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The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism

The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Published online 13 June 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6917–6931 doi: 10.1093/nar/gkz523 The SOXE transcription factors––SOX8, SOX9 and SOX10––share a bi-partite transactivation mechanism Abdul Haseeb and Ver ´ onique Lefebvre Department of Surgery/Division of Orthopaedic Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA Received February 22, 2019; Revised May 23, 2019; Editorial Decision May 31, 2019; Accepted June 03, 2019 ABSTRACT evolved to exert master roles in cell fate determination and differentiation in progenitor and stem cells as well as dif- SOX8, SOX9 and SOX10 compose the SOXE tran- ferentiated cells (1–3). SOX proteins are defined as har- scription factor group. They govern cell fate and boring a high-mobility-group (HMG)-type domain that is differentiation in many lineages, and mutations im- at least 50% identical to that of the family founder, the pairing their activity cause severe diseases, includ- sex-determining region on the Y chromosome (SRY). This ing campomelic dysplasia (SOX9), sex determination domain binds and bends DNA at sequences matching or disorders (SOX8 and SOX9) and Waardenburg-Shah resembling the C[A/T]TTG[T/A][T/A] motif. It also fea- tures nuclear import and export signals and interacts with syndrome (SOX10). However, incomplete knowledge various proteins. Based on sequence identity, SOX pro- of their modes of action limits disease understand- teins are distributed into eight groups, A to H (4). Mem- ing. We here uncover that the proteins share a bipar- bers of the same group share close to 100% identity in the tite transactivation mechanism, whereby a transac- HMG domain and also share a high degree of identity in tivation domain in the middle of the proteins (TAM) other functional domains, including dimerization, transac- synergizes with a C-terminal one (TAC). TAM com- tivation and transrepression motifs, whereas proteins be- prises amphipathic  -helices predicted to form a longing to distinct groups share no or minimal identity protein-binding pocket and overlapping with minimal outside the HMG domain (1). Whereas the HMG domain transactivation motifs (9-aa-TAD) described in many has been characterized in great detail, current knowledge transcription factors. One 9-aa-TAD sequence in- of the structure/function properties of the other cardinal attributes of SOX proteins remains generally meager. We cludes an evolutionarily conserved and functionally here set out to increase knowledge of the transactivation do- required E[D/E]QY motif. SOXF proteins (SOX7, mains of the SOXE proteins. SOX17 and SOX18) contain an identical motif, sug- Humans and most vertebrates possess three SOXE pro- gesting evolution from a common ancestor already teins: SOX8, SOX9 and SOX10. Their genes overlap in ex- harboring this motif, whereas TAC and other trans- pression and are either uniquely, additively, or redundantly activating SOX proteins feature only remotely re- needed in such key processes as chondrogenesis (SOX9), lated motifs. Missense variants in this SOXE/SOXF- sex determination and differentiation (SOX8 and SOX9), specific motif are rare in control individuals, but melanogenesis (SOX9 and SOX10)(5), neural crest devel- have been detected in cancers, supporting its impor- opment (SOX8, SOX9 and SOX10), and neuronal and glial tance in development and physiology. By deepening differentiation (SOX8, SOX9 and SOX10)(6–8). In hu- understanding of mechanisms underlying the cen- mans, SOX8 mutations cause a spectrum of female and tral transactivation function of SOXE proteins, these male reproductive anomalies (9), while SOX9 mutations cause Campomelic Dysplasia, a severe skeletal malforma- findings should help further decipher molecular net- tion syndrome, as well as XY sex reversal (10–12), and works essential for development and health and dys- SOX10 mutations cause Waardenburg-Shah syndrome (13). regulated in diseases. Furthermore, SOX9 and SOX10 overexpression are poor or favorable prognosis markers in many cancers, such as INTRODUCTION glioma, melanoma and breast, colorectal, pancreas and prostate cancer (5,14). These findings point to critical roles The diversification and sophistication of cell types that has for SOXE proteins in various developmental, physiological occurred during evolution has been possible thanks to the and pathological processes. Reaching deep understanding multiplication and specialization of many types of genes of the structural organization and modes of actions of these and regulatory factors. In particular, the SOX family has To whom correspondence should be addressed. Tel: +1 215 590 0146; Email: lefebvrev1@email.chop.edu C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6918 Nucleic Acids Research, 2019, Vol. 47, No. 13 proteins is thus fundamental to uncover how they function These sequences were amplified by PCR using PfuUl- normally and can be dysregulated in diseases. tra High-Fidelity DNA Polymerase (Agilent Technologies, Transactivation is a focal activity of SOXE proteins. For Santa Clara, CA, USA) and human cDNA using forward instance, SOX8 and SOX9 transactivate Sertoli cell-specific and reverse primers containing BamHI and EcoRI sites, genes (15); SOX9 also transactivates chondrocyte-specific respectively (Supplementary Table S2). Plasmids encod- DBD genes (16,17); and SOX10 transactivates oligodendrocyte- ing GAL4 /SOXE fusion proteins were generated by and melanocyte-specific genes ( 18,19). A current conun- cloning SOXE cDNA segments into the pBIND plasmid drum is that compelling evidence of redundant and additive (Promega, Madison, WI, USA). These segments were gen- activities in multiple processes contrasts with data suggest- erated by PCR using custom-made primers (Supplementary ing that SOXE proteins utilize different transactivation do- Table S3). Missense mutations were introduced in SOX se- mains. SOX8 was indeed proposed to transactivate through quences by QuikChange Site-Directed Mutagenesis (Strata- a centrally located sequence (20,21), which we will refer to gene, San Diego, CA, USA) using tailored primers (Supple- as TAM (transactivation domain in the middle of the pro- mentary Table S4). The integrity of all plasmid inserts was tein). In contrast, SOX9 has a key transactivation domain verified by Sanger sequencing. at its C-terminus (22), which we will refer to as TAC, and might enhance its transactivation activity through a PQA- Reporter assays rich domain, which does not exist in SOX8 and SOX10 (23). SOX10, like SOX9, possesses a potent TAC domain (24,25), HEK-293 (CRL-1573; ATCC, Manassas, VA, USA) and and also possesses a so-called K2 domain, matching SOX8 SW-1353 (HTB-94; ATCC) cells were cultured in mono- TAM and contributing to transactivation in an apparently layer in 2 ml DMEM supplemented with 10% FBS (Life cell type-specific manner ( 26). These data raise questions Technologies, Carlsbad, CA, USA). Cells (0.3 million) were on whether SOX8 possesses a functional TAC and SOX9 a plated in each well of six-well plates and transfected 4– functional TAM, and whether the SOX9 PQA and SOX10 6 h later with a mixture made of 100 l DMEM, 3 l TAM have autonomous transactivation activity or only po- FuGENE6 (Promega) and 1 g plasmids. The latter in- tentiate the activity of TAC. cluded 500 ng of reporter plasmid (Col2a1 [5x48]-p89Luc We show here that TAM and TAC are autonomous and (36), Acan [4xA1]-p89Luc (37), pG5Luc (Promega), 6FXO- synergistic transactivation domains in each SOXE protein p89Luc (38) or TOP-Flash (39)), 100 ng of pSVGal and that PQA may help mediate SOX9 transactivation in plasmid (reporter used to measure transfection efficiency) specific contexts. Focusing on TAM, we identify a unique (40), and 400 ng of expression plasmids (various combina- E[D/E]QY sequence that is required for transactivation, tions of empty pCDNA 3.1, pCDNA 3.1-SOXE, pCDNA is remarkably conserved in SOXE and SOXF proteins, and 3.1-SOX17, pCDNA 3.1-SOX5 and pCDNA 3.1-SOX6, DBD is predicted to participate in a binding pocket that likely in- pBind-GAL4 /SOXE, or constitutively stabilized - teracts with transcriptional co-activators or basal transcrip- catenin/CS2 plasmid (37,41)). Cell extracts were prepared tional machinery components. in Tropix lysis buffer (0.2% Triton X-100, 100 mM potas- sium phosphate, pH 7.8, 1 mM DTT) 20–24 h after the start of transfection and assayed for luciferase and - MATERIALS AND METHODS galactosidase activities using the Dual-Light Luciferase & SOX protein sequence analyses -Galactosidase Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA) and a GloMax Explorer SOX protein sequences were downloaded from NCBI (Sup- Multimode Microplate Reader (Promega). Reporter activi- plementary Table S1) and aligned with the ClustalW tool ties were normalized for transfection efficiency by calculat- embedded in MacVector16 software (MacVector, Apex, ing the ratios of luciferase versus -galactosidase activities. NC, USA). Hydropathy plots were generated using the Kyte-Doolittle scale (27). The presence of 9-aa-TAD mo- tifs was determined using the Piskacek tool (28). Secondary Western blot and tertiary structures were predicted for the SOX9 TAM- DBD The levels of SOX and GAL4 /SOX proteins produced CD region using SWISS-MODEL (29), I-TASSER (30) from expression plasmids were determined by subjecting and PEP-FOLD3 (31). The best scoring models were ex- cell extracts to 10% SDS-PAGE