SUMOylated SoxE factors recruit Grg4 and function as transcriptional repressors in the neural crest

Abstract

A growing number of transcriptional regulatory proteins are known to be modified by the small ubiquitin-like protein, SUMO. Posttranslational modification by SUMO may be one means by which transcriptional regulatory factors that play context-dependent roles in multiple processes can be regulated such that they direct the appropriate cellular and developmental outcomes. In early vertebrate embryos, SUMOylation of SoxE transcription factors profoundly affects their function, inhibiting their neural crest-inducing activity and promoting ear formation. In this paper, we provide mechanistic insight into how SUMO modification modulates SoxE function. We show that SUMOylation dramatically altered recruitment of transcriptional coregulator factors by SoxE proteins, displacing coactivators CREB-binding protein/p300 while promoting the recruitment of a corepressor, Grg4. These data demonstrate that SoxE proteins can function as transcriptional repressors in a SUMO-dependent manner. They further suggest a novel multivalent mechanism for SUMO-mediated recruitment of transcriptional coregulatory factors.

Figure 1.

Effects of SoxE and Mitf isoforms on Dct expression in Xenopus embryos and animal caps. (A) Schematic of SoxE and Mitf proteins and SUMOylation mutants. (B) In situ hybridization showing Dct expression in embryos injected with SoxE_2KR, Mitf_2KR, or both. Both Sox9_2KR and Sox10_2KR act synergistically with Mitf_2KR to induce premature and ectopic Dct expression in stage 23 and stage 28 embryos. (C) Animal cap assay showing effects of Sox9_2KR/SUMO on Mitf_2KR-mediated induction of Dct at stage 28. Sox9_2KR enhances, whereas Sox9_2KR/SUMO blocks, Dct induction. Light red staining represents lineage tracer β-gal. Bars, 200 µm. AD, activation domain; LZ, Leucine zipper.

Figure 2.

Effects of Sox9 and Mitf isoforms on Dct promoter activity and subcellular localization. (A) Schematic of the Dct promoter/reporter containing a Mitf binding site, a Lef-1 site, and six SoxE binding sites, S1, S2, S3, S4/4’ (dimeric sites), S5, and S6, from right to left. (B and C) Luciferase reporter assay in stage 17 embryo lysates (B) or human melanoma cells (C) using the Dct reporter. A Renilla reporter was coinjected for normalization. Relative luciferase activity (RLU) is represented as fold activation relative to the normalized activity of control embryos. Coexpression of non-SUMOylable forms of Mitf and Sox9 activate Dct expression synergistically, whereas SUMOylation of either protein significantly reduced reporter activity. (C) Immunofluorescence staining of animal caps dissected from embryos injected with flag-tagged Sox9 isoforms. Nuclei are marked by DAPI. All Sox9 isoforms colocalized with DAPI, suggesting that SUMOylation does not affect cellular localization of Sox9 in Xenopus embryos. Error bars represent the standard deviation of the mean of triplicate assays. Bars, 10 µm.

Figure 3.

SUMOylation does not modulate Dct promoter activity through steric interference. (A) Schematic of Mitf/Sox9-tethered constructs used. (B and C) EMSA demonstrating DNA binding by Mitf isoforms, Mitf/Sox9-tethered proteins (B), and Sox9 isoforms (C). In vitro translated proteins were incubated with ³²P-labeled probes containing S1/M sites (for Mitf and Mitf/Sox9 isoforms) or S4/4′ sites (for Sox9 isoforms) of the Dct promoter. Asterisks denote nonspecific bands. (D) Luciferase reporter assay using stage 17 embryos injected with Mitf/Sox9-tethered constructs or coinjected with Mitf and Sox9 isoforms. SUMO blocks promoter activation even when Mitf and Sox9 are tethered and thus corecruited. Error bars represent the standard deviation of the mean of triplicate assays. RLU, relative luciferase activity; WB, Western blot.

Figure 4.

