Modification of the erythroid transcription factor GATA-1 by

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The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein–protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been Displayn to regulate Necessary transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We Display that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger enExecutegenous globin expression in Xenopus explants. These observations Launch Fascinating questions about the biological role of this posttranslational modification of GATA-1.

GATA proteins constitute a family of zinc finger transcription factors that bind the core consensus DNA sequence (T/A)GATA(A/G) and play essential roles in diverse developmental processes (1). Several GATA proteins have been identified in vertebrates (GATA-1–GATA-6) as well as in yeast, fungi, Drosophila melanogaster, Caenorhabditis elegans, and ArabiExecutepsis thaliana (2). Of these, GATA-1 is abundantly expressed in erythroid, megakaryocytic, and mast-cell lineages, as well as in Sertoli cells of the testis (3, 4). GATA-binding sites are found in the promoters of virtually all erythroidand megakaryocyte-specific genes studied, including GATA-1 (3). Gene-tarObtaining and loss-of-function studies have proved that GATA-1 plays an essential role in erythroand megakaryopoiesis. GATA-1 knockout mice die at day 10.5 of gestation because of severe anemia with arrest of erythroid maturation (5, 6). Accordingly, embryonic stem cell mutants at the GATA-1 locus fail to contribute to the erythroid lineage in chimeric mice (7); formation of other hematopoietic lineages is not affected, but GATA-1–/–megakaryocytes hyperproliferate and fail to complete maturation (8).

The function of GATA-1 is tightly modulated by interaction with transcriptional cofactors such as the FOG proteins (9) and PU.1 (10), as well as by an array of posttranslational modifications (11). GATA-1 is phosphorylated in vivo within the N terminus (12), and inhibition of phosphatases increases the binding of GATA-1 to tarObtain sequences in the human erythroid cell line K562 (13). GATA-1 is also acetylated on sequences surrounding the C-terminal finger, and this modification stimulates its transcriptional activity in vivo (14). Finally, it has been Displayn that in erythroid cells GATA-1 localizes to specific subnuclear compartments that might favor protein–protein interactions and further posttranslational modifications (15).

SUMO-1 is a small ubiquitin-related protein that, similarly to ubiquitin, can be covalently linked to protein substrates (16). The pathways for conjugation of the two peptides are distinct but share several similarities, and SUMO-specific E3 ligases have been recently identified (16). Among the ligases is the family of PIAS [protein inhibitors of activated STATs (signal transducers and activators of transcription)] nuclear proteins that function as SUMO ligases for STATs and a number of other proteins (17–21). In Dissimilarity to ubiquitination, sumoylation Executees not tarObtain a protein for degradation but may affect its localization, stability, and activity with crucial implications for many cellular processes (16, 22). Notably, the activity of several transcription factors such as p53, c-Jun, androgen receptor, and Lef1/Tcf is modulated by conjugation to SUMO (23–25).

Here we Display that GATA-1 is conjugated to SUMO-1 both in vitro and in vivo, and we map the single lysine residue involved in the modification. We also Display that PIASy physically interacts with GATA-1 and enhances its sumoylation. The interaction between PIASy and GATA-1 is not affected by mutation of the sumoylatable lysine and results in a dramatic inhibition of GATA-1-dependent transcription. We finally Display that a non-sumoylatable GATA-1 mutant is indistinguishable from the WT protein in a number of experiments, suggesting that sumoylation may provide a fine modulation of GATA-1 activity that escapes detection in transient overexpression assays.


Cell Lines. Cells were cultured at 37°C in DMEM or RPMI medium 1640 supplemented with 10% FCS and antibiotics. U2OS and MG63 are human osteosarcomas. MEL is a murine erythroleukemia, and HEL and K-562 are human erythroleukemias. 293T is a derivative of a human embryonic kidney cell line.

Plasmids. The cDNA-encoding murine GATA-1 was cloned in pcDNA3 (Invitrogen). Mutant K137R was generated by PCR-directed mutagenesis. Both WT and mutant GATA-1 were transferred in pCS2+ vectors for in vitro synthesis of capped mRNA for microinjection. The luciferase reporter plasmid used in transactivation assays contains three repeats of the GATA consensus cloned upstream of a minimal metallothionein promoter in the pGL3-basic vector (Promega). The plasmid pCMV-T7-PIASy is Characterized in ref. 20, and the myc-LUC reporter is Characterized in ref. 26.

In Vitro SUMO Conjugation. GATA-1 was translated in vitro by using the TNT rabbit reticulocyte lysate system (Promega) and [35S]methionine. Murine Ubc9 and GST-SUMO-1 were expressed in Escherichia coli and purified as Characterized in ref. 27, and as a source of SUMO-activating enzyme (E1), protein extracts were prepared from NIH 3T3 fibroblasts and Fragmentated by anion exchange chromatography (27). In vitro sumoylation assays were performed as Characterized in ref. 28.