and transferring proteins ported in PDB format and processed using UCSF Chimera to PVDF membranes using iBLOT 2 Gel Transfer De- v1.11.2 (32) to generate high-quality images. Synonymous vice (Thermo Fisher Scientific). Membranes were blocked and missense variants in SOXE and SOXF sequences in in Tris-Buffered Saline with 0.1% (v/v) Tween 20 (TBST) control human individuals were downloaded from the gno- and5%(w/v) nonfat dry milk for 1 h and then incu- mAD database (33) and somatic missense variants detected bated overnight at 4 C in blocking solution containing in cancers from the COSMIC database (34). anti-FLAG M2-peroxidase-conjugated antibody (A8592, Sigma-Aldrich, St. Louis, MO, USA) at a 1:12000 dilution Generation of wild-type and mutant SOX protein expression or peroxidase-conjugated GAL4 antibody (sc-510, Santa plasmids Cruz Biotechnology, Dallas, TX, USA) at a 1:500 dilu- Human SOXE and SOX17 expression plasmids were gener- tion. Peroxidase-generated signals were detected using ECL ated by cloning full-length coding sequences in frame with Prime Western Blotting Detection Reagent (GE Healthcare, an N-terminal 3FLAG epitope (35) in the pcDNA3.1(+) Chicago, IL, USA) or SuperSignal West Pico Chemilumi- vector (Thermo Fisher Scientific, Waltham, MA, USA). nescent Substrate (Thermo Fisher Scientific) on a Chemi- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6919 Doc Imaging System (Bio-Rad Laboratories, Hercules, CA, data suggested that PQA is not a transactivation domain USA). and that it interferes with TAC activity. In contrast, Mc- Dowall and colleagues reported that deletion of PQA weak- ened the ability of SOX9 to transactivate a reporter contain- RNA isolation and qRT-PCR assay ing tandemly repeated SOX binding sites, thus suggesting Lipofectamine 3000 (Thermo Fisher Scientific) was used to that PQA could be a weak transactivation domain potenti- transfect mouse chondrogenic ATDC5 cells (42) with vari- ating the activity of TAC (23,43). In view of these results, ous combinations of expression plasmids for mouse SOX5, we attempted to clarify the role of PQA. mouse SOX6, and human full-length or mutant SOX9. To- Comparing SOX9 orthologs, we found that PQA is made tal RNA was prepared 24 h later using TRIzol (Life Tech- of 35–45 residues only consisting of prolines (42%), glu- nologies) and following manufacturer’s instructions. cDNA tamines (39%), and alanines (18%) (Supplementary Fig- was synthesized using the High-Capacity cDNA Reverse ure S2A and B). In contrast, it has only 4–15 residues in Transcription Kit (Thermo Fisher Scientific). qPCR was lower vertebrates, with only a few glutamines in ancient fish. performed using the StepOne Plus Real Time PCR system Only one SOXE protein exists in most invertebrates, and (Thermo Fisher Scientific), SYBR Green PCR Master Mix this protein contains a region poorly enriched in P, Q and (Thermo Fisher Scientific) and custom-designed primers A residues (Supplementary Figure S2C). The PQA domain (Integrated DNA Technologies) (Supplementary Table S5). was thus gradually acquired following SOX9 emergence in Col2a1 and Acan mRNA levels were calculated relative to vertebrates from a SOXE ancestor. those of Hprt according to the Ct method. We investigated the function of PQA using two distinct assays. The first one tested whether the domain is sufficient for transactivation, i.e. capable on its own to interact with Statistical analyses transcriptional co-activators or basal transcription machin- Differences between datasets were evaluated using the Stu- DBD ery. We constructed plasmids encoding GAL4 /SOX9 dent’s t-test. Differences that reached P values lower than fusion proteins and transfected them in HEK-293 cells 0.05 were considered significant. along with pG5Luc, a reporter plasmid containing a tan- DBD dem of vfi e GAL4 -binding sites (Figure 2A). A fusion DBD protein containing GAL4 and the SOX9 TAM-to-TAC RESULTS segment appeared to be about three times more active than Sequence conservation suggests key roles for the SOXE TAM the same protein lacking PQA, but this difference was due and TAC regions in part to differences in protein amount (Figure 2B). A pro- DBD tein made of GAL4 and only PQA was inactive, advo- Alignment of the human SOXE protein sequences showed, cating that PQA lacks autonomous transactivation capa- as expected, that the HMG domain is the most con- bility. Our second assay tested whether PQA is necessary served region among the three proteins, with 97–99% for SOX9 transactivation. We used expression plasmids en- identity/similarity (Figure 1A–C). The homodimerization coding the full-length human SOX9 protein fused to an domain (DIM) comes next, with 81–95% identity/similarity N-terminal 3FLAG epitope and another plasmid encod- among the three proteins. TAM, that is, the reported trans- ing SOX9 without PQA, and we tested them with Col2a1 activation domain of SOX8 and K2 domain of SOX10, is [5x48]-p89Luc, a reporter featuring vfi e tandem copies of third, with 70–84% similarity between the proteins. TAC, a 48-bp Col2a1 enhancer (Figure 2C). This enhancer is a that is, the C-terminal region that includes the main trans- bona fide SOX9 target: it contains a SOX9 consensus bind- activation domain reported for SOX9 and SOX10, is fourth, ing site, i.e. a pair of inverted SOX-like recognition sites sep- with only 45–73% similarity among the proteins. Other re- arated by four nucleotides, and it is directly bound by SOX9 gions are only 7–29% identical/similar. Sequence compar- PQA in chondrocytes in vivo (16,17,44). SOX9 and SOX9 isons for various vertebrate species revealed that SOX8 or- were produced at even levels in HEK-293 cells and trans- thologs are under tighter evolutionary constraint to con- activated the reporter equally potently (Figure 2D). Simi- serve TAM than TAC, whereas SOX9 and SOX10 orthologs lar results were obtained in SW-1353 chondrosarcoma cells are under similar constraints for TAM and TAC (Supple- with Acan [4xA1]-p89Luc, a reporter containing a 359- mentary Figure S1A and B). Overall, SOXE protein or- bp Acan enhancer (Supplementary Figure S2D). This en- thologs are conserved at 34% in TAM, but only 17% in TAC. hancer features SOX9 and SOX5/6 consensus binding sites Together, these data suggest that both TAM and TAC may and is directly bound by the SOX trio in chondrocytes in have key functions in all three SOXE proteins. vivo (16,17,37) In conclusion, these data suggested that PQA has no ma- PQA may contribute to SOX9 stability and transactivation jor role in SOX9 transactivation, but did not rule out that in specific contexts SOX9 acquired this unique domain during evolution to en- In the first investigation of SOX9 domains involved in hance its stability or activity in specific contexts. transactivation, Sudbeck ¨ and colleagues showed that a fu- sion protein made with the GAL4 DNA-binding domain TAM and TAC are synergistic transactivation domains in all DBD (GAL4 ) and a SOX9 segment containing only PQA and three SOXE proteins flanking sequences was unable to activate transcription, and that a fusion protein containing only TAC was 4 times as ac- To determine whether and how TAM and TAC contribute tive as a protein featuring both PQA and TAC (22). These to SOXE transactivation, we first constructed plasmids en- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6920 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 1. Sequence conservation among the human SOXE proteins. (A) Schematic showing the domain organization of the three human SOXE proteins. Conserved domains are shown with boxes and the amino acids at the boundaries of the protein and domains are indicated with numbers. DIM, homod- imerization domain; HMG, DNA-binding domain; TAM, transactivation domain located in the middle of the proteins; PQA, P-, Q- and A-rich domain in SOX9; TAC, carboxy-terminal transactivation domain. (B) ClustalW alignment of the amino acids of the human SOXE proteins. The dimerization (DIM), DNA-binding (HMG), middle transactivation (TAM) and C-terminal transactivation (TAC) domains are boxed. Stars indicate identical residues and dots indicate similar residues. Numbers indicate residue positions within the proteins. (C) Graph showing the degrees of protein conservation. The percentages of sequence identity and similarity were calculated by ClustalW alignment. They are shown for each conserved domain and for the rest of the protein sequences (other). SOX8 is compared to SOX9 (8/9), SOX8 to SOX10 (8/10), SOX9 to SOX10 (9/10) and the three proteins together (E). Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6921 Figure 2. Transactivation capability of the SOX9 PQA domain. (A) Top, schematic of fusion proteins made using the GAL4 DNA-binding domain and SOX9 domains. These proteins are numbered 1 to 4, as in panel D. Bottom, schematic of the pG5Luc reporter used to functionally test the fusion proteins. The reporter has 5 tandem copies of the GAL4 DNA-binding site upstream of a TATA box and the fireyfl luciferase gene. ( B) Left, plot showing the DBD ability of GAL4 /SOX9 fusion proteins to transactivate pG5Luc upon transient transfection in HEK-293 cells. Reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expression plasmid. Right, western blo t showing the levels of the respective proteins present in cell lysates at the end of the experiment. Note that the lower amount of protein in lane 3 compared to lane 2 may explain in part why deletion of PQA reduced the ability of the SOX9 TAM-to-TAC region to activate the pG5Luc reporter. These results were reproduced in multiple experiments. (C) Top, schematic of the SOX9 full-length protein and a mutant protein lacking the PQA domain. Bottom, schematic of the reporter used to functionally test these proteins. The reporter contains vfi e tandem copies of a 48-bp mouse Col2a1 enhancer, which features a SOX9 consensus binding site, and the –89/+6 Col2a1 promoter upstream of the firefly luciferase gene. ( D) Top, plot comparing the ability of SOX9 and PQA SOX9 to transactivate the Col2a1 reporter. HEK-293 cells were transfected with 30 or 100 ng of SOX9 expression plasmids. Reporter activities are presented as described for panel B. Bottom, western blot of cell lysates prepared at the end of the experiment show that deletion of PQA had no obvious effect on SOX9 protein production and stability. DBD Since the relative activities of TAM and TAC greatly dif- coding GAL4 fused to the TAM, TAC or TAM-to-TAC DBD feredinthe GAL4 /SOXE assay depending on their domains of the human proteins (Figure 3A and Supple- TAM SOXE origin, we asked whether swapping SOX9 and mentary Figure S3A) and we transfected them in HEK-293 TAC SOX9 with the corresponding SOX8 and SOX10 do- cells along with pG5Luc (Figure 3B and C). All TAM and mains would affect SOX9 activity. We constructed expres- TAC domains were able to activate transcription, although TAM sion plasmids accordingly and tested them in HEK-293 cells with different performance levels. SOX8 was more po- TAM TAM using the Col2a1 reporter (Supplementary Figure S4A). Al- tent than SOX9 (2.7×) and SOX10 (7.4×). In con- TAC TAC though differential activities were observed that were con- trast, SOX8 was less potent than SOX9 (4.5×)and TAM TAC TAC TAM>TAC sistent with the low activities of SOX10 and SOX8 SOX10 (12×). While SOX8 was less active than TAC DBD TAM TAM>TAC TAM>TAC and high activity of SOX10 in the GAL4 /SOXE as- SOX8 (19.3×), SOX9 and SOX10 say, all chimeric proteins efficiently activated the reporter, were several times more active than their respective TAM indicating that the two domains, regardless of SOXE ori- and TAC domains alone. We next tested the requirement of gin, were able to synergize in the context of the full-length TAM and TAC for transactivation in the natural context of SOX9 protein (Supplementary Figure S4B and C). SOXE proteins. We constructed plasmids encoding the full- length human proteins or proteins lacking TAM or TAC (Figure 3D and Supplementary Figure S3B) and trans- The C-terminal half of TAM (TAM-CD) is a potent transac- fected them in HEK-293 cells (Figure 3E and F). All full- tivation domain length proteins powerfully activated the Col2a1 reporter, Henceforth, we focused on the TAM domain. To reveal but their activities were drastically reduced in the absence which segment of the domain is involved in transactivation, of TAM or TAC (16- to 658-fold). Similar results were ob- DBD we generated plasmids encoding fusions of GAL4 with tained with SW-1353 cells and using the Acan reporter (Sup- halves or quarters of TAM, and plasmids encoding SOX9 plementary Figure S3C–F). In conclusion, the first assay in- proteins lacking most of each TAM quarter (Figure 4A). dicated that TAM and TAC are able to work as indepen- These quarters were named TAM-A, TAM-B, TAM-C and dent transactivation domains, and the second assay indi- TAM-D. In transfection of HEK-293 cells with pG5Luc, cated that the two domains work synergistically in the con- TAM-AB, TAM-C and TAM-D failed to transactivate, text of each SOXE protein. whereas TAM-CD was very potent (Figure 4B). In trans- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6922 Nucleic Acids Research, 2019, Vol. 47, No. 13 DBD Figure 3. Transactivation capabilities of the SOXE TAM and TAC domains. (A) Schematics of fusion proteins containing GAL4 and SOX9 do- DBD mains. See Supplementary Figure S3A for fusion proteins of GAL4 with SOX8 and SOX10 domains. (B) Reporter assay comparing the abilities of DBD GAL4 /SOXE fusion proteins to activate pG5Luc. HEK-293 cells were transfected with pG5Luc and expression plasmids for the fusion proteins shown in panel A. Reporter activities are presented for one experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expressio n plasmid. These results were reproduced multiple times. (C) Western blot of cell lysates prepared at the end of the experiment showing that all protein forms were made DBD TAM>TAC in similar amounts. The blot was made with lysate amounts normalized for transfection efficiency. The lower band seen in the GAL4 /SOX8 lane likely reflects partial degradation of the protein. ( D) Schematics of the SOX9 full-length protein and mutant proteins lacking either TAC or TAM. See Supplementary Figure S3B for equivalent SOX8 and SOX10 schematics. (E) Reporter assay comparing the abilities of the three SOXE proteins to activate the Col2a1 [5x48]-p89Luc reporter in HEK-293 cells, and effects of deleting their TAM or TAC domain. Reporter activities are presented as described in panel B. (F) Western blot of cell lysates prepared at the end of the experiment showing that all protein forms were made. Major differences in reporter activities (panel E) are not due to variations in relative amounts of the proteins and must thus genuinely reflect differences in functional capabilit ies. The blots were made with lysate amounts normalized for transfection efficiency. fection of HEK-293 and SW-1353 cells with the Col2a1 or evidence that both TAM and TAC are critical for SOX9 Acan reporter, TAM-A or TAM-B deletion was inconse- functions. quential, whereas TAM-C or TAM-D deletion virtually ab- rogated SOX9 activity (Figure 4C and Supplementary Fig- ure S5A). Taken together, these data suggested that TAM- TAM-CD exhibits characteristic features of acidic AB is dispensable and that residues within TAM-C and transactivation domains and a unique, highly conserved TAM-D are necessary and sufficient for transactivation. E[D/E]QY motif Since all data were obtained so far using reporter as- Transactivation domains are categorized based on amino says, we next asked whether SOX9 also requires TAM and acid composition (46). For instance, the SOXE TAC is a TAC to activate the endogenous Col2a1 and Acan genes. In non-acidic PQS-rich transactivation domain (22). TAM- transfection of ATDC5 cells, a chondrogenic cell line de- CD examination revealed numerous acidic (Asp and Glu) rived from a mouse teratoma and frequently used to study and other hydrophilic amino acids alternating with hy- chondrocyte differentiation in vitro (45), full-length SOX9 drophobic residues (Ile, Leu, Met, Phe and Val) (Figure successfully cooperated with SOX5 and SOX6 to enhance 5A). This pattern is reminiscent of the minimal nine-amino- Col2a1 and Acan expression (3.7× and 2.5×, respectively) acid-transactivation-domain motif (9-aa-TAD) described (Supplementary Figure S5B). In contrast, SOX9 lacking ei- for acidic transactivation domains in many transcription ther TAC or TAM-D was unable to do so, lending further factors, including GAL4 (yeast), P53, NFAT and NF-kB Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6923 Figure 4. Identification of subdomains of SOX9 TAM mediating transactivation. ( A) From top to bottom, schematic of the SOX9 protein; alignment of DBD the TAM sequences of the three human SOXE proteins; segments of TAM fused to GAL4 ; and TAM-A to TAM-D sequences deleted in the SOX9 DBD protein. (B) Reporter assay comparing the abilities of proteins made by fusing GAL4 with subdomains of SOX9 TAM to transactivate pG5Luc. Reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expre ssion plasmid. The western blot of cell lysates shows that all protein forms were efficiently made in the cells and thus that major differences in reporter act ivities among proteins genuinely reflect intrinsic differences in transactivation capabilities. These results were reproduced in multiple experiments. ( C) Reporter assay comparing the abilities of wild-type SOX9 and SOX9 proteins lacking the whole TAM or TAM segments to transactivate the Col2a1 reporter. Reporter activities are presented as described in panel B. The western blot of cell lysates shows that all protein forms were efficiently made in the cel ls and thus that major differences in reporter activities among proteins genuinely reflect intrinsic differences in transactivation capabilities. These results were reproduced in multiple experiments. proteins (mammals) and VP16 (human herpes virus) (28). ing protein structures directly from amino acid sequences, Accordingly, the Piskacek algorithm identified one such to predict the secondary and tertiary structures of TAM- motif in all SOXE TAM-C regions, one overlapping TAM- CD. The SWISS-MODEL model that reached the high- C and TAM-D in SOX9, and one in SOX9 and SOX10 est quality score (QMEAN, –1.36; sequence identity with TAM-D (Figure 5A). In many cases, 9-aa-TAD sequences the template, 23.53%) was built according to a region of contain a XX core motif (, hydrophobic residue; X, the CdiI Immunity protein from Yersinia kristensenii (PDB any residue) that interacts with basal transcription machin- ID: 4ZQV). The best I-TASSER model (C-score, –1.95; se- ery components, such as hTAF 31 (47). We found a par- quence identity with the template, 19%) was based on a II tially related, but distinct motif in TAM-D in the three hu- glycosylated calcitonin growth factor from Anguilla japon- man SOXE proteins (Figure 5A). This motif responds to ica (PDB ID: 1BYV). Of 10 models proposed by PEP- an E[D/E]QY consensus and is remarkably conserved FOLD3, we retained the best one (sOPEP score: –45.011). not only