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Comparative Study
. 2008 Apr;28(7):2342-57.
doi: 10.1128/MCB.01159-07. Epub 2008 Jan 22.

Identification of a new site of sumoylation on Tel (ETV6) uncovers a PIAS-dependent mode of regulating Tel function

M Guy Roukens  1 , Mariam Alloul-Ramdhani, Alfred C O Vertegaal, Zeinab Anvarian, Crina I A Balog, André M Deelder, Paul J Hensbergen, David A Baker
Affiliations
Comparative Study

Identification of a new site of sumoylation on Tel (ETV6) uncovers a PIAS-dependent mode of regulating Tel function

M Guy Roukens et al. Mol Cell Biol. 2008 Apr.

Abstract

Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.

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Figures

FIG. 1.
FIG. 1.
The highly conserved lysine residue (K11) is the primary substrate for SUMO conjugation to Tel. (A) Endogenous Tel is sumoylated. The left panel shows a Western blot of different amounts of a cell lysate that were prepared from U2OS cells. Tel proteins were detected with a Tel antibody directed against the C terminus of Tel (highlighted with arrows) (20). Endogenous Tel proteins were compared with ectopically expressed Tel sumoylated with SUMO-2 as a control. We established U2OS cell lines stably expressing His epitope-tagged SUMO-1 or SUMO-2. Sumoylated endogenous Tel was recovered from cells lysed in guanidinium, by nickel bead purification (right panel) (sumoylation assay). (B) In vitro sumoylation assay. Both SUMO-1 and SUMO-2 are efficiently, covalently conjugated to Tel almost exclusively on K11 by one of two methods. Fusions between GST and either full-length wild-type Tel or full-length Tel in which lysine at position 11 was mutated to an arginine residue (TelK11-R) were coexpressed in E. coli along with a SUMO E1-ligase (Aos1 or Uba2) and a SUMO E2-ligase (Ubc9) either for SUMO-1 or for SUMO-2 conjugation; proteins were then purified onto glutathione-Sepharose beads. A Coomassie blue-stained gel shows sumoylated Tel, which is absent from TelK11-R preparations (highlighted with asterisks); the results were confirmed by Western blotting (data not shown). A complementary study shows in vitro [35S]methionine-translated Tel proteins that were sumoylated in vitro (37) and then incubated with or without the active site of a SUMO-protease (Lifesensors). (C) MS reveals Tel to be sumoylated on K11. In vitro sumoylated and nonsumoylated Tel were separated by SDS-PAGE and subjected to in-gel digestion with trypsin. MALDI-TOF MS demonstrated an m/z value of 3,800.9 [M+H]+ within the tryptic digest of Tel-SUMO-1 which was absent in unsumoylated Tel. This peptide corresponds to the tryptic peptide of SUMO-1 (trypSUMO-1, ELGMEEEDVIEVYQEQTGG) conjugated to K11 within the N-terminal tryptic peptide of Tel. As a control a nonsumoylated tryptic peptide (T128-160; m/z 3,940.0 [M+H]+) of Tel is shown. The SUMO-1-conjugated peptide was also identified by LC-iontrap MS (m/z 955.0 [M + 4H]4+), and its sequence was subsequently confirmed by MS/MS. (D) K11 forms part of a classic sumoylation consensus site (ψKXE; specifically IKQE, underlined) that is highly conserved in all sequenced vertebrates with the exception of D. rerio. An alternative initiation codon (M43; underlined) is present in all Tel proteins also harboring a K11 sumoylation site (and is absent in D. rerio). Western blotting of cell lysates revealed an additional Tel protein (indicated with an arrow), approximately 11 kDa larger than unmodified Tel that is lost following mutation of the sumoylation consensus site, TelK11-R or TelE13-A. Likewise, mutation of K11 abolishes in vitro sumoylation of Tel. (E to G) In cells, K11 is necessary and sufficient for full (detectable) sumoylation of Tel. HA epitope-tagged versions of wild-type Tel or Tel expressing mutations that disrupt the sumoylation consensus site, TelK11-R or TelE13-A, were cotransfected into 293T cells along with His epitope-tagged SUMO-1 or SUMO-2 (E). Also assayed are two other Tel mutants: TelK99-R, in which the lysine residue at position 99 has been replaced with an arginine residue, and TelD101-A, in which the aspartic acid residue at position 101 has been changed to an alanine residue. A schematic representation of the mutants tested is included. Sumoylated Tel was recovered from cells lysed in guanidinium by nickel bead purification. Conditions for immunoprecipitating sumoylated Tel from cells were optimized (F) to allow in vivo labeling of Tel with [35S]methionine in order to monitor the stability of the pool of sumoylated Tel (G). IP, immunoprecipitation.