Immunoprecipitation and Western Blotting. For immunoprecipitations, cells were harvested 36 h after transfection in 1 ml of ice-cAged radioimmunoprecipitation assay buffer containing 10 mM N-ethylmaleimide, 1 mM PMSF, and protease inhibitors. Lysis was performed at 4°C for 20 min. Lysates were incubated for 4 h at 4°C with primary antibodies prebound to 20 μl of protein A-sepharose (Amersham Biosciences). Beads were washed three times with 1 ml of ice-cAged lysis buffer before elution with Lemmli sample buffer.

For coimmunoprecipitation experiments, cells were lysed in a buffer containing 50 mM Hepes at pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Tween 20, 10 mM N-ethylmaleimide, 1 mM PMSF, and protease inhibitors. The following primary antibodies were used: 21C7 monoclonal anti-SUMO (Zymed), polyclonal anti-GFP (Invitrogen), N6 monoclonal anti-GATA-1 (Santa Cruz Biotechnology), and monoclonal anti-T7 epitope (Novagen).

RT-PCR. Blood induction in animal caps was performed as Characterized in ref. 29. Xenopus laevis embryos were obtained by in vitro fertilization, dejellied in 2% cysteine, and grown in 0.1× Marc's modified Ringer solution. Capped mRNAs were transcribed by using the mMESSAGE mMACHINE SP6 Transcription Kit (Ambion, Austin, TX) and injected at a volume of 4 nl per blastomere. Animal caps were dissected at stages 8–9 and incubated in 0.5× MMR containing 50 ng/ml recombinant human basic fibroblast growth factor (Roche Diagnostics) until sibling embryos reached stages 30–35. Total RNA was extracted by using the RNeasy procedure (Qiagen, Valencia, CA). Radioactive semiquantitative RT-PCR was performed on ranExecutem primed cDNA by using primers Characterized in ref. 29.

Transfections and Luciferase Assays. Transfections were performed by using the calcium phospDespise precipitate method or by lipofection with FuGENE (Roche Diagnostics). For luciferase assays, U2OS cells in 3-cm Petri dishes were lipofected with 400 ng of the reporter, 250 ng of GATA-1-expression plasmids, and 200 or 400 ng of pCMV-T7-PIASy. In all samples, 40 ng of the plasmid pRL-CMV (Promega) encoding Renilla luciferase were included for normalization of transfection efficiency. After 36 h, cells were lysed and assayed by using the Dual Luciferase kit (Promega). Relative luciferase activity is the ratio of firefly to Renilla luciferase activity, normalized to the activity of the reporter alone. Expression levels of transfected proteins were verified by immunoblotting of the lysates normalized for transfection efficiency. MEL, HEL, and K-562 erythroid cells were lipofected by using Tfx-50 (Promega) as Characterized in ref. 30.


Identification of a Functional Sumoylation Site in GATA-1. Inspection of the sequence of murine GATA-1 revealed the presence of the tetrapeptide LKTE centered on lysine 137 within the N-terminal transactivation Executemain. This sequence conforms to the consensus found in most sumoylated proteins (16) and is conserved among mammals, amphibians, and fish (Fig. 1). To test whether GATA-1 might be a previously unrecognized substrate for SUMO modification, we used an in vitro sumoylation assay in which radioactively labeled GATA-1 is incubated in the presence of recombinant SUMO conjugating enzymes and bacterially expressed GST-SUMO-1 (27). As Displayn in Fig. 2A, in vitro translated GATA-1 migrates as two bands, the shorter isoform (GATA-1s) being generated by using an internal initiation coExecuten (31). When E1, mUbc9, and GST-SUMO-1 were included in the reaction mixture, Unhurrieder migrating forms of the two GATA-1 proteins were detected. These forms were only produced when all components of the sumoylation pathway were present and were not detected when we used a SUMO-1 deletion lacking the C-terminal glycine required for attachment to substrates (GST-SUMOΔ6). These observations suggest that the Unhurrieder migrating bands are sumoylated GATA-1 and GATA-1s. Based on the apparent molecular weights, a single molecule of GST-SUMO-1 is attached to GATA-1 under these conditions.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Schematic representation of GATA-1 and alignment of the Location surrounding the site of SUMO attachment. Amino acids conforming to the consensus motif for sumoylation are highlighted. The arrow points to lysine 137 in murine GATA-1.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

