SUMOylation of the transcription factor ZFHX3 at Lys-2806 requires SAE1, UBC9, and PIAS2 and enhances its stability and function in cell proliferation

Abstract

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

Keywords: SUMO-conjugating enzyme UBC9; SUMO-specific peptidase 1 (SENP1); SUMO1-activating enzyme subunit 1 (SAE1); SUMOylation; UBC9; cancer; cell proliferation; posttranslational modification (PTM); proliferation; protein inhibitor of activated STAT2 (PIAS2); protein stability; transcription factor; zinc finger homeobox 3 (ZFHX3).

Figure 1.

Lysine 2806 is the major SUMOylation site of ZFHX3. A, ZFHX3 can be SUMOylated by SUMO1, SUMO2, and SUMO3. HEK293T cells were transfected with expression plasmids of HA-tagged ZFHX3 (HA-ZFHX3) and GFP-tagged SUMO1, SUMO2, or SUMO3, and ZFHX3 was detected by Western blotting with anti-ZFHX3 antibody. B, detection of SUMOylated ZFHX3 in HEK293T cells expressing HA-ZFHX3 and GFP-SUMO1. Cell lysates were precipitated with anti-HA-beads, and eluted proteins were immunoblotted (IB) with anti-SUMO1 (top) or anti-ZFHX3 (middle) antibody. The middle membrane was stripped and reprobed with anti-SUMO1 antibody (bottom). C and D, detection of endogenous ZFHX3 SUMOylation in HeLa cells with RNAi-mediated SENP1 knockdown by IP and Western blotting with ZFHX3 antibody (C) or in HeLa cells by IP with SUMO1 antibody and Western blotting with anti-ZFHX3 and anti-SUMO1 antibodies (D). E, four candidate SUMOylation sites in ZFHX3, including three consensuses and one nonconsensus, were predicted by the SUMOsp software. F, Lys-2806 is the major acceptor site for ZFHX3 SUMOylation. FLAG-ZFHX3, its Lys-to-Arg mutants at predicted SUMOylation sites, and GFP-SUMO1 were expressed in HEK293T cells, and ZFHX3 and its SUMOylated form were detected by Western blotting with anti-ZFHX3 and anti-FLAG antibodies. G, Lys-2806 is SUMOylated by SUMO1, SUMO2, and SUMO3. HEK293T cells expressing GFP-tagged SUMO1, SUMO2, or SUMO3 and HA-tagged ZFHX3 or its K2806R mutant were subjected to Western blotting with anti-HA and anti-GFP antibodies. H, detection of ZFHX3 SUMOylation in HEK293T cells ectopically expressing FLAG-, HA-, and GFP-tagged SUMO1 and Myc-tagged ZFHX3 by Western blotting with the indicated antibodies. I, HEK293T cells transfected with HA-ZFHX3 and different isoforms of SUMO1, SUMO2, or SUMO3 were subjected to Western blotting with the indicated antibodies. J, alignment of ZFHX3 sequences from different species surrounding the human Lys-2806 SUMOylation site, with the conserved lysine shown in boldface type.

Figure 2.

Identification of SAE1 as E1 activating enzyme, UBC9 as E2 conjugating enzyme, and PIAS2 as E3 ligase for ZFHX3 SUMOylation. A and B, ectopic expression of the E1 enzyme SAE1 promotes (A), whereas its knockdown attenuates (B), ZFHX3 SUMOylation, as detected by Western blotting with the indicated antibodies in HEK293T cells expressing HA-ZFHX3 and GFP-SUMO1 in the presence or absence of FLAG-SAE1 or its siRNA. C and D, ectopic expression of UBC9 promotes (C), whereas its knockdown attenuates (D), ZFHX3 SUMOylation, as detected by Western blotting with the indicated antibodies in HEK293T cells expressing HA-ZFHX3 and GFP-SUMO1 in the presence or absence of FLAG-UBC9 or its siRNA. E, ZFHX3 physically associates with UBC9, as detected by co-IP with FLAG M2 beads and Western blotting with anti-HA antibody in HEK293T cells expressing HA-ZFHX3 in the presence or absence of FLAG-UBC9. F–I, identification of PIAS2 as the E3 ligase for ZFHX3 SUMOylation. HEK293T cells expressing FLAG-ZFHX3, GFP-SUMO1, and different HA-tagged PIASs were analyzed by Western blotting with the indicated antibodies (F). HeLa cells ectopically expressing FLAG-UBC9 and HA-PIAS2α were subjected to Western blotting with the indicated antibodies (G). HEK293T cells expressing FLAG-ZFHX3, GFP-SUMO1, and different HA-tagged PIASs were analyzed by co-IP and Western blotting with the indicated antibodies (H). HeLa cells transfected with siRNA against PIAS2α, with ectopic expression of HA-ZFHX3 and GFP-SUMO1, were analyzed by Western blotting with the indicated antibodies (I). J and K, FLAG-UBC9, HA-PIAS2α, and GFP-SUMO1 were ectopically expressed with WT ZFHX3 or its K2806R mutant in HEK293T cells, and SUMOylation of ZFHX3 was analyzed by Western blotting with anti-ZFHX3 antibody.

