The SOXE DIM domain mediates cooperative assembly on flexibly spaced dimer motifs

To test whether SOXE proteins can cooperatively assemble on the melanoma-specific sequence signatures, we conducted electrophoretic mobility shift assays (EMSAs) to quantify the cooperativity factor (ω) using previously described methods³⁶. If the value for ω is>1 we will henceforth refer to ‘cooperative’ binding, if it is ~1 to an additive and if smaller than 1 to a competitive binding mode. To conduct these experiments, we constructed a series of SOXE protein variants and purified them to>95% purity as judged by SDS-PAGE (Fig. 2A,B). These comprise SOXE-HMG constructs consisting of the 79 amino acid DNA binding HMG domain; SOXE-DHMG constructs that contains the 40 amino acid DIM domain preceding the HMG domain and a SOXE-NHMG construct that resembles the a naturally occurring truncation mutant³⁷ that spans N-terminus, DIM and HMG domains (Figs. 1D, ​D,2A,B).2A,B). We designed EMSA probes corresponding to the three motifs enriched in melanoma cell enhancers as well as for DNA elements with more distant or more compressed half-site spacing (Fig. 3A). EMSAs demonstrated that SOX9-NHMG binds to the three elements enriched in melanoma cell DHS in a highly cooperative fashion (Fig. 3B,C). By contrast, experiments using the isolated SOX9 HMG domain shows a marked reduction of the cooperativity reaffirming that the DIM domain facilitates the cooperative assembly (Fig. 3B,D). When control motif configurations that are not enriched in melanoma enhancers (0,1,2bp and 6bp) were tested, a ~10-fold diminished cooperativity factor was measured (Fig. 3B,C). Nevertheless, with a cooperativity factor of ~100, dimers are still formed efficiently. Only the 10bp spacer led to a more pronounced drop of the cooperative binding (Fig. 3B,C). The SOX8-NHMG and SOX10-DHMG constructs cooperate on the 4bp element equally strong as the SOX9-NHMG suggesting that the cooperative recognition of palindromic DNA elements is a conserved property shared by all SOXE TFs (Fig. 3E,F).

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Figure 2

SOXE protein constructs used in this study. (A) Diagrams of the protein constructs used for the binding assays. The DIM domain is shown in white and the 79aa HMG domain in gray. (B) 15% SDS-PAGE of the purified proteins.

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Figure 3

SOXE proteins cooperatively dimerize on flexibly spaced dimer motifs. (A) Sequences of the dsDNA used for EMSAs with SOX half-site in bold face. (B) Barplots showing the mean cooperativity factors (ω) obtained from 3–6 measurements for the the SOX9-NHMG (black) and SOX9-HMG (gray). ω values were calculated as described³⁶. For the 4 and 5bp spacers ω was denoted as infinite as it could not be reliable determined since the fractional contribution of monomer bands is lower than 2% per lane. (C) Representative EMSAs of SOX9-NHMG constructs on differently spaced dimer motifs. (D) EMSAs of SOX9-HMG constructs on 4 and 10bp elements. (E,F) EMSAs of SOX8-NHMG (E) and SOX10-DHMG (F) constructs on the 4bp spacer element.Protein concentrations in nM per lane are indicated and construct and ternary complexes are shown with cartoons to the left of the gel image where lines indicate the DNA and circles the proteins.

SOX8, SOX9 and SOX10 form cooperative heterodimers mediated by a single DIM domain

SOXE proteins can be expressed singly or in different combinations in several cell types but in many instances function non-redundantly^15,17. As transcription factors (TFs) in general, and SOX factors in particular, act combinatorially and switch partners to perform cell-type specific functions³⁸, we decided to study SOXE heterodimerization. Although the HMG as well as the DIM domain is highly conserved between SOX8, SOX9 and SOX10, there are several amino acid substitutions that could cause discriminatory complex formation leading to paralog specific functions (Fig. 1D). Importantly, rather subtle missense mutations within the SOXE-DIM are associated with the CD phenotype^28,29 (Fig. 1D). To be able to distinguish SOXE homodimers and heterodimers in EMSAs, we used protein constructs of different lengths (Fig. 2A,B). When we mixed SOXE constructs with the 4bp spacer element, we found that SOX9-NHMG:SOX10-DHMG, SOX9-DHMG:SOX8-NHMG or SOX8-NHMG:SOX10-DHMG heterodimers migrate as clearly distinguishable bands in EMSAs (Fig. 4A). Moreover, heterodimer bands are as prominent as the respective homodimer bands suggesting that heterodimerization between SOXE pairs occur in a highly cooperative manner (Fig. 4A). This result is consistent with a previous study reporting SOX8-SOX10 heterodimers³⁹.