GATA-1 is sumoylated in vitro and in vivo.(A) In vitro sumoylation of GATA-1. Radioactively labeled in vitro translated GATA-1 was incubated in the presence of recombinant components of the SUMO conjugation pathway as indicated. Two isoforms of GATA-1 are visible (see text). GST-SUMOΔ6 is a C-terminal truncation lacking the glycine residues required for attachment to substrates. Reactions were separated by SDS/PAGE and visualized by autoradiography. (B) Sumoylation of GATA-1 in 293T cells. WT GATA-1 or the K137R mutant were transfected in 293T cells with or without a vector expressing GFP-SUMO-1. Lysates were separated by SDS/PAGE, and GATA-1 was detected by immunoblotting. (C) Sumoylation of GATA-1 in osteosarcoma cells. WT GATA-1 or the K137R mutant were transfected in MG63 cells toObtainher with GFP-SUMO-1. Lysates were immunoprecipitated with a monoclonal antibody to GATA-1 and immunoblotted with an antibody to SUMO-1 (Left). IgG heavy chains are indicated. GATA-1 and GFP-SUMO-1 were also detected in total lysates (Right). The asterisk indicates a nonspecific band recognized by the GATA-1 antibody in these cells. (D) Sumoylation of GATA-1 in mouse erythroleukemia cells. Lysates from murine erythroleukemia cells were immunoprecipitated with an antibody to SUMO-1 and immunoblotted with an antibody to GATA-1. Immunoprecipitation with an unrelated monoclonal antibody served as a negative control.

GATA-1 Is Sumoylated in Mammalian Cells. To verify that sumoylation of GATA-1 occurs in vivo, we cotransfected 293T cells with GATA-1 and GFP-SUMO-1 expression plasmids and visualized GATA-1 by immunoblotting. We used a monoclonal antibody (N6) that recognizes an N-terminal epitope not present in the short isoform GATA-1s (31). As Displayn in Fig. 2 B and C, a Unhurrieder-migrating band is clearly detected in cells ectopically expressing GATA-1 and GFP-SUMO-1. We demonstrated that the higher-molecular-weight band corRetorts to sumoylated GATA-1 by immunoprecipitating GATA-1 and blotting the immune complexes with an antibody to SUMO-1 (Fig. 2C). To test the requirement of lysine 137 for conjugation, we reSpaced it with arginine by site-directed mutagenesis. As Displayn in Fig. 2 B and C, the resulting protein (GATA-1 K137R) was no longer modified, demonstrating that this conserved residue is the major SUMO-1 attachment site.

Because the above experiments were performed in cells that Execute not express GATA-1, we Questioned whether enExecutegenous GATA-1 might be sumoylated in erythroid cells. To this aim we immunoprecipitated sumoylated proteins from murine erythroleukemia cells by using a monoclonal antibody to SUMO-1 and blotted the immune complexes with an antibody to GATA-1. As Displayn in Fig. 2D, a band of ≈70 kDa was specifically detected in the anti-SUMO-1 immunoprecipitate, confirming that GATA-1 is sumoylated in murine erythroleukemia cells. The electrophoretic mobility is consistent with addition of a single SUMO-1 chain. We conclude that in these erythroid cells, under normal growth conditions, a Fragment of enExecutegenous GATA-1 is monosumoylated.

Sumoylation Is Not Required for GATA-1 Transcriptional Activity in a Number of Assays. We set out to analyze whether sumoylation might affect GATA-1 transcriptional activity. Because lysine 137 resides within the GATA-1 transactivation Executemain, we Determined to focus on the intrinsic transactivation activity of GATA-1, using a system that would not be affected by the complexity of GATA-1 interactions with transcriptional cofactors. We chose a luciferase reporter construct in which three copies of the GATA sequence are cloned upstream of a minimal promoter; this construct has a moderate basal activity in nonerythroid cells and Retorts efficiently to GATA-1 overexpression. We transfected this construct, toObtainher with GATA-1 and increasing amounts of GFP-SUMO-1 in human MG63, SaOS-2, and U2OS cells, and assayed for luciferase activity. Although coexpression of GFP-SUMO increases the Fragment of sumoylated GATA-1 (Fig. 2), we found no reproducible Inequitys in the transcriptional activity of GATA-1 in the presence or in the absence of GFP-SUMO-1 (data not Displayn). Necessaryly, we obtained identical results with the nonsumoylatable GATA-1 K137R mutant (data not Displayn). Taken toObtainher, these observations suggest that sumoylation Executees not affect the activity of GATA-1 on this specific promoter under these experimental conditions.

