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Deubiquitylase USP7 and human erythropoiesis
In contrast, the regulation of erythropoiesis by other mechanisms has been less well studied. Notably, our knowledge on post-translational regulation of erythro- poiesis is limited.
Ubiquitination is an enzymatic post-translational modifi- cation. Ubiquitinated proteins are degraded by the ubiqui- tin-proteasome system (UPS). The UPS controls the degra- dation of most intracellular proteins and plays important roles in many cellular processes.5 Although the UPS was first discovered in reticulocytes over 40 years ago,6 to date there are only limited studies on the roles of the UPS in erythro- poiesis. These include the reported role of CUL4A-mediated degradation of p27 in cell proliferation in the early stages of erythropoiesis and cell cycle exit at a later stage of erythro- poiesis.7,8 A recent, exciting study demonstrated that UBE2O remodels the proteome during terminal erythroid differenti- ation, underscoring the importance of the UPS in erythro- poiesis.9
Ubiquitination is a dynamic and reversible process.10 It has been reported that deubiquitylases are capable of removing ubiquitin from their protein substrates and allow proteins to be salvaged from proteasomal degradation.11 USP7 is a de- ubiquitylase that belongs to the ubiquitin-specific protease (USP) family, which constitutes the largest subgroup of de- ubiquitylases. Accumulated evidence has shown that USP7 plays diverse roles in genome stability, epigenetic regulation, the cell cycle, apoptosis, viral infection, immunity and stem cell maintenance.12-17 Recently, USP7 was reported to be an important regulator of osteogenic differentiation and adipo- genesis.18,19 Our RNA-sequencing analyses revealed high- level expression of genes/pathways (including USP7) involved in the ubiquitin system during late stages of termi- nal erythroid differentiation.2 Nevertheless, the function of USP7 in human erythropoiesis remains unexplored.
GATA1 is the key transcription factor for erythropoiesis, controlling the expression of a large series of erythroid genes, including erythropoietin receptor, globins and several
20
membrane proteins. GATA1-deficient mice die in utero due
to severe anemia at embryonic day 10.5-11.5,21 and chimeric mice lacking GATA1 fail to produce mature red blood cells, although the formation of cells of other hematopoietic line- ages is normal.22 In contrast, overexpression of GATA1 in erythroid cells inhibits their differentiation, leading to fatal anemia in mice.23 GATA1 stability is finely regulated by mul- tiple mechanisms,24 since changes in its protein levels will exert a great influence on erythropoiesis. Although GATA1 degradation by the ubiquitin-proteasome pathway has been characterized,24 how GATA1 recycles from the UPS is yet to be defined.
In this study, we demonstrated that USP7 deficiency impairs human terminal erythroid differentiation due to a decreased level of GATA1 protein. We further showed that USP7 interacts directly with GATA1 and catalyzes the removal of poly-ubiquitylation chains on GATA1, thus sta- bilizing GATA1. Our findings have thus not only document- ed the role of a deubiquitylase in erythropoiesis, but also enabled the identification of a novel mechanism by which deubiquitylases regulate GATA1 protein stability.
Methods
Reagents and antibodies
P5091 (S7132) and MG132 (S2619) were obtained from Selleckchem (TX, USA); P22077 (HY-13865) from MCE (NJ,
USA); and cycloheximide was purchased from Sigma-Aldrich (MO, USA). Antibodies used for western blot, immunoprecipita- tion and immunofluorescence studies are detailed in the Online Supplementary Methods. The antibodies used for flow cytometry analysis were glycoprotein A (GPA)-PE-Cy7, GPA-APC, and α4- integrin (CD49d)-PE from BD Pharmingen (NJ, USA). Band 3- APC and 4.1R antibodies were used as previously described.25
Cell culture
Human cord blood samples were obtained from Xiangya Hospital of Central South University or New York Blood Center under Institutional Review Board approval and in accordance with the Declaration of Helsinki. The detailed composition of the culture medium and the cell culture protocol has been described previously.25 HEK293T cells (American Type Culture Collection: CRL-11268) were cultured in Dulbecco modified Eagle medium (Gibco, MA, USA) supplemented with 10% fetal bovine serum (Gibco).
Lentivirus packaging and infection
USP7-specific short hairpin (sh)RNA was purchased from GenePharma (Shanghai, China) (shRNA #1: 5’- AGTCGTTCAGTCGTCGTAT-3’ and #2: 5’-TGGATTTGTG- GTTACGTTACTC-3’, constructed in pGLV3-H1-GFP or pGLV2- U6 vector). GATA1 overexpression (HMD-GATA1-IRES-GFP) and control plasmids have been described previously.26 Lentiviruses were packaged in HEK293T cells according to the manufacturer’s protocol (Invitrogen, MA, USA). A total of 30x107 lentiviral particles were infected using polybrane with 0.5x107 CD34+ cells on day 3 or 4. Puromycin (1 mg/mL) was used for selection of transduced cells.
GATA1 rescue assay
For rescue experiments, erythroid cells were infected with USP7 shRNA or control shRNA lentiviruses for 3 days. On day 7 of culture, erythroid cells were transduced with the control or GATA1 lentivirus. Double-transduced cells were identified fol- lowing puromycin (1 μg/mL) selection and GFP expression from the HMD vector. The extent of terminal erythroid differentiation was monitored beginning on day 9.
RNA isolation, quantitative real-time polymerase chain reaction and western blot analysis.
Standard protocols were used for RNA and protein isolation, polymerase chain reaction (PCR) and western blot analysis. Details are given in the Online Supplementary Methods. The GATA1 primer sequences were described previously.27 USP7 primer sequences were: forward: 5’-AGCGTGGCATCACCATAATC-3’ and reverse: 5’-CGAGGCAACCTTTCAGTTCA-3’.
Immunoprecipitation and glutathione-S-transferase pull-down
Immunoprecipitation studies were performed using M2/Flag or protein A/G-agarose beads. For the glutathione-S-transferase (GST) pull-down assay, purified Flag-USP7 and bacterial expressed GST or GST-GATA1 were used. The methods are described in detail in the Online Supplementary Methods.
In vivo ubiquitylation and deubiquitylation assays
For cell-based deubiquitylation assays, Flag-GATA1 and HA- ubiquitin were co-transfected with an empty vector or a vector expressing USP7 (WT or CS) for 48 h. For USP7 knockdown, the cells were infected with the lentiviruses for 48 h. Additional details of the methods are given in the Online Supplementary
Methods.
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