FIG. 1.
FIG. 1.
The highly conserved lysine residue (K11) is the primary substrate for SUMO conjugation to Tel. (A) Endogenous Tel is sumoylated. The left panel shows a Western blot of different amounts of a cell lysate that were prepared from U2OS cells. Tel proteins were detected with a Tel antibody directed against the C terminus of Tel (highlighted with arrows) (20). Endogenous Tel proteins were compared with ectopically expressed Tel sumoylated with SUMO-2 as a control. We established U2OS cell lines stably expressing His epitope-tagged SUMO-1 or SUMO-2. Sumoylated endogenous Tel was recovered from cells lysed in guanidinium, by nickel bead purification (right panel) (sumoylation assay). (B) In vitro sumoylation assay. Both SUMO-1 and SUMO-2 are efficiently, covalently conjugated to Tel almost exclusively on K11 by one of two methods. Fusions between GST and either full-length wild-type Tel or full-length Tel in which lysine at position 11 was mutated to an arginine residue (TelK11-R) were coexpressed in E. coli along with a SUMO E1-ligase (Aos1 or Uba2) and a SUMO E2-ligase (Ubc9) either for SUMO-1 or for SUMO-2 conjugation; proteins were then purified onto glutathione-Sepharose beads. A Coomassie blue-stained gel shows sumoylated Tel, which is absent from TelK11-R preparations (highlighted with asterisks); the results were confirmed by Western blotting (data not shown). A complementary study shows in vitro [35S]methionine-translated Tel proteins that were sumoylated in vitro (37) and then incubated with or without the active site of a SUMO-protease (Lifesensors). (C) MS reveals Tel to be sumoylated on K11. In vitro sumoylated and nonsumoylated Tel were separated by SDS-PAGE and subjected to in-gel digestion with trypsin. MALDI-TOF MS demonstrated an m/z value of 3,800.9 [M+H]+ within the tryptic digest of Tel-SUMO-1 which was absent in unsumoylated Tel. This peptide corresponds to the tryptic peptide of SUMO-1 (trypSUMO-1, ELGMEEEDVIEVYQEQTGG) conjugated to K11 within the N-terminal tryptic peptide of Tel. As a control a nonsumoylated tryptic peptide (T128-160; m/z 3,940.0 [M+H]+) of Tel is shown. The SUMO-1-conjugated peptide was also identified by LC-iontrap MS (m/z 955.0 [M + 4H]4+), and its sequence was subsequently confirmed by MS/MS. (D) K11 forms part of a classic sumoylation consensus site (ψKXE; specifically IKQE, underlined) that is highly conserved in all sequenced vertebrates with the exception of D. rerio. An alternative initiation codon (M43; underlined) is present in all Tel proteins also harboring a K11 sumoylation site (and is absent in D. rerio). Western blotting of cell lysates revealed an additional Tel protein (indicated with an arrow), approximately 11 kDa larger than unmodified Tel that is lost following mutation of the sumoylation consensus site, TelK11-R or TelE13-A. Likewise, mutation of K11 abolishes in vitro sumoylation of Tel. (E to G) In cells, K11 is necessary and sufficient for full (detectable) sumoylation of Tel. HA epitope-tagged versions of wild-type Tel or Tel expressing mutations that disrupt the sumoylation consensus site, TelK11-R or TelE13-A, were cotransfected into 293T cells along with His epitope-tagged SUMO-1 or SUMO-2 (E). Also assayed are two other Tel mutants: TelK99-R, in which the lysine residue at position 99 has been replaced with an arginine residue, and TelD101-A, in which the aspartic acid residue at position 101 has been changed to an alanine residue. A schematic representation of the mutants tested is included. Sumoylated Tel was recovered from cells lysed in guanidinium by nickel bead purification. Conditions for immunoprecipitating sumoylated Tel from cells were optimized (F) to allow in vivo labeling of Tel with [35S]methionine in order to monitor the stability of the pool of sumoylated Tel (G). IP, immunoprecipitation.