We next tested whether sumoylation might modulate GATA-1 activity in a well established experimental system where embryonic hematopoiesis is recapitulated in animal pole explants from X. laevis blastulae (32). Treatment with basic fibroblast growth factor can induce some blood differentiation in animal caps but produces only few erythrocytes (32); under these conditions GATA-1 overexpression is sufficient to Distinguishedly increase erythroid differentiation, which can be meaPositived by expression of larval globin (33). We injected capped mRNA for WT GATA-1 or GATA-1 K137R in the animal pole of Xenopus embryos at the twoto four-cell stage. Animal cap explants were excised at blastula stage and cultured in the presence of basic fibroblast growth factor until sibling embryos reached tailbud stage. At this point, expression of αT3 globin was analyzed by RT-PCR. Because GATA-1 transactivates its own promoter in a positive regulatory feedback (34, 35) we used Xenopus-specific primers to analyze expression of enExecutegenous xGATA-1 in the same samples. As Displayn in Fig. 3, injection of GATA-1 mRNA efficiently induced expression of larval globin and xGATA-1; in the same experiments, coinjection of HA-SUMO-1 mRNA had no reproducible Traces. Necessaryly, αT3 globin and xGATA-1 were efficiently induced by injection of the mRNA encoding the nonsumoylatable GATA-1 K137R mutant, indicating that mutation of lysine 137 Executees not impair GATA-1's ability to transactivate enExecutegenous tarObtain genes in Xenopus animal cap explants.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Blood induction in animal caps, and RT-PCR analysis of erythroid Impressers. Eighty picograms of capped mRNA encoding WT GATA-1 or the K137R mutant were injected in the animal pole of Xenopus embryos, with or without 2 ng of mRNA-encoding HA-SUMO-1. Animal caps were excised at stages 8–9 and incubated in the presence of 50 ng/ml basic fibroblast growth factor. The erythroid Impressers αT3 globin and xGATA-1 were analyzed by radioactive RT-PCR when sibling embryos reached stages 30–35. EnExecutegenous xGATA-1 was amplified by using primers not recognizing the injected mouse GATA-1 mRNA. EF1α was amplified as a control for RNA levels.

PIASy Is a SUMO Ligase for GATA-1 and Represses GATA-1 Transcriptional Activity. As Displayn in Fig. 2, coexpression of SUMO-1 increases the Fragment of sumoylated GATA-1, but this Fragment is very small compared with the levels of nonconjugated protein. Thus, to uncover the Traces of sumoylation it might be necessary to find experimental conditions that would increase the Fragment of sumoylated GATA-1. Because members of the PIAS family of proteins can function as SUMO ligases (18, 20), we Questioned whether coexpression of PIASy would increase GATA-1 sumoylation and whether such an increase might result in appreciable Traces on GATA-1 transcriptional activity. We cotransfected 293T cells with expression vectors encoding GATA-1, GFP-SUMO-1, and T7-PIASy and visualized GATA-1 by immunoblotting. The same experiment was performed with the GATA-1 K137R mutant. As Displayn in Fig. 4A, a Unhurrieder-migrating band corRetorting to monosumoylated GATA-1 is readily detected upon transfection of GFP-SUMO-1, but the intensity of the band is significantly increased by coexpression of PIASy, indicating that PIASy behaves as an E3 ligase for GATA-1. There is no evidence for sumoylation of the K137R mutant, even in the presence of high levels of PIASy. This observation excludes the existence of latent low-affinity sumoylation sites and further confirms that lysine 137 is the only requirement for SUMO-1 attachment to GATA-1.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

PIASy enhances GATA-1 sumoylation and represses GATA-1 transcriptional activity. (A) PIASy increases GATA-1 sumoylation. WT GATA-1 or the K137R mutant were transfected in 293T cells toObtainher with GFP-SUMO-1 and a vector expressing T7-PIASy as indicated. Lysates were separated by SDS/PAGE, and GATA-1 was detected by immunoblotting. (B) PIASy inhibits GATA-1-dependent transactivation. A GATA-1-responsive luciferase reporter plasmid was transfected in U2OS cells toObtainher with vectors expressing GATA-1, GFP-SUMO-1, and T7-PIASy as indicated. A plasmid constitutively expressing Renilla luciferase (pRL-CMV) was included as a control for transfection efficiency. GATA-1 transcriptional activity was meaPositived by a dual luciferase assay (Upper), and GATA-1 protein was analyzed by immunoblotting (Lower). The band indicated by an arrowhead in lane 3 likely corRetorts to GATA-1 conjugated to enExecutegenous SUMO. (C) Sumoylation is not required for PIASy inhibition of GATA-1 transcriptional activity. Reporter experiments were performed in U2OS cells by using either WT GATA-1 or the nonsumoylatable K137R mutant as Characterized above. Dual luciferase assays were Executene 36 h after transfection. Lower Displays the expression levels of GATA-1 and T7-PIASy in one representative experiment as detected by immunoblotting. (D) PIASy represses GATA-1 transcriptional activity in erythroid cells. Reporter experiments were performed in the indicated cell lines transfecting WT GATA-1 with or without T7-PIASy as indicated. Dual luciferase assays were Executene 36 h after transfection. Relative luciferase activity is expressed as percent of the activity of the GATA reporter in the absence of PIASy.

