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L. Liang al.
Figure 6. A schematic model of post-transcriptional regulation of GATA1. Post-transcriptional regulation of GATA1 includes the translational and post-translational levels. The translational level of GATA1 is mainly controlled by RPS19. Decrease or mutation of RPS19 results in reduced translation of GATA1. At the post-transla- tional level, the nuclear HSP70 protects GATA1 from caspase 3 cleavage. In addition, acetylation and phosphorylation of GATA1 cooperate as the signal for ubiquity- lation of GATA1 to degradation. USP7 interacts directly with GATA1 and maintains stability of GATA1 by removing the poly-ubiquitylation.
is known about the mechanisms by which the proteome is remodeled. Previous studies demonstrated that the bal- ance between ubiquitination and deubiquitination plays important roles in homeostasis of cellular protein pools.45 In the present study, we documented, for the first time, the role of a deubiquitylase, USP7, in erythroid differenti- ation. We further documented that the mechanism is sta- bilization of the erythropoiesis master regulator GATA1.
USP7 is a member of a deubiquitinating enzyme family that contains more than 90 genes.46 USP7 expression is ubiquitous in different cell types: mice with knocked out USP7, which are homozygous for a null allele, show embryonic growth arrest and die between embryonic day 6.5 and 7.5.47 Furthermore, conditional knockout mice showed that USP7 is required for development of the cen- tral nervous system and functional regulatory T cells.48,49 We expect that deletion of USP7 in erythroid cells in vivo will led to altered erythropoiesis. We are in the process of generating such conditional knockout mice to define the function of USP7 in vivo. Besides USP7, many other de- ubiquitinating enzymes are also expressed in erythroid cells, although at lower levels than USP7. It will be inter- esting in future studies to identify the functional roles of other deubiquitylases during erythroid differentiation. Since each deubiquitinating enzyme has different sub- strate specificity,47 it is likely that members of the deubiq- uitinating enzyme family may regulate different aspects of erythropoiesis via different mechanisms.
As the key transcriptional factor for erythropoiesis, GATA1 protein expression is tightly regulated at several levels. These include translational control by ribosome lev-
els,26,41 stabilization by HSP70 from caspase 3 cleavage,27,38-40 and degradation by acetylation and phosphorylation-asso- ciated ubiquitination.24 Here we show that knockdown of USP7 by shRNA or inhibition of USP7 activity by USP7- specific inhibitors led to dramatic decreases in GATA1 pro- tein levels. Interestingly, USP7 knockdown or inhibition had no effects on the protein levels of RPS19 or HSP70, strongly suggesting that USP7 affects the stability of GATA1 in a direct manner. This notion is supported by our findings that USP7 binds GATA1 directly and stabilizes GATA1 by de-ubiquitination. Specifically, USP7 catalyzes the removal of K48-linked poly-ubiquitin which is a pro- teasome degradation signal for proteins. Based on our find- ings and that of others, we propose a schematic model for post-transcriptional regulation of GATA1 (Figure 6). GATA1 functions in the context of multi-protein complex- es that include interacting proteins such as FOG1.50-53 Although USP7 knockdown or inhibition did not affect the level of FOG1 or NuRD complex (Online Supplementary Figure S8), we cannot exclude the possibility that these important GATA1 cofactors or modifications can modulate GATA1-USP7 interactions during erythroid development. Further study is therefore warranted to investigate whether USP7 binds other cofactors such as FOG1 and/or different modifications of GATA1.
Altered expression of GATA1 has been reported in myelodysplastic syndromes39,54 and β-thalassemia.40 However, the mechanisms of the altered GATA1 expres- sion remains to be fully defined. It has been reported that USP7 is associated with several human diseases.55-57 Given the close relationship between USP7 and GATA1, demon-
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haematologica | 2019; 104(11)