in all SOX8, SOX9, and SOX10 vertebrate se- All models concurred that TAM-CD could form two - quences (Supplementary Figure S1), but also in the lamprey helices, one using the TAM-C 9-aa-TAD motif and the SOXE3 protein and in the sole SOXE protein existing in in- other one using most of the TAM-D 9-aa-TAD sequence vertebrates (Figure 5B). Since the P53 XX motif was and E[D/E]QY motif (Figure 5A, C and D). These he- shown to transit from a random coil to an -helix upon lices would fold into a protein-binding pocket coated exter- binding to hTAF 31 (47), we used SWISS-MODEL and nally with polar residues and internally with hydrophobic II I-TASSER, which are template-based structure prediction and aromatic residues. software, and PEP-FOLD3, a de novo program predict- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6924 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 5. In silico analysis of transactivation domain features of the SOXE TAM-CD region. (A) Plots of the hydropathy scores of TAM-CD and flanking residues in human SOX8, SOX9 and SOX10. Amino acids are typed in colors according to the nature of their side chains, as indicated underneath the plots. Two predicted -helices are shaded in the plots; 9-aa-TAD motifs are delineated with brown brackets underneath the sequences; and the conserved E[D/E]QY motif is highlighted with a green box. (B) ClustalW sequence alignment showing a high degree of conservation of the E[D/E]QY motif in SOXE proteins from various vertebrate and invertebrate species. (C and D) Binding-pocket structure of the SOX9 TAM-CD domain predicted by SWISS- MODEL, I-TASSER and PEP-FOLD3. Top, ball-and-stick representation showing amino acid cores and side chains. Bottom, cartoon representations. The N- and C-termini of the domain are marked. The -helices are indicated as H1 and H2. The E[D/E]QY motif is highlighted with a green bubble. The color code is otherwise the same as for the sequences in the panel A. Specific residues in the E [D/E]QY motif are critical for cantly change the hydropathy index. These findings further transactivation supported the conclusion that hydrophobic residues pro- jecting inside the binding pocket are critical for transacti- We introduced a series of missense mutations in TAM- vation and that even non-hydrophobic residues composing CD to test the importance of highly conserved residues in the E[D/E]QY motif are critical too. transactivation (Figure 6A). These mutations were selected to significantly alter the hydropathicity, polarity or size of amino acid side chains. Overall, mutations in residues par- The E[D/E]QY motif is conserved in both SOXE and ticipating in the -helix 1 had no drastic effect on SOX9 SOXF proteins activity (Figure 6B). In the GAL4 assay, where transacti- The discovery that the E[D/E]QY motif of TAM-CD vation is only driven by TAM-CD and is thus more sen- is fully evolved in invertebrates prompted us to determine sitive, mutations of residues with hydrophobic side chains whether an identical or similar motif and its associated fea- protruding inside the binding pocket (Leu278 and Val282) tures are also present in the SOXE TAC domain and in were deleterious, whereas mutations in residues with acidic other transactivating SOX proteins. We used three criteria: side chains projecting outwards (Glu277 and Asp281) were (i) sequence conservation between group members; (ii) pres- inconsequential (Figure 6C). ence of 9-aa-TAD domains and (iii) presence of XX or Replacing the first residue of the E [D/E]QY motif E[D/E]QY-like motifs. The SOXE TAC domains con- with the residues present in the XX motif of VP16 tained 9-aa-TAD motifs and a VYXXL sequence resem- or P53 (Glu293Met or Glu293Thr, respectively) impaired bling a XX, but no E[D/E]QY-like sequence (Sup- the activity of full-length SOX9 and one of the mutations plementary Figure S6A). SOXB1 (SOX1, SOX2 and SOX3) also affected TAM-CD activity, explaining that this acidic and SOXC (SOX4, SOX11 and SOX12) proteins featured residue (or Asp) is conserved in all SOXE proteins (Figure 9-aa-TAD, and XX/E[D/E]QY-like sequences in 6D and E). All mutations introduced in other residues of their transactivation domains, but these sequences were the E[D/E]QY motif dramatically reduced SOX9 and very different from those of the SOXE TAM-CD domains TAM-CD activities, except F294L, which resulted in an aro- (Supplementary Figure S6B). Interestingly, SOXF proteins matic to aliphatic side chain change, but did not signifi- (SOX7, SOX17 and SOX18) featured a 9-aa-TAD sequence Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6925 Figure 6. Test of the effects of amino acid substitutions in TAM-CD on transactivation. (A) Schematic showing the SOX9 TAM-CD residues substi- DBD TAM-CD tuted in GAL4 /SOX9 and SOX9 expression plasmids. (B–E) Reporter assays comparing the abilities of wild-type and variant SOX9 and DBD TAM-CD GAL4 /SOX9 proteins to transactivate their respective Col2a1 and pG5Luc target reporters upon transfection in HEK-293 cells. Normalized reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures and in percentage of the activities obtained with wild-type proteins. Western blots of cell lysates prove that major differences in reporter activities are not due to differences in relative amounts of the various proteins. These results were reproduced in multiple experiments. with an E[D/E]QYL motif fully matching the SOXE stitutively stabilized -catenin from activating TOP-Flash, E[D/E]QY consensus in their transactivation domains a reporter gene classically used as a readout of canoni- (Figure 7A). This motif is also remarkably conserved from cal WNT signaling (51). SOX9 was also shown to inhibit invertebrates to humans (Figure 7B). This finding pairs with -catenin transcriptional activity, but to use its TAC do- the fact that the HMG domains of the SOXE and SOXF main to bind to -catenin (52). We therefore decided to di- proteins are more closely related to one another than to rectly compare the contributions of the SOX9 and SOX17 those of other SOX proteins (48). Altogether, the data sug- E[D/E]QYL motifs to the protein activities. As expected, gest that the SOXE and SOXF groups emerged from a com- deletion of the motif significantly reduced the abilities of mon ancestor that was already featuring an E[D/E]QY SOX9 and SOX17 to activate reporter genes (Supplemen- motif. We looked for the presence of an E[D/E]QY mo- tary Figure S7A and B). When tested with TOP-Flash, tif in all other SOX proteins, but did not find any (data not wild-type SOX9 and SOX17 inhibited the activity of con- shown). stitutively stabilized -catenin in a dose-dependent manner The E[D/E]QYL motif was previously recognized in (Supplementary Figure S7C and D). Deletion of the whole SOXF proteins, and SOX17 was shown to require it to ac- TAM or only its EFDQYL motif slightly reversed SOX9 tivate endodermal genes on its own and in synergy with - inhibition of -catenin, whereas deletion of TAC totally re- catenin (49) as well as to reprogram somatic cells into in- versed this inhibition, and whereas deletion of the EFE- duced pluripotent stem cells (50). Further, this domain was QYL motif from SOX17 effectively reversed the inhibition. shown to help SOX17 bind to -catenin and prevent con- These data suggest that the E[D/E]QY motif might con- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6926 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 7. Identification of an E [D/E]QY motif in SOXF proteins. (A) Schematics showing the locations of the HMG and transactivation regions (TA, pale green boxes) of SOXF proteins and alignment of TA regions that show a high degree of conservation among group members, a 9-aa-TAD motif (brown bracket) and an E[D/E]QYL motif. The TA regions were previously delineated for SOX7 (63), SOX17 (64) and SOX18 (59,65). (B) ClustalW sequence alignment showing the high degree of conservation of the E[D/E]QYL motif in SOXF proteins from various vertebrate and invertebrate species. tribute to inhibition of canonical WNT signaling by both cancer and participates in tumorigenesis. The occurrence of SOXE and SOXF proteins, but that TAC has a dominant missense variants was lower in the TAM than in the HMG role in SOXE proteins and that SOXF proteins might fea- domain in COSMIC samples, but the difference did not ture specific sequences around EFEQYL that potentiate its reach statistical significance. We therefore closely examined inhibitory activity. The latter proposition is supported by the types of missense variants present in the SOXE and evidence that deletion of its entire C-terminus prevented SOXF E[D/E]QY motifs in gnomAD and COSMIC SOX17 from binding to -catenin, whereas the sole dele- samples. tion of the EFEQYL motif (located in the C-terminus) only Interestingly, gnomAD missense variants affected many had a partial effect (49). residues around the E[D/E]QY motifs of SOXE and SOXF proteins, but none occurred within the SOX9, SOX10 and SOX18 E[D/E]QY motifs and only a few occurred The SOXE/SOXF E[D/E]QY motif is highly conserved within the SOX8, SOX7 and SOX17 E[D/E]QY mo- in the human population tifs (Figure 8A and B). The SOX8 variants detected in The outstanding degree of conservation of the this motif represented conservative changes (D286E and SOXE/SOXF E[D/E]QY motif suggests that mu- Q287R) in the residues that occupy the X positions in tations in this motif would be incompatible with healthy the related XX sequence of other transcription fac- development and adult life. To test this hypothesis, we tors. The D286E change is unlikely to be consequential