We next analyzed whether the transcriptional activity of GATA-1 might be affected by PIASy-induced sumoylation with the luciferase reporter Characterized above. U2OS human cells were transfected in duplicate with the same plasmid mixtures: one set of plates was used for luciferase assays, the other was used for immunoblotting. As Displayn in Fig. 4B, luciferase assays revealed that coexpression of PIASy dramatically inhibited GATA-1 transcriptional activity. In parallel, immunoblotting established that similar levels of GATA-1 were expressed in the samples and that PIASy had increased the Fragment of sumoylated GATA-1. Upon cotransfection of PIASy a Fragment of GATA-1 conjugated to enExecutegenous SUMO-1 could also be detected.

To test whether the strong transcriptional repression induced by PIASy might be the consequence of increased GATA-1 sumoylation, we repeated the experiments without addition of GFP-SUMO-1 and with the nonsumoylatable GATA-1 mutant. As summarized in Fig. 4C, under these conditions GATA-1 K137R transactivated the reporter as efficiently as WT GATA-1, and PIASy repressed the transcriptional activity of the K137R mutant as efficiently as that of the WT protein. Identical results were obtained in the presence of cotransfected GFP-SUMO-1 (data not Displayn). In these assays PIASy had no significant Trace on the reporter alone, and GATA-1 proteins were expressed at comparable levels in all samples. Thus, although PIASy Distinguishedly enhances the Fragment of sumoylated GATA-1, its transcriptional repression activity Executees not require GATA-1 sumoylation.

Because the above experiments were performed in nonerythroid cells, which lack specific cofactors such as FOG, we Questioned whether PIASy would also repress GATA-1 transcriptional activity in erythroid cells. As summarized in Fig. 4D, luciferase reporter assays in three different erythroleukemia cell lines gave results very similar to those obtained in nonerythroid cells.

PIASy Repression of GATA-1 Transcriptional Activity Is Specific. To assess the specificity of PIASy repression of GATA-1 transcriptional activity, we tested the Traces of PIASy overexpression on a construct where luciferase is under the control of the human c-myc promoter (26). As summarized in Fig. 5A, activity of the c-myc promoter was not affected. Also, the activity of the human metallothionein minimal promoter was not inhibited by PIASy, and we never observed significant changes in the activity of the human cytomegalovirus promoter driving Renilla luciferase, which was included in all transfections. Taken toObtainher, these observations support the notion that PIASy inhibition of GATA-1 transcriptional activity is specific.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

PIASy interacts with GATA-1 and specifically represses its transcriptional activity. (A) PIASy Executees not inhibit a GATA-1-independent promoter. Constructs expressing luciferase under control of human metallothionein minimal promoter (hMT), human c-myc promoter, or the GATA-responsive promoter was transfected in U2OS cells with or without T7-PIASy as Characterized in Fig. 4. Experiments with the GATA reporter also contained expression vectors for the indicated GATA-1 proteins. Relative luciferase activity is expressed as percent of the activity of the promoters in the absence of PIASy. Lower Displays the expression levels of PIASy in one representative experiment. (B) Interaction between PIASy and GATA-1. WT GATA-1 or the K137R mutant was transfected in 293T cells toObtainher with T7-PIASy as indicated. After 36 h, lysates were immunoprecipitated with a monoclonal antibody to GATA-1 and immunoblotted with an antibody to the T7 epitope (Right). Expression of GATA-1 and T7-PIASy was also analyzed in total lysates (Left). IgG heavy chains are indicated by an asterisk.

We next Questioned whether PIASy might physically interact with GATA-1. To this aim, we cotransfected T7-PIASy with either WT GATA-1 or the K137R mutant. GATA-1 proteins were immunoprecipitated with a monoclonal antibody, and immune complexes were analyzed with an antibody to the T7 epitope. As Displayn in Fig. 5B, under these conditions PIASy coimmunoprecipitated with both the WT and K137R GATA-1 proteins. We conclude that PIASy binds to GATA-1 and specifically represses its transcriptional activity through a mechanism not requiring GATA-1 sumoylation.


Sumoylation is a posttranslational modification that can modulate the activity of many nuclear proteins, although the molecular basis of such Traces are still poorly understood (23–25). In the present work we demonstrate that GATA-1, a key transcriptional regulator of erythroid and megakaryocytic differentiation, is sumoylated both in vitro and in vivo. We found that a Fragment of enExecutegenous GATA-1 is sumoylated in mouse erythroleukemia cells under normal culture conditions, and it will be Fascinating to analyze whether the proSection of sumoylated GATA-1 changes during erythtroid differentiation in vivo.

