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. 2016 May 27;291(22):11619-34.
doi: 10.1074/jbc.M115.713370. Epub 2016 Apr 11.

Transcription Factor hDREF Is a Novel SUMO E3 Ligase of Mi2α

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Free PMC article

Transcription Factor hDREF Is a Novel SUMO E3 Ligase of Mi2α

Daisuke Yamashita et al. J Biol Chem. .
Free PMC article

Abstract

The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.

Keywords: E3 SUMO ligase; Mi2alpha; gene regulation; hDREF; nucleosome remodeling deacetylase (NuRD); sumoylation; transcription factor; transcription regulation.

Figures

FIGURE 1.
FIGURE 1.
hDREF is SUMOylated. A, endogenous hDREF is conjugated with SUMO-1. Proteins in HeLa cell extract were immunoprecipitated (IP) with anti-SUMO-1 polyclonal antibody and subjected to immunoblotting analysis (IB) with anti-hDREF antibody. The data are representative of two independent experiments with similar results. B, HeLa cells were cotransfected with HA-hDREF and CFP-SUMO expression plasmids as indicated. HA-hDREF polypeptides with or without SUMO conjugation and SUMOylated endogenous proteins were detected by immunoblotting analysis with anti-HA and anti-GFP antibodies, respectively. The data are representative of two independent experiments with similar results. C, HeLa cells were cotransfected with HA-hDREF plasmid and CFP-SUMO-1 plasmids (wild-type (wt) or G97A mutant (mt) SUMO-1) as indicated. HA-hDREF polypeptides were precipitated with anti-HA antibody and analyzed by immunoblotting using an anti-HA antibody (top). SUMOylated proteins coimmunoprecipitated with HA-hDREF were also detected using anti-GFP antibody (bottom). The data are representative of two independent experiments with similar results. Arrowheads, represent HA-hDREF with CFP-SUMO-1 conjugation. D, in vitro SUMOylation assay using a set of hDREF deletion mutants labeled with [35S]methionine. hDREF was synthesized by cell-free coupled in vitro transcription/translation in the presence of [35S]methionine and subjected to an in vitro SUMOylation reaction containing 2 μg of GST-SUMO-1(GG), 1 μg of GST-Ubc9, 0.5 μg of GST-Uba2, 0.5 μg of GST-Aos1 in 50 mm Tris-HCl (pH 8.0), 5 mm MgCl2, 5 mm ATP, and 1 mm dithiothreitol at 37 °C for 30 min. Reactions were terminated by heating the samples in Laemmli's sample buffer at 85 °C for 3 min. The samples were resolved by SDS-PAGE, and signals were detected by autoradiography. *, SUMOylated 35S-hDREF. The data are representative of two independent experiments with similar results.
FIGURE 2.
FIGURE 2.
hDREF increases the amount of SUMO-conjugated protein in vivo. A, 35S-labeled full-length hDREF was synthesized by a cell-free coupled in vitro transcription/translation reaction in the presence of [35S]methionine and subjected to GST pull-down using GST fusion proteins as indicated. As a positive control, GST-hDREF was used because hDREF forms a homodimer. The data are representative of two independent experiments with similar results. B, HeLa cells were cotransfected with HA-hDREF plasmid and Myc-SUMO-1, Myc-SUMO-2, Myc-SUMO-3, Myc-Ubc9, Myc-PIAS1, or Myc-Pc2 plasmid as indicated. At 24 h after DNA transfection, the cells were fixed with 3.7% PFA and stained using anti-Myc and anti-HA antibodies. Single confocal optical sections (n > 10) are shown. Scale bar, 5 μm. C, HeLa cells were transduced with lentivirus expressing shRNA against hDREF or scramble control. At 72 h after transduction, the cells were transfected with CFP-SUMO-1, CFP-SUMO-2, or CFP-SUMO-3 and cultured for an additional 24 h. Whole cell lysates were prepared, and protein samples (20 μg of protein) were analyzed by immunoblotting (IB) using anti-hDREF antibody and anti-GFP antibody. Two independent experiments validated >85% knockdown of hDREF by shRNA. The data are representative of two independent experiments with similar results. D, schematic representation of the hDREF structural domain and amino acid residues required for SUMO conjugation. Mutations resided in the N-terminal BED finger domain (C47A, C50A, C47A/C50A, H61A, and H71A), putative SUMOylation domain (ΔM360 and ΔL401), or the C-terminal hATC (hAT family C-terminal dimerization: pfam05699) domain (ΔATC). HeLa cells were transfected with plasmid expressing WT or mutant HA-hDREF as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and hDREF and endogenous proteins conjugated with SUMO-1 were analyzed by immunoblotting using anti-HA and anti-GFP antibodies, respectively. The data are representative of three independent experiments with similar results.
FIGURE 3.
FIGURE 3.