Figure 3.

SENP1 deSUMOylates ZFHX3. A, SENP1 and SENP2, but not SENP3, -5, -6, and -7a, deSUMOylate ZFHX3. HEK293T cells were transfected with HA-ZFHX3, GFP-SUMO1, and the indicated isoforms of SENPs, and SUMOylation of ZFHX3 was analyzed by Western blotting with antibody against ZFHX3. B, ZFHX3 interacts with SENP1 and SENP2. HEK293T cells expressing HA-ZFHX3 and FLAG-SENP1 or FLAG-SENP2 were subjected to immunoprecipitation and Western blotting with the indicated antibodies. C, depletion of SENP1 enhanced SUMOylation of ZFHX3. HEK293T cells were transfected with control or siRNA against SENP1, with ectopic expression of HA-ZFHX3 and GFP-SUMO1 or FLAG-SENP1 for Western blotting detection as indicated. D, SENP1 deSUMOylates ZFHX3 dependent on its catalytic activity. HA-ZFHX3 and GFP-SUMO1/SUMO2/SUMO3 were ectopically expressed in the presence of WT SENP1 or the SENP1 C603A mutant in HEK293T cells, and SUMOylation of ZFHX3 was examined by Western blotting with the indicated antibodies. E, SENP1 reduces SUMOylation of ZFHX3 in a dose-dependent manner. HEK293T cells were transfected with HA-ZFHX3 and GFP-SUMO1 with different amounts of FLAG-SENP1, and SUMOylation of ZFHX3 was analyzed by Western blotting with the indicated antibodies. F and G, SENP1-mediated ZFHX3 deSUMOylation mainly occurs at Lys-2806 of ZFHX3. WT ZFHX3 or its K2806R mutant was transfected with GFP-SUMO1 and FLAG-SENP1 into HEK293T cells (F), and siRNAs against SENP1 were transfected into HeLa cells with expression plasmids of GFP-SUMO1 and WT ZFHX3 or its K2806R mutant (G), and SUMOylation of ZFHX3 was analyzed by Western blotting with anti-HA antibody.

Figure 4.

SENP1 interacts with ZFHX3. A and B, ZFHX3 and SENP1 interact with each other. HEK293T cells expressing HA-ZFHX3 and FLAG-SENP1 were subjected to immunoprecipitation and Western blotting with the indicated antibodies (A). HeLa cells were subjected to immunoprecipitation with ZFHX3 antibody in the presence of NEM and Western blotting with anti-ZFHX3 and SENP1 antibodies to detect the interaction of endogenous ZFHX3 and SENP1 (B). C and D, SENP1 binds to ZFHX3 independent of its catalytic activity. HEK293T cells transfected with HA-ZFHX3 and FLAG-SENP1 C603A mutant (C) or the same cells transfected with HA-ZFHX3 and FLAG-SENP1 in the presence or absence of NEM (D) were subjected to immunoprecipitation and Western blotting with the indicated antibodies. E, SENP1 interacts with the central region of ZFHX3. HEK293T cells transfected with FLAG-SENP1 and WT HA-ZFHX3 or its deletion mutants were subjected to immunoprecipitation and Western blotting with the indicated antibodies. F, ZFHX3 interacts with the central region of SENP1. HEK293T cells transfected with HA-ZFHX3 and WT FLAG-SENP1 or its deletion mutants were subjected to immunoprecipitation and Western blotting with the indicated antibodies. G and H, diagrams of ZFHX3 and its deletion mutants (G) as well as SENP1 and its deletion mutants (H).

Figure 5.