FIG. 2.
FIG. 2.
K11 of Tel polymers, but not Tel monomers, is found conjugated with SUMO in cells. (A) Disruption of the SAM domain prevents Tel self-association. HA epitope-tagged and Flag epitope-tagged fusions of wild-type Tel or Tel expressing mutations that disrupt the SAM domain (A*, which contains an arginine residue in place of an alanine residue; deletion of the SAM domain is shown as ΔSAM) were expressed in 293T cells in the indicated combinations. A schematic representation of the mutants tested is included. (B and C) Monomeric forms of Tel exhibit low levels of sumoylation in cells. (B) HA epitope-tagged versions of wild-type Tel or Tel expressing mutations that disrupt the SAM domain (described above) were cotransfected into 293T cells along with His epitope-tagged SUMO-1 or SUMO-2, and a sumoylation assay was performed. (C) The experiment in panel B was performed using Tel mutants in which each of the five helices that comprise the SAM domain were individually deleted. A schematic representation of the mutants tested is included. (D) Tel monomers are efficiently sumoylated in vitro on K11. Fusions between GST and either full-length wild-type Tel or full-length Tel harboring mutations that disrupt the SAM domain (see above) were sumoylated in E. coli as described in the legend of Fig. 1A. Sumoylated forms of Tel are highlighted (*) and were confirmed by Western blotting and also MS (data not shown). Shown also is an HA Western blot to detect Tel following an in vitro sumoylation assay (37) using the indicated Tel proteins that were made by in vitro translation in the presence of 1 mM unlabeled methionine. Sumoylated proteins that are absent from TelA* proteins containing a mutation of K11 to an arginine (TelA*/K11-R) are highlighted with arrows. (E) Inhibiting proteasome function stabilizes the pool of Tel monomers sumoylated on K11 in cells. Cells were cotransfected with the indicated Tel constructs along with His epitope-tagged SUMO-2. Following incubation with or without MG132, a sumoylation assay was performed. IP, immunoprecipitation.
FIG. 3.
FIG. 3.
PIAS3 stimulates sumoylation of Tel on K11. (A) Ectopic expression of PIAS3 strongly stimulates sumoylation of K11 of Tel. Tel or TelK11-R was transfected into 293T cells with or without His epitope-tagged SUMO-2, either alone or together with the indicated PIAS constructs. Sumoylated Tel was recovered from cells by a sumoylation assay. (B) Endogenous PIAS3 is essential for normal Tel sumoylation. Cells were transfected with the indicated constructs and a sumoylation assay was performed 2 days later. In the absence of an effective antibody specific for human PIAS3, the efficiency of PIAS3 knockdown was assessed by targeting Flag epitope-tagged PIAS3 expressed in U2OS cells. A nonspecific siRNA was used as a control. (C) Endogenous PIAS3 is required for sumoylation of endogenous Tel. We established U2OS cell lines stably expressing either His epitope-tagged SUMO-1 or SUMO-2. The indicated cell lines were transfected with either a control siRNA (directed against GFP) or siRNAs directed against PIAS3, and a sumoylation assay was performed 2 days later. The efficiency of PIAS3 knockdown was assessed by targeting Flag epitope-tagged PIAS3 expressed in U2OS cells. A nonspecific siRNA was used as a control (right panel). (D) Colocalization of Tel and PIAS3 in the nucleus. Cells were transfected with the indicated constructs and proteins were detected with the indicated antibodies. TelΔEDBD is the same as wild-type Tel except it lacks the EDBD. (E) Tel interacts with PIAS3. 293T cells were transfected with the indicated constructs. Tel complexes were immunopurified from cell lysates, made using radioimmunoprecipitation assay-SDS lysis buffer, using an antibody directed against the HA epitope, and associated PIAS protein was detected using an antibody directed against the Flag epitope. (F) Tel binding to PIAS3 requires its EDBD in cells. The indicated HA epitope-tagged Tel proteins were coexpressed with or without PIAS3 (not shown since the background was clear). Tel complexes were immunopurified from cell lysates using an antibody directed against the HA epitope, and associated PIAS3 protein was detected using an antibody directed against the Flag epitope. Tel M43 lacks the N-terminal 42 amino acids; Tel A* contains an amino substitution (arginine residue in place of an alanine residue) at position 93; TelΔSAM lacks the SAM domain; and Tel ΔC34, TelΔC27, and TelΔC12 have deletions of the C terminus of the indicated lengths. A schematic representation of the mutants tested is included. (G) Mutations that disrupt the EDBD or the C terminus of Tel lead to mislocalization of Tel to the cytoplasm. Cells were transfected with the indicated constructs, and immunohistochemistry was performed with the antibodies shown. (H) Mislocalization strongly abrogates sumoylation. Cells were cotransfected with the indicated constructs, and a sumoylation assay was performed. DAPI, 4′,6′-diamidino-2-phenylindole; IP, immunoprecipitation.