hDREF directs Mi2α SUMOylation in vivo. A, HeLa cells were cotransfected with HA-Mi2α with or without of FLAG-hDREF and CFP-SUMO-1 plasmids as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and HA-Mi2α, FLAG-hDREF, and CFP or CFP-SUMO-1 were detected by immunoblotting analysis (IB) using anti-HA, anti-FLAG, and anti-GFP antibodies, respectively. The data are representative of two independent experiments with similar results. B, HeLa cells were cotransfected with HA-Mi2α and CFP-SUMO paralogs with or without FLAG-hDREF plasmid as indicated. Whole cell lysates were prepared at 24 h after DNA transfection and HA-Mi2α and CFP or CFP-SUMOs were detected by immunoblotting analysis using anti-HA and anti-GFP antibodies, respectively. The data are representative of two independent experiments with similar results. C, Mi2α is SUMOylated at Lys-1971 by hDREF. Left, alignment of putative SUMOylation sites on Mi2α. Right, HeLa cells were cotransfected with HA-Mi2α WT or KR mutants and CFP-SUMO-1 with or without FLAG-hDREF plasmid. At 24 h after transfection, whole cell lysates were prepared, and HA-Mi2α was detected by immunoblotting analysis using anti-HA antibody. The data are representative of two independent experiments with similar results. D, HeLa cells were cotransfected with HA-Mi2α and CFP-SUMO-1 with or without FLAG-hDREF (shRNA-resistant) expression plasmid and plasmid expressing shRNA against hDREF mRNA (KD) or scramble shRNA plasmid as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and HA-Mi2α was detected by immunoblotting analysis using anti-HA antibody. Knockdown of endogenous hDREF and expression of shRNA-resistant hDREF were evidently detected on an immunoblot by using anti-hDREF antibody. The data are representative of two independent experiments with similar results. E, HeLa cells were expressed with HA-Mi2α, CFP-SUMO-1, and WT or SUMOylation-defective hDREF mutants (W500A/W501A and LLVL/AAAA) as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and Mi2α polypeptides were detected by immunoblotting analysis using anti-HA antibody. The data are representative of two independent experiments with similar results.
FIGURE 4.
FIGURE 4.
hDREF is a SUMO E3 ligase. A, 35S-labeled full-length Mi2α was synthesized by cell-free coupled in vitro transcription/translation and subjected to in vitro SUMOylation reaction. The reaction mixture contained GST-hDREF, GST-PIAS1, or GST-Pc2 as an E3 enzyme in addition to GST-SUMO-1, GST-Uba2, GST-Aos1, and GST-Ubc9 and ATP, as described under “Experimental Procedures.” Proteins were separated on an SDS-polyacrylamide gel, and 35S-Mi2α polypeptide was detected by autoradiography. The data are representative of three independent experiments with similar results. B, Coomassie Brilliant Blue (CBB) staining of purified recombinant proteins used in A and C. C, in vitro SUMOylation using a recombinant His-Mi2α(1617–2000). In vitro SUMOylation in the reaction mixture containing GST-SUMO-1, GST-Aos1 (E1), GST-Uba2 (E1), GST-Ubc9 (E2), GST-hDREF, and His-Mi2α(1617–2000), as indicated, was performed. Proteins were separated on an SDS-polyacrylamide gel, and His-Mi2α polypeptide was detected by immunoblotting analysis (IB) using anti-His antibody. The data are representative of three independent experiments with similar results.
FIGURE 5.
FIGURE 5.
SUMO-1 enhances the association between hDREF and Mi2α. A, 293FT cells were cotransfected with HA-Mi2α (WT or K1971R mutant) with or without CFP-SUMO-1 and FLAG-hDREF plasmids as indicated. At 24 h after DNA transfection, whole cell lysates were prepared and subjected to immunoprecipitation (IP) using anti-HA antibody. hDREF and its SUMOylated form co-immunoprecipitated with Mi2α were detected by immunoblotting analysis (IB) using anti-FLAG antibody. The data are representative of two independent experiments with similar results. B, 293FT cells were cotransfected with HA-Mi2α (WT or K1971R mutant) with and without CFP-SUMO-1 and FLAG-hDREF plasmids as indicated. At 24 h after DNA transfection, whole cell lysates were prepared and subjected to immunoprecipitation using anti-FLAG antibody. Mi2α and its SUMOylated form in the immunoprecipitated material were detected by immunoblotting analysis using anti-HA antibody. The data are representative of two independent experiments with similar results. C, 293FT cells were transfected with FLAG-HDAC1 and HA-Mi2α (WT or K1971R mutant) with or without CFP-SUMO-1 and HA-hDREF plasmids as indicated. At 24 h after DNA transfection, cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. Components of NuRD complex (HDAC1, Mi2α, RbAp48, MTA2, and MBD3) in the immunoprecipitated samples were detected with anti-FLAG, anti-HA antibodies, and specific antibodies against RbAp48, MTA2, and MBD3, respectively. The data are representative of three independent experiments with similar results.