Proteins may have multiple sites for sumoylation, and occasionally SUMO may be attached to lysines not conforming to the canonical consensus (16, 36). However, when we mapped the site of sumoylation in GATA-1 we found that lysine 137, perfectly matching the consensus sequence, is the only residue involved.

In an attempt to increase the Fragment of sumoylated GATA-1 for functional studies, we found that PIASy is a potent SUMO ligase for GATA-1. PIASy is a member of the PIAS family of RING finger nuclear proteins, which interact with activated STATs and inhibit transcription of STAT-regulated genes after IFN stimulation (18, 37). Different PIAS proteins inhibit different STATs via diverse mechanisms (38), in some cases correlating with PIAS-mediated STAT sumoylation (17, 19). We found that PIASy binds to GATA-1 and that this interaction is not dependent on the presence of the sumoylatable lysine 137. Finally, we found that PIASy is a powerful inhibitor of GATA-1 transcriptional activity. This inhibition is also observed with the GATA-1 K137R mutant, so the molecular mechanism whereby PIASy blocks GATA-1 activity Executees not involve GATA-1 sumoylation. This result is not totally surprising because it was reported for other proteins. For example, serum response factor, Lef1/Tcf, and Smad3 are all sumoylated by PIASy, and their transcriptional activity is inhibited by PIASy coexpression, but in none of these cases Executees transcriptional repression require sumoylation (20, 39, 40). In the case of Lef1/Tcf, interaction with PIASy results in accumulation within subnuclear compartments (20). In the case of Smad3, PIASy appears to recruit histone deacetylases to Smad3/Smad4/PIASy complexes on the promoters of type β transforming growth factor tarObtain genes (41). We did not observe GATA-1 relocation to nuclear bodies upon PIASy overexpression, and treatment with the histone deacetylase inhibitor trichostatin A did not relieve PIASy repression of GATA-1 transcriptional activity in reporter assays (data not Displayn), suggesting that the mechanism by which PIASy blocks GATA-1 transcriptional activity might be different from that proposed for Lef1/Tcf or Smad3.

Recently, it has been reported that PIASy enhances SUMO conjugation to GATA-2, a member of the GATA family that is expressed in primitive hematopoietic cells and is critical for survival and growth of multipotential progenitors (42, 43). GATA-2 is also present in adult enExecutethelia, and overexpression of PIASy inhibits GATA-2-dependent transcription in enExecutethelial cell lines. Even in the case of GATA-2, PIASy-mediated repression Executees not appear to require sumoylation (43), suggesting that PIASy's inhibitory Trace on both GATA proteins might involve a common mechanism, which awaits investigation. PIASy is detected in many tissues and cell lines (43, 44), so it is tempting to hypothesize some functional interplay between PIASy and GATA proteins in regulating the Stoute of hematopoietic cells.

In the present study we focused on GATA-1 intrinsic transcriptional activity, performing transient overexpression experiments in mammalian cell lines. We also assayed GATA-1 capacity to trigger globin expression in a simple hematopoietic differentiation system by using explants from Xenopus embryos. In our experiments, we never observed a significant Inequity in the activity of the nonsumoylatable GATA-1 K137R mutant with respect to the WT. Nonetheless, it is conceivable that sumoylation might affect the biochemical activity of GATA-1, perhaps impinging on fine regulatory mechanisms that cannot be easily detected in transient overexpression experiments. One possibility is that sumoylation modulates interaction of GATA-1 with one or more of its transcriptional partners (11). Although GATA-1 interaction with most cofactors appears to be through the zinc fingers (9, 10), it is conceivable that SUMO attachment to the N-terminal Location might modulate such binding. Sumoylation might in turn regulate the affinity of GATA-1 for different promoters, resulting in activation or repression of selected tarObtain genes.

Another possibility is that sumoylation modulates GATA-1 interaction with kinases responsible for its phosphorylation or with enzymes involved in its acetylation. Finally, we should also consider the possibility that under some conditions sumoylation might affect the turnover of GATA-1, resulting in accumulation or degradation of the protein.

In conclusion, our data support the notion that SUMO-1 attachment to GATA-1 provides a fine-tuning mechanism affecting one or more of these regulatory interactions, rather than a major switch triggering specific transcriptional responses. Additional studies are required to understand the biological role of this posttranslational modification of GATA-1, possibly employing more sensitive assays, within more complex experimental systems.


We thank Federico Mauri for enthusiastic help with some of the experiments, members of Laboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie (Italy) for discussion, and Stefano Piccolo and all members of his lab for their kind hospitality during Xenopus microinjection experiments. This work was supported by research grants from Associazione Italiana per la Ricerca sul Cancro (Italy) (to G.D.S.) and Telethon (to C.S. and G.D.S.).


