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L. Liang al.
USP7 stabilizes GATA1 protein through K48 deubiquitylation
Since USP7 is a deubiquitylase, it is reasonable to spec- ulate that USP7 regulates the stability of GATA1 via de- ubiquitylation. To test this hypothesis, we investigated the effect of USP7 on the poly-ubiquitylation of GATA1. As expected, knockdown of USP7 resulted in a significant increase in the poly-ubiquitylation of GATA1 (Figure 5A). In contrast, ectopic expression of USP7-WT but not the catalytic inactive mutant USP7-CS reduced the level of poly-ubiquitylation of GATA1 (Figure 5B and Online Supplementary Figure S6A). Moreover, USP7-mediated
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decrease of GATA1 poly-ubiquitylation was blocked by the USP7 inhibitors P5091 and P22077 (Figure 5C and Online Supplementary Figure S6B), demonstrating that the enzymatic activity of USP7 is essential for the USP7-medi- ated deubiquitylation of GATA1. To verify that GATA1 is a direct substrate of USP7, the purified USP7 protein was incubated with ubiquitylated GATA1 in a cell-free system. As shown in Figure 5D, GATA1 poly-ubiquitylation was decreased in the presence USP7, indicating that USP7 de- ubiquitylates GATA1 directly. To determine which poly- ubiquitin chain on GATA1 is removed by USP7, we employed a series of ubiquitin mutants that contain only
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Figure 4. USP7 interacts directly with GATA1. (A) Representative western blot analysis of USP7 after immunoprecipitation of endogenous GATA1 at the indicated time-points (days 9, 11 and 13) from erythroblasts. (B) Representative western blot analysis of GATA1 after immunoprecipitation of endogenous USP7 at the indicated time-points (days 9, 11 and 13) from erythroblasts. (C) Representative western blot analysis of pull-down of purified Flag-USP7 with purified GST-GATA1. (D) Schematic representation of the USP7 deletion mutants used for domain mapping (left panel). HEK293T cells ectopically expressing GATA1 were co-transfected with USP7 deletion mutants. Interactions were analyzed using a co-immunoprecipitation assay (right panel). (E) Schematic representation of full length and various deletion mutants of GATA1 used for domain mapping (left panel). HEK293T cells ectopically expressing USP7 were co-transfected with GATA1 deletion mutants. Interactions were analyzed using a co-immunoprecipitation assay (right panel).
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haematologica | 2019; 104(11)


































































































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