FIG. 3.
FIG. 3.
PIAS3 stimulates sumoylation of Tel on K11. (A) Ectopic expression of PIAS3 strongly stimulates sumoylation of K11 of Tel. Tel or TelK11-R was transfected into 293T cells with or without His epitope-tagged SUMO-2, either alone or together with the indicated PIAS constructs. Sumoylated Tel was recovered from cells by a sumoylation assay. (B) Endogenous PIAS3 is essential for normal Tel sumoylation. Cells were transfected with the indicated constructs and a sumoylation assay was performed 2 days later. In the absence of an effective antibody specific for human PIAS3, the efficiency of PIAS3 knockdown was assessed by targeting Flag epitope-tagged PIAS3 expressed in U2OS cells. A nonspecific siRNA was used as a control. (C) Endogenous PIAS3 is required for sumoylation of endogenous Tel. We established U2OS cell lines stably expressing either His epitope-tagged SUMO-1 or SUMO-2. The indicated cell lines were transfected with either a control siRNA (directed against GFP) or siRNAs directed against PIAS3, and a sumoylation assay was performed 2 days later. The efficiency of PIAS3 knockdown was assessed by targeting Flag epitope-tagged PIAS3 expressed in U2OS cells. A nonspecific siRNA was used as a control (right panel). (D) Colocalization of Tel and PIAS3 in the nucleus. Cells were transfected with the indicated constructs and proteins were detected with the indicated antibodies. TelΔEDBD is the same as wild-type Tel except it lacks the EDBD. (E) Tel interacts with PIAS3. 293T cells were transfected with the indicated constructs. Tel complexes were immunopurified from cell lysates, made using radioimmunoprecipitation assay-SDS lysis buffer, using an antibody directed against the HA epitope, and associated PIAS protein was detected using an antibody directed against the Flag epitope. (F) Tel binding to PIAS3 requires its EDBD in cells. The indicated HA epitope-tagged Tel proteins were coexpressed with or without PIAS3 (not shown since the background was clear). Tel complexes were immunopurified from cell lysates using an antibody directed against the HA epitope, and associated PIAS3 protein was detected using an antibody directed against the Flag epitope. Tel M43 lacks the N-terminal 42 amino acids; Tel A* contains an amino substitution (arginine residue in place of an alanine residue) at position 93; TelΔSAM lacks the SAM domain; and Tel ΔC34, TelΔC27, and TelΔC12 have deletions of the C terminus of the indicated lengths. A schematic representation of the mutants tested is included. (G) Mutations that disrupt the EDBD or the C terminus of Tel lead to mislocalization of Tel to the cytoplasm. Cells were transfected with the indicated constructs, and immunohistochemistry was performed with the antibodies shown. (H) Mislocalization strongly abrogates sumoylation. Cells were cotransfected with the indicated constructs, and a sumoylation assay was performed. DAPI, 4′,6′-diamidino-2-phenylindole; IP, immunoprecipitation.
FIG. 4.
FIG. 4.