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Figure 4

Cooperative SOX8/9/10 heterodimerization is promoted by a single DIM domain. (A) EMSAs after mixing DIM containing SOXE proteins lead to the formation of a prominent heterodimer band in addition to homodimer bands. (B) Mixing of the SOX9-NHMG with the isolated HMGs of SOX8, SOX9 and SOX10 results in the formation of SOXE-HMG:SOX9-NHMG heterodimers that are more abundant than the SOX9-NHMG homodimers or SOXE-HMG homodimers. (C,D) Likewise, when the SOX9-NHMG was replaced by the SOX10-DHMG (C) or SOX8-NHMG (D) constructs to monitor heterodimer formation the SOX10-DHMG:SOXE-HMG and SOX8-NHMG:SOXE-HMG heterodimers were found to be the most prominent complexes.

Several TF families harbor dimerization or multimerization domains including the Smad family⁴⁰, SCAN C2H2 zinc fingers⁴¹ and nuclear receptors (NRs)⁴². In those cases, the dimerization (SCAN-C2H2 TFs and NRs) or trimerization (Smads) is mediated by reciprocal interactions between the multimerization domains (mad homology 2 (MH2) for Smads, the SCAN domain for SCAN-C2H2 zinc fingers, and the ligand binding domain (LBD) for NRs). We therefore assumed that the DIM of SOXE promotes dimerization predominantly through DIM:DIM interactions. However, to our surprise, when we mixed DIM containing SOX9-NHMG constructs with isolated SOX8-HMG, SOX9-HMG and SOX10-HMG proteins, we observed distinctive SOX9-NHMG:SOXE-HMG complexes (Fig. 4B). In fact, SOX9-NHMG:SOXE-HMG complexes migrate as more prominent bands than homodimeric SOX9-NHMG:SOX9-NHMG complexes suggesting the preferential formation of SOX9-NHMG:SOXE-HMG heterodimers (Fig. 4B). Similar observations were made when the SOX10-DHMG or SOX8-NHMG constructs were mixed with SOX8-HMG, SOX9-HMG or SOX10-HMG constructs, respectively (Fig. 4C,D). Therefore, a single DIM domain is sufficient to support cooperative dimer formation between SOXE TFs suggesting that DIM:HMG rather than DIM:DIM interactions facilitate cooperative DNA recognition.

The HMG box mediates selective dimerization of SOXE factors

The finding that only one DIM domain is necessary to mediate effective DNA dependent dimerization with HMG boxes raises the intriguing question as to whether SOXE proteins cooperate with other members of the SOX family. To assess whether such interactions are in principle possible, we first asked whether SOXE factors are co-expressed with non-SOXE TFs. To this end we inspected the expression of all 20 SOX proteins in 315 primary human tissues as reported by the FANTOM5 consortium¹⁴. We found that SOX9 is broadly expressed in the majority of the 315 cell types while the expression domains of SOX8 and SOX10 are slightly more restricted (Fig. 5A). As most other SOX factors (exceptions being SOX14 and SOX30) show an equally widespread tissue distribution, SOXE factors are co-expressed with most non-SOXE in some cell types giving rise to a plethora of theoretically possible heterodimer combinations (Fig. 5A). When the expression data are clustered in a correlation heatmap (Fig. 5B) or represented as a network based on expression correlation (Fig. 5C), we found that the three SOXE TFs frequently co-occur but they also cluster with the SOXB group proteins SOX1, SOX2 and SOX21. Given the prevalent co-expression of SOXE and non-SOXE TFs, we decided to test whether SOXE factors can heterodimerize with isolated HMG boxes of SOX2 (SOXB), SOX4 (SOXC), SOX6 (SOXD) and SOX18 (SOXF) representing the major subgroups of the SOX family. However, SOX9-NHMG (Fig. 5D) and SOX10-DHMG (Fig. 5E) homodimers predominate on all EMSAs containing SOXE/non-SOXE-HMG mixtures and SOXE/non-SOXE-HMG heterodimers are barely detectable. Rather, the HMG boxes of SOX2, SOX6, SOX4 and SOX18 form prominent monomeric complex or weakly homodimerize. Collectively, these results show that SOXE factors evolved a unique protein module, the DIM domain, accompanied by co-evolving HMG box features to promote selective dimerization.

