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. 2005 Feb;25(4):1549-59.
doi: 10.1128/MCB.25.4.1549-1559.2005.

Role for SUMO modification in facilitating transcriptional repression by BKLF

José Perdomo  1 , Alexis Verger, Jeremy Turner, Merlin Crossley
Affiliations

Role for SUMO modification in facilitating transcriptional repression by BKLF

José Perdomo et al. Mol Cell Biol. 2005 Feb.

Abstract

Small ubiquitin-like modifier (SUMO) is a protein moiety that is ligated to lysine residues on a variety of target proteins. Many known SUMO substrates are transcription factors or coregulators of transcription, and in most cases, modification with SUMO leads to the attenuation of transcriptional activation. We have examined basic Kruppel-like factor/Kruppel-like factor 3 (BKLF), a zinc finger transcription factor that is known to function as a potent transcriptional repressor. We show that BKLF recruits the E2 SUMO-conjugating enzyme Ubc9 and can be modified by the addition of SUMO-1 in vitro and in vivo. The SUMO E3 ligases PIAS1, PIASgamma, PIASxalpha, and PIASxbeta but not Pc2 enhance the sumoylation of BKLF. Site-directed mutagenesis identified two lysines (K10 and K197) of BKLF as the sumoylation sites. Sumoylation does not detectably affect DNA binding by BKLF, but mutation of the sumoylation sites reduces transcriptional repression activity. Most interestingly, when mutations preventing sumoylation are combined with an additional mutation that eliminates contact with the C-terminal binding protein (CtBP) corepressor, BKLF becomes an activator of transcription. These results link SUMO modification to transcriptional repression and demonstrate that both recruitment of CtBP and sumoylation are required for full repression by BKLF.