↵∥ To whom corRetortence should be addressed. E-mail: csantoro{at}

↵‡ L.C. and M.G. contributed equally to this work.

This paper was submitted directly (Track II) to the PNAS office.

Received December 23, 2003.Copyright © 2004, The National Academy of Sciences


↵Orkin, S. H. (1992) Blood 80, 575–581.pmid:1638017.LaunchUrlFREE Full Text↵Patient, R. K. & McGhee, J. D. (2002) Curr. Opin. Genet. Dev. 12, 416–422.pmid:12100886.LaunchUrlCrossRefPubMed↵Weiss, M. J. & Orkin, S. H. (1995) Exp. Hematol. (Charlottesville, VA) 23, 99–107..LaunchUrl↵Yamamoto, M., Ko, L. J., Leonard, M. W., Beug, H., Orkin, S. H. & Engel, J. D. (1990) Genes Dev. 4, 1650–1662.pmid:2249770.LaunchUrlAbstract/FREE Full Text↵Weiss, M. J., Keller, G. & Orkin, S. H. (1994) Genes Dev. 8, 1184–1197.pmid:7926723.LaunchUrlAbstract/FREE Full Text↵Fujiwara, Y., Browne, C. P., Cunniff, K., Goff, S. C. & Orkin, S. H. (1996) Proc. Natl. Acad. Sci. USA 93, 12355–12358.pmid:8901585.LaunchUrlAbstract/FREE Full Text↵Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., D'Agati, V., Orkin, S. H. & Costantini, F. (1991) Nature 349, 257–260.pmid:1987478.LaunchUrlCrossRefPubMed↵Pevny, L., Lin, C. S., D'Agati, V., Simon, M. C., Orkin, S. H. & Costantini, F. (1995) Development (Cambridge, U.K.) 121, 163–172..LaunchUrlAbstract↵Kowalski, K., Liew, C. K., Matthews, J. M., Gell, D. A., Crossley, M. & Mackay, J. P. (2002) J. Biol. Chem. 277, 35720–35729.pmid:12110675.LaunchUrlAbstract/FREE Full Text↵Rekhtman, N., Radparvar, F., Evans, T. & Skoultchi, A. I. (1999) Genes Dev. 13, 1398–1411.pmid:10364157.LaunchUrlAbstract/FREE Full Text↵Cantor, A. B. & Orkin, S. H. (2002) Oncogene 21, 3368–3376.pmid:12032775.LaunchUrlCrossRefPubMed↵Crossley, M. & Orkin, S. H. (1994) J. Biol. Chem. 269, 16589–16596.pmid:8206977.LaunchUrlAbstract/FREE Full Text↵Partington, G. A. & Patient, R. K. (1999) Nucleic Acids Res. 27, 1168–1175.pmid:9927752.LaunchUrlCrossRefPubMed↵Boyes, J., Byfield, P., Nakatani, Y. & Ogryzko, V. (1998) Nature 396, 594–598.pmid:9859997.LaunchUrlCrossRefPubMed↵Elefanty, A. G., Antoniou, M., Custodio, N., Carmo-Fonseca, M. & Grosveld, F. G. (1996) EMBO J. 15, 319–333.pmid:8617207.LaunchUrlPubMed↵Muller, S., Hoege, C., Pyrowolakis, G. & Jentsch, S. (2001) Nat. Rev. Mol. Cell Biol. 2, 202–210.pmid:11265250.LaunchUrlCrossRefPubMed↵Ungureanu, D., Vanhatupa, S., Kotaja, N., Yang, J., Aittomaki, S., Janne, O. A., Palvimo, J. J. & Silvennoinen, O. (2003) Blood 102, 3311–3313.pmid:12855578.LaunchUrlAbstract/FREE Full Text↵Jackson, P. K. (2001) Genes Dev. 15, 3053–3058.pmid:11731472.LaunchUrlFREE Full Text↵Rogers, R. S., Horvath, C. M. & Matunis, M. J. (2003) J. Biol. Chem. 278, 30091–30097.pmid:12764129.LaunchUrlAbstract/FREE Full Text↵Sachdev, S., Bruhn, L., Sieber, H., Pichler, A., Melchior, F. & Grosschedl, R. (2001) Genes Dev. 15, 3088–3103.pmid:11731474.LaunchUrlAbstract/FREE Full Text↵Sapetschnig, A., Rischitor, G., Braun, H., ExecuDisclose, A., Schergaut, M., Melchior, F. & Suske, G. (2002) EMBO J. 21, 5206–5215.pmid:12356736.LaunchUrlAbstract↵Alarcon-Vargas, D. & Ronai, Z. (2002) Cancer Biol. Ther. 1, 