Posttranscriptional regulation of Tel sumoylation. (A and B) An alternative Tel isoform produced through use of an alternative initiation codon (M43) escapes sumoylation. TelM43 can be translated from the full-length Tel cDNA. 293T cells were separately transfected with three different Tel constructs (A). One expressed an HA epitope at the N terminus, another expressed the HA epitope at the C terminus enabling visualization of both Tel and TelM43 isoforms, and a final construct contained an HA epitope at the C terminus but expressed an isoleucine residue in place of the methionine at position 43 (TelM43-I). A separate panel highlights endogenous Tel proteins detected following Western blotting of lysates prepared from primary human hemopoietic blast cells. Sumoylation of the TelM43 isoform is strongly abrogated (B). Cells were cotransfected with the indicated constructs, and a sumoylation assay was performed. (C) Sumoylation of K11 is independent of the TelM43 isoform. Cells were cotransfected with the indicated constructs, and a sumoylation assay was performed. TelM43-I is described for panel A. TelM43-I/K11-R is identical to TelM43-I except that lysine at position 11 has been mutated into an arginine residue. A schematic representation of the mutants tested is included. (D) Nonsumoylated Tel isoforms adopt a more speckled subcellular distribution. Cells were transfected with the indicated constructs, and proteins were detected with the indicated antibodies.
FIG. 5.
FIG. 5.
Sumoylation of K11 inhibits repression by Tel. (A) Either 293T, U2OS, or NIH 3T3 cells were cotransfected with the indicated Tel constructs along with a luciferase reporter in which an engineered minimal Tel/Yan regulatable promoter controls TK-driven luciferase expression. Equivalent transfection efficiencies of the indicated constructs were determined by a β-galactosidase assay for expression of a cotransfected LacZ reporter, and protein levels of ectopically expressed Tel constructs were determined by Western blotting of lysates. Shown is a representative experiment using 293T cells. Experiments were performed in triplicate. (B) Tel repression of expression of endogenous human stromelysin-1 (MMP3) is inhibited by sumoylation of K11. The upper panel shows that the promoters of rat and human stromelysin-1 share a number of conserved, potential Tel DNA binding sites. Highlighted in bold are previously characterized Tel binding sites (5). Additional highly conserved putative Tel binding sites are underlined. In the lower left panel, we established stable cell lines expressing the indicated Tel constructs. We prepared cDNA from these lines and performed quantitative PCR to assess the levels of expression of endogenous stromelysin-1. Shown graphically are the relative repression levels normalized against a control human gapdh gene. Shown in the right panel is a luciferase reporter assay. Luciferase expression was placed under the control of the human stromelysin promoter. Equivalent transfection efficiencies of the indicated constructs were determined by a β-galactosidase assay for expression of a cotransfected LacZ reporter, and protein levels of ectopically expressed Tel constructs were determined by Western blotting of lysates. Shown is a representative experiment using U2OS cells. Experiments were performed in triplicate. (C) D. rerio Tel does not express an N-terminal site of sumoylation but is sumoylated at position K441. Cells were transfected with the indicated constructs together with SUMO-2, and Tel sumoylation was assessed by Western blotting. D. rerio TelK12-R is the same as wild-type D. rerio Tel except that the lysine residue at position 12 has been replaced with an arginine residue. D. rerio TelR14-E is identical to wild-type D. rerio Tel except that the N-terminal sumoylation consensus site has been created by replacing the arginine residue with a glutamic acid residue at position 14. D. rerio TelK441-R is the same as wild-type D. rerio Tel except that the lysine residue at position 441 has been replaced with an arginine residue. D. rerio TelK441-R/R14-E is the same as D. rerio TelK441-R except that the arginine residue at position 14 has been replaced with a glutamic acid residue. A schematic representation of the mutants tested is included. (D) Cells were transfected with the indicated constructs, and a luciferase repression assay was performed as described in panel A. (E) Sumoylation of K11 impedes the ability of Tel to associate with DNA. Tel proteins were efficiently sumoylated following in vitro translation in reticulocyte lysates as previously described (37). Biotinylated Ets DNA-binding sites were coupled to streptavidin beads following the manufacturer's advice (Invitrogen) and incubated with the indicated proteins. Associated Tel proteins were recovered by SDS-PAGE. Tel-Tel interactions were assessed by incubating GST Tel fusion proteins or GST proteins alone, along with sumoylated and nonsumoylated in vitro translated forms of Tel. (F and G) PIAS3 enhancement of repression by Tel is sumoylation independent. The experiment shown in panel F is the same as that described for panel A except that cells were cotransfected with or without PIAS3. In the experiment in panel G, cells were transfected with the indicated constructs, and a luciferase reporter assay was performed as described for panel A. PIAS3ΔRING is the same as PIAS3 except that the RING domain has been deleted.