Blocking HDAC activity does not relieve SUMO-mediated inhibition of the Dct promoter. (A and B) Luciferase reporter assays in stage 17 embryo lysates (A) or human melanoma cells (B) using the Dct reporter. Sox9 and Mitf isoforms were coexpressed in the presence or absence of HDAC inhibitor trichostatin A (TSA). Treatment with TSA did not relieve Dct inhibition mediated by either Sox9_2KR/SUMO or Mitf_2KR/SUMO. Error bars represent the standard deviation of the mean of triplicate assays. (C) In situ hybridization showing TSA treatment was effective, as it caused expected loss of Slug expression in stage 17 embryos. Bars, 200 µm. RLU, relative luciferase activity.

Figure 5.

SUMOylation of Sox9 alters recruitment of transcriptional cofactors. (A) Coimmunoprecipitation from embryo lysates expressing flag-tagged Sox9 isoforms. Immunoprecipitation used antibodies against endogenous CBP or p300 followed by Western analysis with α-flag. A mock antibody treatment (−) was used as a negative control. SUMOylation blocks interaction with CBP or p300. (B) Coimmunoprecipitation from embryos injected with Myc-tagged Sox9 isoforms and flag-tagged Grg4. Immunoprecipitation used a flag antibody followed by Western analysis with α-Myc. Sox9_2KR/SUMO, but not Sox9_2KR, interacts with corepressor Grg4. (C) Schematic summarizing results of interaction experiments using Grg4 and SoxE/SUMO deletion constructs. The Grg4-WD40 domain and the activation domain plus SUMO moiety of SoxE/SUMO are sufficient for interaction. (D) GST pull-down experiment demonstrating direct interaction between the Grg4-WD40 domain and the Sox9 activation domain (AD)–SUMO fusion. IB, immunoblot; IP, immunoprecipitation.

Figure 6.

Grg4-mediated repression of the Dct promoter and neural crest formation are dependent on SoxE SUMOylation. (A) Luciferase assay measuring Dct reporter activity in embryos expressing either wild-type Sox9 or Sox9_2KR. Grg4 represses Dct promoter activity mediated by wild-type Sox9 but not the form that cannot be SUMOylated. (B) Luciferase assay measuring Dct reporter activity in embryos expressing wild-type Sox9 either in the presence or absence of SUMOylase Senp1b. Grg4 can repress Dct promoter activity mediated by wild-type Sox9 only in the absence of Senp1b. (C, top) In situ hybridization of showing Grg4-mediated inhibition of neural crest markers Slug and Sox10 at stage 17. (bottom) Ectopic ear formation was observed in Grg4-injected embryos at stage 28. (D) In situ hybridization showing effects of Grg4, Sox9_2KR, or both on Sox10 expression. Grg4 cannot repress Sox9_2KR-mediated neural crest formation. (E, top) Schematic of Sox9_2KR-Grg4–tethered construct. In situ hybridization showing effects of Sox9_2KR-Grg4, Sox9_2KR, or Sox9_2KR/SUMO on Sox10 expression. Tethered Sox9_2KR-Grg4 phenocopies the effects of Sox9_2KR/SUMO. Arrowheads denote the injected side of embryos. Error bars represent the standard deviation of the mean of triplicate assays. Bars, 200 µm. RLU, relative luciferase activity.

Figure 7.

SoxE-SUMO recruits Grg4 by a novel bivalent mechanism. (A) Mutation of an essential SIM-interacting residue on the SUMO moiety (SUMO_mut and Sox9_2KR/SUMO_mut) abolishes interaction with UBC9 but not with Grg4QW, which includes the poly-Q and WD domains. (B) Coimmunoprecipitation from Xenopus lysates showing that neither SUMO nor Sox9 is sufficient for robust interaction with Grg4. Strong interaction requires surfaces on both SUMO and Grg4. (A and B) Asterisks denote IgG bands. (C) Model describing the mechanism underlying SUMO-mediated modulation of SoxE function. SUMOylation of SoxE converts it from a transcriptional activator to a repressor by displacing coactivator CBP/p300 and recruiting corepressor Grg4 via a multivalent interaction.