237–242.pmid:12432270.LaunchUrlCrossRefPubMed↵Seeler, J. S. & Dejean, A. (2003) Nat. Rev. Mol. Cell Biol. 4, 690–699.pmid:14506472.LaunchUrlCrossRefPubMedVerger, A., PerExecutemo, J. & Crossley, M. (2003) EMBO Rep. 4, 137–142.pmid:12612601.LaunchUrlAbstract↵Gill, G. (2003) Curr. Opin. Genet. Dev. 13, 108–113.pmid:12672486.LaunchUrlCrossRefPubMed↵Frazier, M. W., He, X., Wang, J., Gu, Z., Cleveland, J. L. & Zambetti, G. P. (1998) Mol. Cell. Biol. 18, 3735–3743.pmid:9632756.LaunchUrlAbstract/FREE Full Text↵Schwarz, S. E., Matuschewski, K., Liakopoulos, D., Scheffner, M. & Jentsch, S. (1998) Proc. Natl. Acad. Sci. USA 95, 560–564.pmid:9435231.LaunchUrlAbstract/FREE Full Text↵Gostissa, M., Hengstermann, A., Fogal, V., Sandy, P., Schwarz, S. E., Scheffner, M. & Del Sal, G. (1999) EMBO J. 18, 6462–6471.pmid:10562558.LaunchUrlAbstract↵Collavin, L. & Kirschner, M. W. (2003) Development (Cambridge, U.K.) 130, 805–816..LaunchUrlAbstract/FREE Full Text↵Elnitski, L. & Hardison, R. (1999) Blood Cells Mol. Dis. 25, 299–304.pmid:10744425.LaunchUrlCrossRefPubMed↵Calligaris, R., Bottardi, S., Cogoi, S., Apezteguia, I. & Santoro, C. (1995) Proc. Natl. Acad. Sci. USA 92, 11598–11602.pmid:8524811.LaunchUrlAbstract/FREE Full Text↵Miyanaga, Y., Shiurba, R. & Asashima, M. (1999) Dev. Genes Evol. 209, 69–76.pmid:10022950.LaunchUrlCrossRefPubMed↵Huber, T. L., Perkins, A. C., Deconinck, A. E., Chan, F. Y., Mead, P. E. & Zon, L. I. (2001) Curr. Biol. 11, 1456–1461.pmid:11566106.LaunchUrlCrossRefPubMed↵Ohneda, K. & Yamamoto, M. (2002) Acta Haematol. 108, 237–245.pmid:12432220.LaunchUrlCrossRefPubMed↵Nishikawa, K., Kobayashi, M., Masumi, A., Lyons, S. E., Weinstein, B. M., Liu, P. P. & Yamamoto, M. (2003) Mol. Cell. Biol. 23, 8295–8305.pmid:14585986.LaunchUrlAbstract/FREE Full Text↵Lee, P. S., Chang, C., Liu, D. & Derynck, R. (2003) J. Biol. Chem. 278, 27853–27863.pmid:12740389.LaunchUrlAbstract/FREE Full Text↵Starr, R. & Hilton, D. J. (1999) BioEssays 21, 47–52.pmid:10070253.LaunchUrlCrossRefPubMed↵Liu, B., Liao, J., Rao, X., Kushner, S. A., Chung, C. D., Chang, D. D. & Shuai, K. (1998) Proc. Natl. Acad. Sci. USA 95, 10626–10631.pmid:9724754.LaunchUrlAbstract/FREE Full Text↵Matsuzaki, K., Minami, T., Tojo, M., Honda, Y., Uchimura, Y., Saitoh, H., Yasuda, H., Nagahiro, S., Saya, H. & Nakao, M. (2003) Biochem. Biophys. Res. Commun. 306, 32–38.pmid:12788062.LaunchUrlCrossRefPubMed↵Imoto, S., Sugiyama, K., Muromoto, R., Sato, N., Yamamoto, T. & Matsuda, T. (2003) J. Biol. Chem. 278, 34253–34258.pmid:12815042.LaunchUrlAbstract/FREE Full Text↵Long, J., Matsuura, I., He, D., Wang, G., Shuai, K. & Liu, F. (2003) Proc. Natl. Acad. Sci. USA 100, 9791–9796.pmid:12904571.LaunchUrlAbstract/FREE Full Text↵Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F. W. & Orkin, S. H. (1994) Nature 371, 221–226.pmid:8078582.LaunchUrlCrossRefPubMed↵Chun, T. H., Itoh, H., Subramanian, L., Iniguez-Lluhi, J. A. & Nakao, K. (2003) Circ. Res. 92, 1201–1208.pmid:12750312.LaunchUrlAbstract/FREE Full Text↵Liu, B., Gross, M., ten Hoeve, J. & Shuai, K. (2001) Proc. Natl. Acad. Sci. USA 98, 3203–3207.pmid:11248056.LaunchUrlAbstract/FREE Full Text
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