These results imply that USP7 regulates erythroid differentiation through GATA1.
USP7 regulates the stability of the GATA1 protein
GATA1 protein levels are regulated by several proteins, including HSP70, a GATA1 chaperone38-40 and RPS19, nec- essary for GATA1 translation.26,41 To examine whether HSP70 and RPS19 are involved in the regulation of GATA1 mediated by USP7, we analyzed the effect of USP7 knock- down on their expression levels. As shown in Online Supplementary Figure S5A, B, USP7 knockdown or inhibi- tion had no effects on HSP70 or RPS19 protein levels. Moreover, USP7 knockdown did not affect the transloca- tion of HSP70 into the nucleus (Online Supplementary Figure S5C, D). The above findings strongly suggest that USP7 regulates the stability of GATA1 protein directly. We per- formed several additional studies to confirm this hypoth- esis and to define the underlying mechanisms. First, co- expression of wildtype USP7 (USP7-WT) with GATA1 increased the GATA1 level (Figure 3A). Importantly, cat- alytically inactive mutant USP7 (USP7-CS, C233S) did not increase GATA1 protein levels (Figure 3B). Second, down- regulation of GATA1 by USP7 knockdown or the USP7 inhibitors P5091 and P22077 was reversed by the protea- some inhibitor MG132 (Figure 3C-E), implying that USP7 maintains the steady-state levels of GATA1 by blocking its proteasomal degradation. To further examine the relation- ship between USP7 and GATA1, we measured the half-life of intracellular GATA1 after cells had been treated with cycloheximide to inhibit protein biosynthesis. As shown in Figure 3F, knockdown of USP7 significantly shortened the half-life of the GATA1 protein. Conversely, overex- pression of USP7-WT, but not USP7-CS, prolonged the half-life of GATA1 (Figure 3G). Taken together, our data demonstrate that USP7 stabilizes GATA1 by preventing its proteasomal degradation.
USP7 interacts directly with GATA1
Having demonstrated that USP7 stabilizes GATA1, we then examined whether this effect is through their direct interaction by performing co-immunoprecipitation exper- iments. USP7 or GATA1 was separately immunoprecipi- tated from cultured primary erythroblasts and the recipro- cal protein was detected by western blot analysis. As
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haematologica | 2019; 104(11)