searched for literature reports of missense mutations or since both D and E are highly acidic and occupy the third other in-frame micro-alterations in this domain in SOXE position of the SOXE/SOXF E[D/E]QY motif. The and SOXF genes that were linked to a human disease, but other variant, Q287R, might be consequential since Q is did not find any. We then searched GnomAD, a database highly conserved in SOXE and SOXF proteins and since of genomic sequences from >140 000 unrelated control it is uncharged whereas R is positively charged. To test individuals, and COSMIC, a catalog of somatic mutations whether this variant could affect protein activity, we intro- in cancer. Detailed analysis of SOX9 revealed that synony- duced an equivalent mutation (Q296R) in the SOX9 and DBD TAM-CD mous and missense variants affected similar proportions GAL4 /SOX9 proteins. We observed that both of residues throughout all domains of the protein, except proteins still retained significant activity in their respective the HMG domain, where significantly fewer missense assays (Figure 8C), suggesting that the SOX8 Q287R vari- variants were detected in gnomAD individuals compared ant is not detrimental enough to cause a disease. to synonymous variants in the same cohort and compared Unlike the gnomAD database, the COSMIC database to missense variants in cancers (Supplementary Figure did contain missense variants in the SOX9 EFDQYL mo- S8A and B). This finding suggests that a particularly tight tif (Supplementary Figure S8C). We tested them along with sequence conservation constraint exists for this domain two variants in the -helix 1. The latter almost doubled the in healthy individuals and that this constraint is lifted in activity of SOX9, whereas the two variants affecting the E Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6927 Figure 8. Analysis of SOXE and SOXF missense variants in the human control population and model for the SOXE bipartite transactivation mechanism. (A) Missense variants listed in gnomAD in SOXE TAM-CD are presented underneath the domain sequences. The numbers of alleles detected in over 140 000 unrelated individuals are indicated for each variant. (B) Missense variants listed in gnomAD in the SOXF E[D/E]QYL motif and flanking residues. (C) Test of the effect of a Q296R variant detected in SOX8 in healthy human individuals on transactivation. The variant was introduced in the DBD TAM-CD GAL /SOX9 and SOX9 proteins. The proteins were then tested in HEK-293 cells upon co-transfection with the pG5Luc or Col2a1 reporter. Other missense variants (as described in Figure 6) were tested in parallel for comparison. Data were calculated and are presented as in similar assays in previous figures. They were reproduced in multiple experiments. ( D) Model of the current view for the mechanism used by SOXE proteins to activate target genes. Previous studies demonstrated that SOXE proteins homodimerize upon binding to inverted recognition sites present in the enhancers or promoter of their target genes. These events involve their HMG and dimerization domains. The present study uncovered that they transactivate these genes through a bi-partite mechanism involving a centrally located domain (TAM) synergizing with the protein C-terminal region (TAC). The functional core of TAM is predicted to fold into binding pocket-like structure upon interaction with specific transcriptional co-activators or components of the basal tra nscription machinery. It contains a highly conserved E[D/Q]Y motif that is critical for transactivation and is thus likely involved in recognizing and firmly binding functional partners. Not shown here is a PQA-rich domain present solely in SOX9 that may facilitate transactivation in specific contexts. residue of the EFDQYL motif greatly decreased SOX9 ac- this motif could underlie congenital or acquired human dis- tivity and the variant affecting the D residue of the motif eases. modestly decreased SOX9 activity (Supplementary Figure S8D). Beside confirming the importance of EFDQYL for DISCUSSION SOX9 activity, these data add support to the notion that This study has brought unity and new information on the the SOXE/SOXF E[D/E]QY motif is critical for the mechanisms whereby the SOXE proteins achieve transacti- function of SOXE proteins and suggest that mutations in vation. It has provided evidence that each protein carries Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6928 Nucleic Acids Research, 2019, Vol. 47, No. 13 two synergistic transactivation domains, TAM and TAC domain, and was mentioned to interact also with SOX8 (Figure 8D). By deploying a bipartite transactivation mech- TAM and SOX10 TAC (55,56). P53 and other proteins con- anism, SOXE proteins may engage exclusive sets of tran- tact CBP/p300 through 9-aa-TAD domains (57), but other scriptional partners to effectively activate target genes. The transcriptional partners have also been identified for 9-aa- C-terminal half of TAM is predicted to form a protein- TAD and XX domains, such hTAF 31 (47). All this II binding pocket. Its E[D/E]QY core motif is functionally suggests that SOXE proteins may use their two transacti- crucial and is highly conserved evolutionarily and in human vation domains to contact various partners and to do so healthy individuals not only in SOXE proteins but also in cooperatively or independently of one another. SOXF proteins. Dissection of functional segments revealed that the C- The unique PQA domain of SOX9 gained in length and terminal half of TAM (TAM-CD) is both needed and suffi- P-, Q- and A-enrichment upon mammalian evolution, but cient for transactivation. TAM-CD has characteristic fea- its role remains unclear. Glutamine-rich sequences exist in tures of acidic transactivation domains: enrichment for many types of proteins, especially in transactivation do- acidic residues that may promote an unstructured confor- mains, and there is evidence that they help stabilize pro- mation prone to encounter transcriptional partners, and en- teins and strengthen protein-protein interactions (53). The richment for aromatic and bulky hydrophobic residues that SOX9 PQA domain was not found in our study nor in could favor the formation of a specific structure upon inter- a previous study by Sudbeck ¨ et al. to have autonomous action with these partners (58). Protein modeling tools sup- transactivation function (22). McDowall et al. reported that ported this view by proposing that TAM-CD would indeed PQA enhanced the ability of SOX9 to transactivate an fold into a binding pocket. Functional assays added fur- artificial pS10E1bCat reporter, which likely bound SOX9 ther support to this view by showing that non-conservative monomers (23). We failed to replicate this finding using substitutions were most deleterious when occurring in hy- bona fide Col2a1 and Acan reporters that bind SOX9 drophobic residues predicted to line the inner surface of the DBD TAM>TAC homodimers, but observed that GAL4 /SOX9 binding pocket. was less abundant and less active in transactivating pG5Luc We found an E[D/E]QY sequence to be the most con- when it lacked PQA. This reporter is also likely to bind served segment of TAM-CD among all SOXE proteins from DBD GAL4 /SOX9 monomers. One can thus envision that invertebrates to humans and also to be the most critical PQA enhances SOX9 stability and transactivation efficiency segment for transactivation. Its conservation in SOXF pro- when the protein binds target genes as a monomer, as in Ser- teins unsettles a longstanding concept that proteins from toli cells, but may not be necessary when the protein binds different SOX groups share no significant identity out- targets as a homodimer, as in other cell types. side the HMG domain and strongly suggests that the two Different transactivation mechanisms were previously SOX groups recently evolved from a common ancestor. suggested for the three SOXE proteins, mostly because most It is intriguing, however, that the two groups diverged in DBD studies only used the GAL4 assay to map transactiva- their expression patterns and gene targetome to control dif- tion domains and did not test the consequences of dele- ferent cell types, but nevertheless conserved a core motif tions or point mutations in the protein sequences. The use for transactivation. Missense mutations introduced in the of both assays led us to find two functional domains in each E[D/E]QY sequence of SOX18 were previously shown protein. This finding was not unexpected considering the to impair transactivation, showing that this motif is critical high degree of sequence conservation and redundancy of in SOX18 too (59). Previous studies also showed that this se- SOXE proteins in many processes. What was unexpected quence facilitates interaction of SOX17 with -catenin and was to find that the two domains synergize with one an- may thereby allow SOX17 to cooperate with -catenin in other. Schreiner et al. indeed showed that deletion of SOX10 the activation of endodermal genes in Xenopus embryos TAM in the mouse led to a milder disease than total inac- (49) and to reprogram fibroblasts into pluripotent stem cells tivation of Sox10 (26). Melanogenesis and enteric nervous with higher efficiency than SOX2 ( 50). These data, how- system development were significantly impaired, but neural ever, contrast with evidence that SOX17 can also repress crest and oligodendrocyte development were only slightly -catenin/TCF activity and interact with -catenin to pro- affected. This led the authors to suggest that SOX10 TAM mote its degradation (60). Similarly, SOX9 and -catenin contributes to SOX10 activity in a cell type-specific con- were shown to physically interact with one another and text rather than being mandatory in all processes to syner- to have reciprocal antagonistic activities, including induc- gize with TAC. It remains possible, however, that TAM con- tion of mutual degradation (52). TAC was required for in- tributes to SOX10 activity in all cell types, but that its dele- teraction of SOX9 with -catenin. In direct comparisons, tion is pathogenic only in cells critically dependent on its we found that the EF[D/E]QYL motif is more critical for dosage. Supporting this model is the fact that humans with transactivation by SOX9 than by SOX17, but is more po- heterozygous mutations inactivating one SOX10 allele have tent in SOX17 than SOX9 to inhibit -catenin. Altogether, deficiencies primarily in melanogenesis and enteric nervous conservation, structural and functional data point to the system development. Synergy implies that TAC and TAM E[D/E]QY motif as being a pivotal sequence that com- domains may cooperatively contact the same protein or bines with other sequences, likely distinct in SOXE and may contact different components of transcriptional com- SOXF proteins, to engage in protein interactions with ei- plexes. CBP/p300, a major transcriptional co-activator, was ther transcriptional or non-transcriptional consequences. shown to interact with SOX9 via TAC, but apparently not SOXE and SOXF mutations cause severe developmen- through TAM (54). TRAP230, a member of the transcrip- tal diseases, namely Campomelic Dysplasia and XY sex re- tional mediator, was shown to interact with the SOX9 TAC versal for SOX9, Waardenburg-Shah syndrome for SOX10, Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6929 and hypotrichosis-lymphedema-telangiectasia-renal defect FUNDING syndrome for SOX18 (61). The mutations vary from en- National Institute of Arthritis and Musculoskeletal and tire gene deletions to point mutations. Among the latter, Skin Diseases (NIAMS) [AR046249, AR072649 to V.L.]. nonsense mutations within the SOXE TAC domain have Funding for open access charge: NIAMS [AR072649 to demonstrated the critical importance of this domain. Mis- V.L.]. sense mutations are almost always located in the HMG do- Conflict of interest statement. None declared. main or SOXE dimerization domain. 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Tsuchiya,T., Suzuki,E., Miyado,M., Hata,K., Nakabayashi,K. et al. (2015) Testicular dysgenesis/regression without campomelic http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism

Haseeb,, Abdul; Lefebvre,, Véronique
Nucleic Acids Research , Volume 47 (13) – Jul 26, 2019
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Oxford University Press
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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Abstract

Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Published online 13 June 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6917–6931 doi: 10.1093/nar/gkz523 The SOXE transcription factors––SOX8, SOX9 and SOX10––share a bi-partite transactivation mechanism Abdul Haseeb and Ver ´ onique Lefebvre Department of Surgery/Division of Orthopaedic Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA Received February 22, 2019; Revised May 23, 2019; Editorial Decision May 31, 2019; Accepted June 03, 2019 ABSTRACT evolved to exert master roles in cell fate determination and differentiation in progenitor and stem cells as well as dif- SOX8, SOX9 and SOX10 compose the SOXE tran- ferentiated cells (1–3). SOX proteins are defined as har- scription factor group. They govern cell fate and boring a high-mobility-group (HMG)-type domain that is differentiation in many lineages, and mutations im- at least 50% identical to that of the family founder, the pairing their activity cause severe diseases, includ- sex-determining region on the Y chromosome (SRY). This ing campomelic dysplasia (SOX9), sex determination domain binds and bends DNA at sequences matching or disorders (SOX8 and SOX9) and Waardenburg-Shah resembling the C[A/T]TTG[T/A][T/A] motif. It also fea- tures nuclear import and export signals and interacts with syndrome (SOX10). However, incomplete knowledge various proteins. Based on sequence identity, SOX pro- of their modes of action limits disease understand- teins are distributed into eight groups, A to H (4). Mem- ing. We here uncover that the proteins share a bipar- bers of the same group share close to 100% identity in the tite transactivation mechanism, whereby a transac- HMG domain and also share a high degree of identity in tivation domain in the middle of the proteins (TAM) other functional domains, including dimerization, transac- synergizes with a C-terminal one (TAC). TAM com- tivation and transrepression motifs, whereas proteins be- prises amphipathic  -helices predicted to form a longing to distinct groups share no or minimal identity protein-binding pocket and overlapping with minimal outside the HMG domain (1). Whereas the HMG domain transactivation motifs (9-aa-TAD) described in many has been characterized in great detail, current knowledge transcription factors. One 9-aa-TAD sequence in- of the structure/function properties of the other cardinal attributes of SOX proteins remains generally meager. We cludes an evolutionarily conserved and functionally here set out to increase knowledge of the transactivation do- required E[D/E]QY motif. SOXF proteins (SOX7, mains of the SOXE proteins. SOX17 and SOX18) contain an identical motif, sug- Humans and most vertebrates possess three SOXE pro- gesting evolution from a common ancestor already teins: SOX8, SOX9 and SOX10. Their genes overlap in ex- harboring this motif, whereas TAC and other trans- pression and are either uniquely, additively, or redundantly activating SOX proteins feature only remotely re- needed in such key processes as chondrogenesis (SOX9), lated motifs. Missense variants in this SOXE/SOXF- sex determination and differentiation (SOX8 and SOX9), specific motif are rare in control individuals, but melanogenesis (SOX9 and SOX10)(5), neural crest devel- have been detected in cancers, supporting its impor- opment (SOX8, SOX9 and SOX10), and neuronal and glial tance in development and physiology. By deepening differentiation (SOX8, SOX9 and SOX10)(6–8). In hu- understanding of mechanisms underlying the cen- mans, SOX8 mutations cause a spectrum of female and tral transactivation function of SOXE proteins, these male reproductive anomalies (9), while SOX9 mutations cause Campomelic Dysplasia, a severe skeletal malforma- findings should help further decipher molecular net- tion syndrome, as well as XY sex reversal (10–12), and works essential for development and health and dys- SOX10 mutations cause Waardenburg-Shah syndrome (13). regulated in diseases. Furthermore, SOX9 and SOX10 overexpression are poor or favorable prognosis markers in many cancers, such as INTRODUCTION glioma, melanoma and breast, colorectal, pancreas and prostate cancer (5,14). These findings point to critical roles The diversification and sophistication of cell types that has for SOXE proteins in various developmental, physiological occurred during evolution has been possible thanks to the and pathological processes. Reaching deep understanding multiplication and specialization of many types of genes of the structural organization and modes of actions of these and regulatory factors. In particular, the SOX family has To whom correspondence should be addressed. Tel: +1 215 590 0146; Email: lefebvrev1@email.chop.edu C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6918 Nucleic Acids Research, 2019, Vol. 47, No. 13 proteins is thus fundamental to uncover how they function These sequences were amplified by PCR using PfuUl- normally and can be dysregulated in diseases. tra High-Fidelity DNA Polymerase (Agilent Technologies, Transactivation is a focal activity of SOXE proteins. For Santa Clara, CA, USA) and human cDNA using forward instance, SOX8 and SOX9 transactivate Sertoli cell-specific and reverse primers containing BamHI and EcoRI sites, genes (15); SOX9 also transactivates chondrocyte-specific respectively (Supplementary Table S2). Plasmids encod- DBD genes (16,17); and SOX10 transactivates oligodendrocyte- ing GAL4 /SOXE fusion proteins were generated by and melanocyte-specific genes ( 18,19). A current conun- cloning SOXE cDNA segments into the pBIND plasmid drum is that compelling evidence of redundant and additive (Promega, Madison, WI, USA). These segments were gen- activities in multiple processes contrasts with data suggest- erated by PCR using custom-made primers (Supplementary ing that SOXE proteins utilize different transactivation do- Table S3). Missense mutations were introduced in SOX se- mains. SOX8 was indeed proposed to transactivate through quences by QuikChange Site-Directed Mutagenesis (Strata- a centrally located sequence (20,21), which we will refer to gene, San Diego, CA, USA) using tailored primers (Supple- as TAM (transactivation domain in the middle of the pro- mentary Table S4). The integrity of all plasmid inserts was tein). In contrast, SOX9 has a key transactivation domain verified by Sanger sequencing. at its C-terminus (22), which we will refer to as TAC, and might enhance its transactivation activity through a PQA- Reporter assays rich domain, which does not exist in SOX8 and SOX10 (23). SOX10, like SOX9, possesses a potent TAC domain (24,25), HEK-293 (CRL-1573; ATCC, Manassas, VA, USA) and and also possesses a so-called K2 domain, matching SOX8 SW-1353 (HTB-94; ATCC) cells were cultured in mono- TAM and contributing to transactivation in an apparently layer in 2 ml DMEM supplemented with 10% FBS (Life cell type-specific manner ( 26). These data raise questions Technologies, Carlsbad, CA, USA). Cells (0.3 million) were on whether SOX8 possesses a functional TAC and SOX9 a plated in each well of six-well plates and transfected 4– functional TAM, and whether the SOX9 PQA and SOX10 6 h later with a mixture made of 100 l DMEM, 3 l TAM have autonomous transactivation activity or only po- FuGENE6 (Promega) and 1 g plasmids. The latter in- tentiate the activity of TAC. cluded 500 ng of reporter plasmid (Col2a1 [5x48]-p89Luc We show here that TAM and TAC are autonomous and (36), Acan [4xA1]-p89Luc (37), pG5Luc (Promega), 6FXO- synergistic transactivation domains in each SOXE protein p89Luc (38) or TOP-Flash (39)), 100 ng of pSVGal and that PQA may help mediate SOX9 transactivation in plasmid (reporter used to measure transfection efficiency) specific contexts. Focusing on TAM, we identify a unique (40), and 400 ng of expression plasmids (various combina- E[D/E]QY sequence that is required for transactivation, tions of empty pCDNA 3.1, pCDNA 3.1-SOXE, pCDNA is remarkably conserved in SOXE and SOXF proteins, and 3.1-SOX17, pCDNA 3.1-SOX5 and pCDNA 3.1-SOX6, DBD is predicted to participate in a binding pocket that likely in- pBind-GAL4 /SOXE, or constitutively stabilized - teracts with transcriptional co-activators or basal transcrip- catenin/CS2 plasmid (37,41)). Cell extracts were prepared tional machinery components. in Tropix lysis buffer (0.2% Triton X-100, 100 mM potas- sium phosphate, pH 7.8, 1 mM DTT) 20–24 h after the start of transfection and assayed for luciferase and - MATERIALS AND METHODS galactosidase activities using the Dual-Light Luciferase & SOX protein sequence analyses -Galactosidase Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA) and a GloMax Explorer SOX protein sequences were downloaded from NCBI (Sup- Multimode Microplate Reader (Promega). Reporter activi- plementary Table S1) and aligned with the ClustalW tool ties were normalized for transfection efficiency by calculat- embedded in MacVector16 software (MacVector, Apex, ing the ratios of luciferase versus -galactosidase activities. NC, USA). Hydropathy plots were generated using the Kyte-Doolittle scale (27). The presence of 9-aa-TAD mo- tifs was determined using the Piskacek tool (28). Secondary Western blot and tertiary structures were predicted for the SOX9 TAM- DBD The levels of SOX and GAL4 /SOX proteins produced CD region using SWISS-MODEL (29), I-TASSER (30) from expression plasmids were determined by subjecting and PEP-FOLD3 (31). The best scoring models were ex- cell extracts to 10% SDS-PAGE and transferring proteins ported in PDB format and processed using UCSF Chimera to PVDF membranes using iBLOT 2 Gel Transfer De- v1.11.2 (32) to generate high-quality images. Synonymous vice (Thermo Fisher Scientific). Membranes were blocked and missense variants in SOXE and SOXF sequences in in Tris-Buffered Saline with 0.1% (v/v) Tween 20 (TBST) control human individuals were downloaded from the gno- and5%(w/v) nonfat dry milk for 1 h and then incu- mAD database (33) and somatic missense variants detected bated overnight at 4 C in blocking solution containing in cancers from the COSMIC database (34). anti-FLAG M2-peroxidase-conjugated antibody (A8592, Sigma-Aldrich, St. Louis, MO, USA) at a 1:12000 dilution Generation of wild-type and mutant SOX protein expression or peroxidase-conjugated GAL4 antibody (sc-510, Santa plasmids Cruz Biotechnology, Dallas, TX, USA) at a 1:500 dilu- Human SOXE and SOX17 expression plasmids were gener- tion. Peroxidase-generated signals were detected using ECL ated by cloning full-length coding sequences in frame with Prime Western Blotting Detection Reagent (GE Healthcare, an N-terminal 3FLAG epitope (35) in the pcDNA3.1(+) Chicago, IL, USA) or SuperSignal West Pico Chemilumi- vector (Thermo Fisher Scientific, Waltham, MA, USA). nescent Substrate (Thermo Fisher Scientific) on a Chemi- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6919 Doc Imaging System (Bio-Rad Laboratories, Hercules, CA, data suggested that PQA is not a transactivation domain USA). and that it interferes with TAC activity. In contrast, Mc- Dowall and colleagues reported that deletion of PQA weak- ened the ability of SOX9 to transactivate a reporter contain- RNA isolation and qRT-PCR assay ing tandemly repeated SOX binding sites, thus suggesting Lipofectamine 3000 (Thermo Fisher Scientific) was used to that PQA could be a weak transactivation domain potenti- transfect mouse chondrogenic ATDC5 cells (42) with vari- ating the activity of TAC (23,43). In view of these results, ous combinations of expression plasmids for mouse SOX5, we attempted to clarify the role of PQA. mouse SOX6, and human full-length or mutant SOX9. To- Comparing SOX9 orthologs, we found that PQA is made tal RNA was prepared 24 h later using TRIzol (Life Tech- of 35–45 residues only consisting of prolines (42%), glu- nologies) and following manufacturer’s instructions. cDNA tamines (39%), and alanines (18%) (Supplementary Fig- was synthesized using the High-Capacity cDNA Reverse ure S2A and B). In contrast, it has only 4–15 residues in Transcription Kit (Thermo Fisher Scientific). qPCR was lower vertebrates, with only a few glutamines in ancient fish. performed using the StepOne Plus Real Time PCR system Only one SOXE protein exists in most invertebrates, and (Thermo Fisher Scientific), SYBR Green PCR Master Mix this protein contains a region poorly enriched in P, Q and (Thermo Fisher Scientific) and custom-designed primers A residues (Supplementary Figure S2C). The PQA domain (Integrated DNA Technologies) (Supplementary Table S5). was thus gradually acquired following SOX9 emergence in Col2a1 and Acan mRNA levels were calculated relative to vertebrates from a SOXE ancestor. those of Hprt according to the Ct method. We investigated the function of PQA using two distinct assays. The first one tested whether the domain is sufficient for transactivation, i.e. capable on its own to interact with Statistical analyses transcriptional co-activators or basal transcription machin- Differences between datasets were evaluated using the Stu- DBD ery. We constructed plasmids encoding GAL4 /SOX9 dent’s t-test. Differences that reached P values lower than fusion proteins and transfected them in HEK-293 cells 0.05 were considered significant. along with pG5Luc, a reporter plasmid containing a tan- DBD dem of vfi e GAL4 -binding sites (Figure 2A). A fusion DBD protein containing GAL4 and the SOX9 TAM-to-TAC RESULTS segment appeared to be about three times more active than Sequence conservation suggests key roles for the SOXE TAM the same protein lacking PQA, but this difference was due and TAC regions in part to differences in protein amount (Figure 2B). A pro- DBD tein made of GAL4 and only PQA was inactive, advo- Alignment of the human SOXE protein sequences showed, cating that PQA lacks autonomous transactivation capa- as expected, that the HMG domain is the most con- bility. Our second assay tested whether PQA is necessary served region among the three proteins, with 97–99% for SOX9 transactivation. We used expression plasmids en- identity/similarity (Figure 1A–C). The homodimerization coding the full-length human SOX9 protein fused to an domain (DIM) comes next, with 81–95% identity/similarity N-terminal 3FLAG epitope and another plasmid encod- among the three proteins. TAM, that is, the reported trans- ing SOX9 without PQA, and we tested them with Col2a1 activation domain of SOX8 and K2 domain of SOX10, is [5x48]-p89Luc, a reporter featuring vfi e tandem copies of third, with 70–84% similarity between the proteins. TAC, a 48-bp Col2a1 enhancer (Figure 2C). This enhancer is a that is, the C-terminal region that includes the main trans- bona fide SOX9 target: it contains a SOX9 consensus bind- activation domain reported for SOX9 and SOX10, is fourth, ing site, i.e. a pair of inverted SOX-like recognition sites sep- with only 45–73% similarity among the proteins. Other re- arated by four nucleotides, and it is directly bound by SOX9 gions are only 7–29% identical/similar. Sequence compar- PQA in chondrocytes in vivo (16,17,44). SOX9 and SOX9 isons for various vertebrate species revealed that SOX8 or- were produced at even levels in HEK-293 cells and trans- thologs are under tighter evolutionary constraint to con- activated the reporter equally potently (Figure 2D). Simi- serve TAM than TAC, whereas SOX9 and SOX10 orthologs lar results were obtained in SW-1353 chondrosarcoma cells are under similar constraints for TAM and TAC (Supple- with Acan [4xA1]-p89Luc, a reporter containing a 359- mentary Figure S1A and B). Overall, SOXE protein or- bp Acan enhancer (Supplementary Figure S2D). This en- thologs are conserved at 34% in TAM, but only 17% in TAC. hancer features SOX9 and SOX5/6 consensus binding sites Together, these data suggest that both TAM and TAC may and is directly bound by the SOX trio in chondrocytes in have key functions in all three SOXE proteins. vivo (16,17,37) In conclusion, these data suggested that PQA has no ma- PQA may contribute to SOX9 stability and transactivation jor role in SOX9 transactivation, but did not rule out that in specific contexts SOX9 acquired this unique domain during evolution to en- In the first investigation of SOX9 domains involved in hance its stability or activity in specific contexts. transactivation, Sudbeck ¨ and colleagues showed that a fu- sion