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Figures

FIG. 1.
FIG. 1.
BKLF interacts with Ubc9 and is modified by SUMO-1 in vitro and in vivo. (A) mBKLF interacts with mUbc9. The results of yeast two-hybrid assays using mUbc9 as prey are shown. The interaction of mUbc9 with hSUMO-1 (positive control) and with mBKLF (1-268) is indicated by growth on −Leu −Trp −His plates. Control cells were transfected with empty bait vector. (B) In vitro sumoylation of BKLF. [35S]-methionine-labeled BKLF was expressed in rabbit reticulocyte lysate (lane 1), and SUMO modification was assayed in the presence or absence of purified GST-E1 (SAE1/SAE2), GST-E2 (Ubc9), GST-SUMO-1, or GST alone as indicated. Apparent molecular mass markers (in kilodaltons), full-length BKLF (double arrow), and BKLF-SUMO-1 conjugates (thick arrows) are indicated. Protein bands marked with asterisks appear to correspond to SUMO-modified forms of BKLF translation products of lower molecular mass produced in the in vitro reaction. (C) In vivo sumoylation of BKLF. COS-1 cells were transfected with constructs encoding full-length BKLF and HA-tagged SUMO-1 (500 ng). At 48 h posttransfection, nuclear extracts were prepared in the presence of N-ethylmaleimide (NEM) to inhibit desumoylating enzymes. Proteins were separated on SDS-12% polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were subsequently incubated with BKLF-specific (αBKLF, top panel) or HA (αHA, bottom panel) antibodies as indicated. Thick arrows point to the covalently modified wild-type BKLF protein. The asterisk depicts an endogenous sumoylated protein(s) (possibly RanBP2/Nup358) that was detectable with the HA antiserum but not with the BKLF antibody.
FIG. 2.
FIG. 2.
Identification of amino acids essential for SUMO-1 modification of BKLF. (A) Schematic representation of wild-type BKLF protein and the BKLF mutants used in this study. The black box indicates the repression domain of BKLF, the grey box indicates the PXDLT motif that recruits the corepressor mCtBP2, and three black arcs represent the zinc fingers of the DNA-binding domain of BKLF. K10 and K were mutated to 197 or alanine (A) or arginine (R). E199 was mutated to alanine. The mutant proteins were subjected to in vitro and in vivo SUMO modification. Only results obtained with lysine-to-alanine mutations are shown. (B) Mouse BKLF is sumoylated on residues 10 and 197. In vitro sumoylation was performed as described for Fig. 1B. Thick arrows indicate the major SUMO-1-modified forms of BKLF. (C) K197 is the major SUMO-1 acceptor site in BKLF. COS cells were cotransfected with the indicated BKLF mutants and a plasmid expressing HA-SUMO-1 (500 ng). Nuclear extractions and Western blotting were performed as in Fig. 1C. Thick arrows indicate the three SUMO-1-modified forms of BKLF.
FIG. 3.
FIG. 3.
PIAS family members stimulate SUMO conjugation to BKLF. (A) E3 activity in vitro. 35S-methionine-labeled BKLF was sumoylated in vitro as described for Fig. 1B but using only 100 ng of GST-SUMO-1. A total of 500 ng of purified GST-PIAS1, GST-ARIP3, or GST-Miz1 was added as indicated. Molecular mass markers (in kilodaltons), full-length BKLF (double arrows), and BKLF-SUMO-1 conjugates (thick arrows) are indicated. Protein bands marked with asterisks appear to correspond to SUMO-modified forms of BKLF translation products of lower molecular mass or to a multiply sumoylated form. (B) E3 activity in vivo. (Left panel) COS-1 cells were transfected with 500 ng of BKLF and HA-SUMO-1 expression vectors (lane 1) or 50 ng of HA-SUMO-1 expression vector (lanes 2 to 7) and 2 μg of expression vector for Miz1, PIASy, ARIP3, PIAS1, and Pc2 as indicated. The proteins were detected by Western blotting using anti-BKLF antibody. (Right panel) Experiments were also performed with a mutant BKLF that prevents CtBP2 binding (ΔDL).
FIG. 4.
FIG. 4.
SUMO-modified BKLF retains DNA-binding activity. A gel mobility shift experiment using BKLF or mutants BKLF K10A, K197A, or K10A/197A and GFP-SUMO-1 coexpressed in COS-1 cells shows that SUMO-modified BKLF binds a CACCC-box element. Lanes 1 to 15 contained COS-1 cell nuclear extracts prepared from cells transfected with the following expression vectors: lane 1, empty vector; lanes 2 to 3, BKLF-expressing vector; lanes 4 to 15, BKLF or indicated BKLF mutants and GFP-SUMO-1. The asterisk depicts an endogenous protein that binds to the probe. Antibodies were used as follows: anti-BKLF (lanes 3, 5, 8, 11, and 14) or anti-SUMO-1 (lanes 6, 9, 12, and 15). The arrows indicate the sites of migration of the BKLF-DNA complex, the SUMO-modified BKLF-DNA complexes supershifted with anti-SUMO-1 antisera, and the BKLF-DNA complex supershifted with anti-BKLF antisera, as indicated. The free probe is not shown.
FIG. 5.
FIG. 5.
SUMO modification sites are required for BKLF-mediated transcriptional repression. (A) A schematic diagram of the reporter construct used is shown at the top of the panel. A total of 50 ng of BKLF and the mutants indicated was cotransfected with 500 ng of glucocorticoid receptor expression plasmid and 500 ng of the CAT reporter into SL-2 cells. The addition of dexamethasone (Dex.) is indicated by plus signs. CAT activity was determined as described in Materials and Methods. Severalfold activation values were calculated from CAT activity levels in relation to glucocorticoid receptor activity, which has been given the arbitrary value of 100. CAT activity values are representative of at least three independent experiments. Adh, alcohol dehydrogenase promoter. (B) DNA-binding activity of BKLF mutants. Nuclear extracts from transfected SL-2 were used for a gel mobility assay using a CACCC-element-containing probe. The arrows indicate the BKLF/DNA complex. The free probe is not shown. C, untransfected SL-2 cells. The asterisk indicates an endogenous protein that binds to the probe.
FIG. 6.
FIG. 6.
Influence of the promoter context on BKLF repression activity. A schematic diagram of the Aγ-globin reporter construct used is shown at the top. Transient transfections into SL-2 cells were performed as described for Fig. 5.
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