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Figure 5

The SOXE-HMG encodes selectivity determinants (A) Expression data for human SOX proteins reported by the FANTOM5 consortium¹⁴ was hierarchically clustered in glbase⁶⁰ showing the widespread co-expression of SOX proteins in many cell types. Expression data were transformed into a correlation heatmap (B) or into a network using r2 correlation coefficients based on the co-expression similarity of SOX genes (C) further illustrating the frequent co-occurrence of SOXE factors (in particular SOX8 and SOX10) but also highlights the correlation of SOXE factors with SOXB family members SOX1, SOX2 and SOX21. Network nodes are SOX TFs and edges are drawn between nodes if the r2 correlation across the FANTOM5 expression dataset is >0.4 (bold lines) or >0.1 (dotted lines). (D,E) The SOX9-NHMG (D) or the SOX10-DHMG (E) was mixed with the HMG boxes of SOX2, SOX4, SOX6 and SOX18 to assess whether SOXE proteins have the capacity to interact with non-SOXE factors. SOX9-NHMG/non-SOXE-HMG heterodimers are barely visible and the SOX9-NHMG or SOX10-DHMG homodimers and non-SOXE-HMG monomers pre-dominate. Non-SOXE-HMG boxes are indicated using black filled circles and the SOX9-NHMG as white circles and the SOX10-DHMG as gray circles in the cartoons marking the microstates to the right of the gel images. A 109aa HMG construct was used for SOX2 and 79aa HMG constructs for SOX4, SOX6 and SOX18 explaining the different mobility.

Protein production

The 79aa HMG boxes SOX4, SOX6, SOX8, SOX9, SOX10 (with N-terminal His6-tag), SOX18 and the 109aa SOX2 HMG box (without N-terminal His6-tag) were prepared as described^47,59. Constructs containing the DIM domain termed SOX9-DHMG and SOX10-DHMG and constructs starting at the N-terminal ATG and ending after the HMG box termed SOX8-NHMG and SOX9-NHMG were cloned into pETG60A or pDEST-hisMBP expression plasmids using the Gateway BP and LR cloning system (Invitrogen) using primers listed in Table 1. Proteins were expressed in BL21(DE3) or BL21(DE3)pLys and grown in Terrific Broth (TB) in the presence of 1mM IPTG at 18°C for 18h. Cells were pelleted and resuspended in lysis buffer (20mM Tris-HCl pH 8.0; 300mM NaCl; 20mM Imidazole) and lysed by high-pressure disruption (Guangzhou JuNeng Biology & Technology , JN3000 PLUS) at 4°C. Fusing protein were extracted from cellular lysates using Ni-NTA Agarose (Qiagen), eluted with elution buffer (20mM Tris-HCl pH 8.0; 100mM NaCl; 300mM Imidazole) and then cleaved by adding 1/30 (weight per weight) tobacco etch virus (TEV) protease at 4°C for 24h. SOXE constructs were then purified using a 6mL Resource S column (GE Healthcare) connected to an AKTAxpress system (GE Healthcare) with a NaCl gradient from 100mM to 1M. Proteins were desalted using PD-10 columns (GE healthcare) and a buffer containing 20mM Tris-HCl pH 8.0; 100mM NaCl. The concentration was determined by measuring the absorbance at 280nm with a Nanodrop 2000 spectrophotometer and proteins were aliquoted and stored at −80°C.

Electrophoretic mobility shift assay

DNA oligos were procured from Life Technologies and dsDNA probes were generated by mixing cy5-labelled forward and unlabeled reverse strands in 1X annealing buffer (20mM Tris–HCl, pH 8.0; 50mM MgCl2; 50mM KCl) and heating to 95°C for 5min and subsequent cooling to 4°C at with 1°C /min in a PCR block. Each EMSA reaction was carried out using a 1X EMSA buffer (10 mM Tris–HCl pH 8.0, 0.1mg/ml bovine serum albumin, 50μM ZnCl2, 100mM KCl, 10% (v/v) glycerol, 0.1% (v/v) Igepal CA630 and 2mM beta-mercaptoethanol), 50nM dsDNA and varying protein concentrations for 4h at 4°C in the dark. For heterodimer assays the 4bp spacer probe was selected. After incubation, samples were loaded onto 12% 1X Tris-glycine (25mM Tris-HCl pH 8.0, 192mM glycine) native PAGE gels and run at 200V for 35–40min in 1X TG buffer in the cold room. Bands were visualized using a Typhoon FLA-7000 PhosphorImager (FUJIFILM) and quantified using the Image Quant software (GE Healthcare). Cooperativity factors were calculated as described previously³⁶.

R.J. is supported by a 2013 MOST China-EU Science and Technology Cooperation Program, Grant No. 2013DFE33080, by the National Natural Science Foundation of China (Grant No. 31471238) and a 100 talent award of the Chinese Academy of Sciences. A.J. is supported by the National Science Centre (Poland), grant no. 2011/03/N/NZ2/03177. K.S.E.C. is supported by the University Grants Council (grant AoE/M-04/04) and Research Grants Council of HK (T12-708/12-N). S.P. was supported by funds from the Agency for Science Technology and Research (A*STAR Singapore). We thank Calista Keow Leng Ng for providing HMG domain proteins and Saravanan Vivekanandan and Prasanna Kolatkar for providing pDESTHisMBP-SOX9-NHMG expression plasmid. We are grateful to Andrew Hutchins for comments on the manuscript.