SUMOylation of ZFHX3 enhances its protein stability. A, degradation of ZFHX3 by the EFP E3 ligase is partially rescued by SUMO1 expression in HeLa cells transfected with HA-ZFHX3, FLAG-SENP1, and SUMO1, as detected with anti-ZFHX3 antibody. B, SUMOylation prevents the degradation of ZFHX3 induced by ubiquitin. HeLa cells transfected with Myc-ZFHX3, HA-Ub, and SUMO1 were subjected to Western blotting with the indicated antibodies. C, knockdown of endogenous EFP increases the levels of ZFHX3 and SUMOylated ZFHX3. HeLa cells were transfected with siRNA against EFP with HA-ZFHX3 and GFP-SUMO1, and ZFHX3 and SUMOylated ZFHX3 were detected with anti-ZFHX3 antibody. D, SUMO1 weakens the ZFHX3-EFP interaction. HEK293T cells transfected with HA-ZFHX3 and FLAG-EFP in the presence or absence of GFP-SUMO1 were treated with MG132 (20 μm, 6 h) and then subjected to immunoprecipitation and Western blotting with the indicated antibodies. E, SENP1 enhances the interaction between ZFHX3 and EFP. HeLa cells were transfected with siRNA against SENP1 with HA-ZFHX3 and FLAG-EFP, treated with MG132, and then subjected to immunoprecipitation with HA-beads and Western blotting with the indicated antibodies. F, loss of SUMOylation enhances the ubiquitination of ZFHX3. WT ZFHX3 or its K2806R mutant was ectopically expressed with HA-Ub in HEK293T cells, which were treated with MG132 and then subjected to immunoprecipitation and Western blotting with anti-FLAG and anti-HA antibodies, respectively. G, half-life of ZFHX3-K2806R is shorter than that of ZFHX3-WT. HEK293T cells were transfected with WT ZFHX3 or the ZFHX3-K2806R mutant, treated with 100 μg/ml CHX for the indicated times, and then subjected to Western blotting with anti-HA antibody. Representative blots are shown at the top, and the quantitation of relative protein levels is shown at the bottom (data were from three independent experiments). *, p < 0.05; **, p < 0.01. H and I, half-life of endogenous ZFHX3 was determined by the CHX assay in HeLa cells transfected with siRNA against UBC9 (H) or PIAS2α (I) and treated with CHX for the indicated times. Western blotting, protein quantification, and statistical analysis were performed the same way as in G except that data were from two independent experiments. Error bars, S.E.

Figure 6.

SUMOylation does not change the subcellular localization of ZFHX3. A, subcellular localization of ZFHX3. WT ZFHX3 or its K2806R mutant plasmid was transfected into HeLa cells, and ZFHX3 was immunostained with anti-HA antibody (red) and visualized by fluorescence microscopy. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 5 μm. B, nuclear expression of ZFHX3 was increased by NEM treatment. Cytoplasmic and nuclear fractions were collected from HeLa cells treated with or without NEM and then subjected to Western blotting with antibodies against the lamin B1 nuclear protein, the tubulin cytoplasmic protein, and ZFHX3. C, GA attenuates ZFHX3 SUMOylation. Hela cells were transfected with HA-ZFHX3 and GFP-SUMO1, treated with different concentrations of GA for 4 h, and then subjected to Western blotting with the indicated antibodies. D, GA attenuates the nuclear localization of ZFHX3. HeLa cells were treated with 50 μm GA for 4 h and then analyzed as in B.

Figure 7.

SUMOylation is required for ZFHX3 to promote cell proliferation and tumor growth of MDA-MB-231 breast cancer cells. A, the K2806R mutation of ZFHX3 dramatically decreases cell growth, as measured by the SRB assay. B, interruption of ZFHX3 SUMOylation prevents ZFHX3 from promoting sphere formation in Matrigel. The data are presented as the number of spheres (>75 μm) per well (1600 seeded cells) after culture for 12 days (top) or 15 days (bottom). Representative bright field images for spheres are shown for each group. Scale bar, 200 μm. ***, p < 0.001. C, SUMOylation deficiency of ZFHX3 significantly reduces cell proliferation in Hs 578T cells. Growth of cells with ectopic expression of control vector, WT ZFHX3, or ZFHX3 K2806R mutant plasmid were measured by crystal violet staining and Matrigel assays described as above. Scale bar, 200 μm. *, p < 0.05; ***, p < 0.001. D, interruption of SUMOylation prevents ZFHX3 from promoting xenograft tumor growth. Cells stably expressing control vector, WT ZFHX3, and the K2806R mutant were inoculated into nude mice subcutaneously (1.5 × 10⁶ cells/site). Tumor volumes were measured at the indicated times (middle), and tumors were isolated at 35 days (images are shown at the left) and weighted (right). ***, p < 0.001. E, detection of the indicated molecules in cells stably transfected with control vector, WT ZFHX3, or its K2806R mutant by Western blotting with respective antibodies. F, detection of Ki-67, MYC, and cyclin D1 by IHC staining in xenograft tumors from D (left), and the rates of staining positivity for each of the molecules are shown at the right. Scale bar, 100 μm. **, p < 0.01; ***, p < 0.001. Error bars, S.E.