FIGURE 6.
FIGURE 6.
SUMOylation by hDREF promotes release of Mi2α from chromatin. A, HeLa cells were transfected with FLAG-hDREF, HA-Mi2α (WT or K1971R mutant), or Myc-SUMO-1 as indicated. At 24 h after DNA transfection, cells were treated with or without 40 μm digitonin for 6 min at 20 °C, fixed with 2% PFA, and stained with anti-FLAG and anti-HA antibodies followed by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. In the case of no treatment with digitonin, cells were permeabilized with 0.3% Triton X-100 after fixation. Signals of FLAG-hDREF and HA-Mi2α were shown in confocal images using Zeiss LSM 510 microscope. Scale bars, 5 μm. The images are representative of cells (n > 50) examined in three independent experiments. B, HeLa cells simultaneously expressing FLAG-hDREF, HA-Mi2α (WT), and Myc-SUMO-1 were treated with 40 μm digitonin for 7 min at 20 °C, fixed with 2% paraformaldehyde, and immunostained with anti-FLAG and anti-PML antibodies (top) or with anti-FLAG and anti-Myc antibodies (bottom) as indicated. Confocal images of signals were obtained using a Zeiss LSM 510 microscope. Scale bars, 5 μm. The images are representative of cells (n > 50) examined in three independent experiments. C, 293FT cells were transfected with FLAG-hDREF or HA-Mi2α (WT or K1971R mutant) with or without CFP-SUMO-1 plasmid as indicated. At 24 h after DNA transfection, soluble and chromatin-bound fractions were prepared by biochemical fractionation as described under “Experimental Procedures.” hDREF and Mi2α in each fraction were detected by immunoblotting analysis (IB) using anti-FLAG and anti-HA antibodies, respectively. Data are representative of three independent experiments. D, signals of Mi2α in the soluble and chromatin-bound fractions were quantified by densitometry scanning of immunoblots. Graphs show the percentage of Mi2α in the soluble fraction and in the chromatin-bound fraction recovered from total cell lysates. Error bars, S.E. *, p < 0.05, n = 3.
FIGURE 7.
FIGURE 7.
hDREF and SUMO-1 coexpression overcomes transcriptional repression by Mi2α. A, HeLa cells were transfected with plasmid expressing FLAG-hDREF, HA-Mi2α (WT or K1971R), or CFP-SUMO-1 as indicated. At 48 h after transfection, total RNA was extracted, and qRT-PCR was performed for RPS6 and GAPDH. The intensity of individual bands was quantified and expressed relative to the control (no transfection). Amounts of RPS6 mRNA were normalized to those of GAPDH as an internal control. Data are the mean of three independent experiments. Error bars, S.E. **, p < 0.02; n = 3. B, HeLa cells were transfected with FLAG-hDREF, HA-Mi2α (WT or K1971R), or CFP-SUMO-1 in the indicated combinations. At 48 h after transfection, total RNA was extracted, and qRT-PCR was performed for RPS6 and GAPDH. Amounts of RPS6 mRNA were normalized by those of GAPDH as an internal control. Data are the average of three independent experiments. Error bars, S.E. *, p < 0.05; **, p < 0.02; n = 3. C, HeLa cells were transfected with FLAG-hDREF, HA-Mi2α (WT or K1971R), and CFP-SUMO-1 as indicated. At 48 h after transfection, total RNA was extracted, and qRT-PCR was performed for RPS6, RPL10, RPL12, and GAPDH. Amounts of mRNA of RPS6, RPL10, and RPL12 were normalized by those of GAPDH as an internal control. Data are expressed as the average value relative to a no transfection control. n = 3. D, schematic illustration of the RPS6 gene. Arrowhead, a hDREF binding site. Solid bars, the region amplified by PCR in ChIP analysis. E, 293FT cells were transfected with FLAG-hDREF, HA-Mi2α, and CFP-SUMO-1 as indicated. At 36 h after transfection, cells were cross-linked with 1.1% formaldehyde for 5 min, lysed, and sonicated. After centrifugation, the cleared supernatant containing chromatin was subjected to immunoprecipitation using antibodies (control IgG, anti-FLAG, anti-HA, anti-RNA polymerase II (phospho-CTD), anti-histone H3 Lys-4-trimethyl, and anti-histone H3 Lys-9-trimethyl). PCR was quantitatively performed in the reaction mixture containing 32P-dCTP using genomic DNA from the input extract and DNA recovered from the immunoprecipitates as indicated. PCR products were separated by electrophoresis on 6% acrylamide gels and detected by autoradiography. The intensity of individual bands was quantified and expressed as a percentage of input. Data are the average of three independent experiments. Error bars, S.E. (*, p < 0.05; **, p < 0.02). F, ChIP assays were performed using antibodies as described. Instead of WT Mi2α, SUMOylation-defective K1971R mutant was expressed in 293FT cells. Data are the average of three independent experiments. Error bars, S.E. (*, p < 0.05; **, p < 0.02).

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