FIG. 5.
FIG. 5.
Sumoylation of K11 inhibits repression by Tel. (A) Either 293T, U2OS, or NIH 3T3 cells were cotransfected with the indicated Tel constructs along with a luciferase reporter in which an engineered minimal Tel/Yan regulatable promoter controls TK-driven luciferase expression. Equivalent transfection efficiencies of the indicated constructs were determined by a β-galactosidase assay for expression of a cotransfected LacZ reporter, and protein levels of ectopically expressed Tel constructs were determined by Western blotting of lysates. Shown is a representative experiment using 293T cells. Experiments were performed in triplicate. (B) Tel repression of expression of endogenous human stromelysin-1 (MMP3) is inhibited by sumoylation of K11. The upper panel shows that the promoters of rat and human stromelysin-1 share a number of conserved, potential Tel DNA binding sites. Highlighted in bold are previously characterized Tel binding sites (5). Additional highly conserved putative Tel binding sites are underlined. In the lower left panel, we established stable cell lines expressing the indicated Tel constructs. We prepared cDNA from these lines and performed quantitative PCR to assess the levels of expression of endogenous stromelysin-1. Shown graphically are the relative repression levels normalized against a control human gapdh gene. Shown in the right panel is a luciferase reporter assay. Luciferase expression was placed under the control of the human stromelysin promoter. Equivalent transfection efficiencies of the indicated constructs were determined by a β-galactosidase assay for expression of a cotransfected LacZ reporter, and protein levels of ectopically expressed Tel constructs were determined by Western blotting of lysates. Shown is a representative experiment using U2OS cells. Experiments were performed in triplicate. (C) D. rerio Tel does not express an N-terminal site of sumoylation but is sumoylated at position K441. Cells were transfected with the indicated constructs together with SUMO-2, and Tel sumoylation was assessed by Western blotting. D. rerio TelK12-R is the same as wild-type D. rerio Tel except that the lysine residue at position 12 has been replaced with an arginine residue. D. rerio TelR14-E is identical to wild-type D. rerio Tel except that the N-terminal sumoylation consensus site has been created by replacing the arginine residue with a glutamic acid residue at position 14. D. rerio TelK441-R is the same as wild-type D. rerio Tel except that the lysine residue at position 441 has been replaced with an arginine residue. D. rerio TelK441-R/R14-E is the same as D. rerio TelK441-R except that the arginine residue at position 14 has been replaced with a glutamic acid residue. A schematic representation of the mutants tested is included. (D) Cells were transfected with the indicated constructs, and a luciferase repression assay was performed as described in panel A. (E) Sumoylation of K11 impedes the ability of Tel to associate with DNA. Tel proteins were efficiently sumoylated following in vitro translation in reticulocyte lysates as previously described (37). Biotinylated Ets DNA-binding sites were coupled to streptavidin beads following the manufacturer's advice (Invitrogen) and incubated with the indicated proteins. Associated Tel proteins were recovered by SDS-PAGE. Tel-Tel interactions were assessed by incubating GST Tel fusion proteins or GST proteins alone, along with sumoylated and nonsumoylated in vitro translated forms of Tel. (F and G) PIAS3 enhancement of repression by Tel is sumoylation independent. The experiment shown in panel F is the same as that described for panel A except that cells were cotransfected with or without PIAS3. In the experiment in panel G, cells were transfected with the indicated constructs, and a luciferase reporter assay was performed as described for panel A. PIAS3ΔRING is the same as PIAS3 except that the RING domain has been deleted.
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