protein made with the GAL4 DNA-binding domain TAM and TAC are synergistic transactivation domains in all DBD (GAL4 ) and a SOX9 segment containing only PQA and three SOXE proteins flanking sequences was unable to activate transcription, and that a fusion protein containing only TAC was 4 times as ac- To determine whether and how TAM and TAC contribute tive as a protein featuring both PQA and TAC (22). These to SOXE transactivation, we first constructed plasmids en- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6920 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 1. Sequence conservation among the human SOXE proteins. (A) Schematic showing the domain organization of the three human SOXE proteins. Conserved domains are shown with boxes and the amino acids at the boundaries of the protein and domains are indicated with numbers. DIM, homod- imerization domain; HMG, DNA-binding domain; TAM, transactivation domain located in the middle of the proteins; PQA, P-, Q- and A-rich domain in SOX9; TAC, carboxy-terminal transactivation domain. (B) ClustalW alignment of the amino acids of the human SOXE proteins. The dimerization (DIM), DNA-binding (HMG), middle transactivation (TAM) and C-terminal transactivation (TAC) domains are boxed. Stars indicate identical residues and dots indicate similar residues. Numbers indicate residue positions within the proteins. (C) Graph showing the degrees of protein conservation. The percentages of sequence identity and similarity were calculated by ClustalW alignment. They are shown for each conserved domain and for the rest of the protein sequences (other). SOX8 is compared to SOX9 (8/9), SOX8 to SOX10 (8/10), SOX9 to SOX10 (9/10) and the three proteins together (E). Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6921 Figure 2. Transactivation capability of the SOX9 PQA domain. (A) Top, schematic of fusion proteins made using the GAL4 DNA-binding domain and SOX9 domains. These proteins are numbered 1 to 4, as in panel D. Bottom, schematic of the pG5Luc reporter used to functionally test the fusion proteins. The reporter has 5 tandem copies of the GAL4 DNA-binding site upstream of a TATA box and the fireyfl luciferase gene. ( B) Left, plot showing the DBD ability of GAL4 /SOX9 fusion proteins to transactivate pG5Luc upon transient transfection in HEK-293 cells. Reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expression plasmid. Right, western blo t showing the levels of the respective proteins present in cell lysates at the end of the experiment. Note that the lower amount of protein in lane 3 compared to lane 2 may explain in part why deletion of PQA reduced the ability of the SOX9 TAM-to-TAC region to activate the pG5Luc reporter. These results were reproduced in multiple experiments. (C) Top, schematic of the SOX9 full-length protein and a mutant protein lacking the PQA domain. Bottom, schematic of the reporter used to functionally test these proteins. The reporter contains vfi e tandem copies of a 48-bp mouse Col2a1 enhancer, which features a SOX9 consensus binding site, and the –89/+6 Col2a1 promoter upstream of the firefly luciferase gene. ( D) Top, plot comparing the ability of SOX9 and PQA SOX9 to transactivate the Col2a1 reporter. HEK-293 cells were transfected with 30 or 100 ng of SOX9 expression plasmids. Reporter activities are presented as described for panel B. Bottom, western blot of cell lysates prepared at the end of the experiment show that deletion of PQA had no obvious effect on SOX9 protein production and stability. DBD Since the relative activities of TAM and TAC greatly dif- coding GAL4 fused to the TAM, TAC or TAM-to-TAC DBD feredinthe GAL4 /SOXE assay depending on their domains of the human proteins (Figure 3A and Supple- TAM SOXE origin, we asked whether swapping SOX9 and mentary Figure S3A) and we transfected them in HEK-293 TAC SOX9 with the corresponding SOX8 and SOX10 do- cells along with pG5Luc (Figure 3B and C). All TAM and mains would affect SOX9 activity. We constructed expres- TAC domains were able to activate transcription, although TAM sion plasmids accordingly and tested them in HEK-293 cells with different performance levels. SOX8 was more po- TAM TAM using the Col2a1 reporter (Supplementary Figure S4A). Al- tent than SOX9 (2.7×) and SOX10 (7.4×). In con- TAC TAC though differential activities were observed that were con- trast, SOX8 was less potent than SOX9 (4.5×)and TAM TAC TAC TAM>TAC sistent with the low activities of SOX10 and SOX8 SOX10 (12×). While SOX8 was less active than TAC DBD TAM TAM>TAC TAM>TAC and high activity of SOX10 in the GAL4 /SOXE as- SOX8 (19.3×), SOX9 and SOX10 say, all chimeric proteins efficiently activated the reporter, were several times more active than their respective TAM indicating that the two domains, regardless of SOXE ori- and TAC domains alone. We next tested the requirement of gin, were able to synergize in the context of the full-length TAM and TAC for transactivation in the natural context of SOX9 protein (Supplementary Figure S4B and C). SOXE proteins. We constructed plasmids encoding the full- length human proteins or proteins lacking TAM or TAC (Figure 3D and Supplementary Figure S3B) and trans- The C-terminal half of TAM (TAM-CD) is a potent transac- fected them in HEK-293 cells (Figure 3E and F). All full- tivation domain length proteins powerfully activated the Col2a1 reporter, Henceforth, we focused on the TAM domain. To reveal but their activities were drastically reduced in the absence which segment of the domain is involved in transactivation, of TAM or TAC (16- to 658-fold). Similar results were ob- DBD we generated plasmids encoding fusions of GAL4 with tained with SW-1353 cells and using the Acan reporter (Sup- halves or quarters of TAM, and plasmids encoding SOX9 plementary Figure S3C–F). In conclusion, the first assay in- proteins lacking most of each TAM quarter (Figure 4A). dicated that TAM and TAC are able to work as indepen- These quarters were named TAM-A, TAM-B, TAM-C and dent transactivation domains, and the second assay indi- TAM-D. In transfection of HEK-293 cells with pG5Luc, cated that the two domains work synergistically in the con- TAM-AB, TAM-C and TAM-D failed to transactivate, text of each SOXE protein. whereas TAM-CD was very potent (Figure 4B). In trans- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6922 Nucleic Acids Research, 2019, Vol. 47, No. 13 DBD Figure 3. Transactivation capabilities of the SOXE TAM and TAC domains. (A) Schematics of fusion proteins containing GAL4 and SOX9 do- DBD mains. See Supplementary Figure S3A for fusion proteins of GAL4 with SOX8 and SOX10 domains. (B) Reporter assay comparing the abilities of DBD GAL4 /SOXE fusion proteins to activate pG5Luc. HEK-293 cells were transfected with pG5Luc and expression plasmids for the fusion proteins shown in panel A. Reporter activities are presented for one experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expressio n plasmid. These results were reproduced multiple times. (C) Western blot of cell lysates prepared at the end of the experiment showing that all protein forms were made DBD TAM>TAC in similar amounts. The blot was made with lysate amounts normalized for transfection efficiency. The lower band seen in the GAL4 /SOX8 lane likely reflects partial degradation of the protein. ( D) Schematics of the SOX9 full-length protein and mutant proteins lacking either TAC or TAM. See Supplementary Figure S3B for equivalent SOX8 and SOX10 schematics. (E) Reporter assay comparing the abilities of the three SOXE proteins to activate the Col2a1 [5x48]-p89Luc reporter in HEK-293 cells, and effects of deleting their TAM or TAC domain. Reporter activities are presented as described in panel B. (F) Western blot of cell lysates prepared at the end of the experiment showing that all protein forms were made. Major differences in reporter activities (panel E) are not due to variations in relative amounts of the proteins and must thus genuinely reflect differences in functional capabilit ies. The blots were made with lysate amounts normalized for transfection efficiency. fection of HEK-293 and SW-1353 cells with the Col2a1 or evidence that both TAM and TAC are critical for SOX9 Acan reporter, TAM-A or TAM-B deletion was inconse- functions. quential, whereas TAM-C or TAM-D deletion virtually ab- rogated SOX9 activity (Figure 4C and Supplementary Fig- ure S5A). Taken together, these data suggested that TAM- TAM-CD exhibits characteristic features of acidic AB is dispensable and that residues within TAM-C and transactivation domains and a unique, highly conserved TAM-D are necessary and sufficient for transactivation. E[D/E]QY motif Since all data were obtained so far using reporter as- Transactivation domains are categorized based on amino says, we next asked whether SOX9 also requires TAM and acid composition (46). For instance, the SOXE TAC is a TAC to activate the endogenous Col2a1 and Acan genes. In non-acidic PQS-rich transactivation domain (22). TAM- transfection of ATDC5 cells, a chondrogenic cell line de- CD examination revealed numerous acidic (Asp and Glu) rived from a mouse teratoma and frequently used to study and other hydrophilic amino acids alternating with hy- chondrocyte differentiation in vitro (45), full-length SOX9 drophobic residues (Ile, Leu, Met, Phe and Val) (Figure successfully cooperated with SOX5 and SOX6 to enhance 5A). This pattern is reminiscent of the minimal nine-amino- Col2a1 and Acan expression (3.7× and 2.5×, respectively) acid-transactivation-domain motif (9-aa-TAD) described (Supplementary Figure S5B). In contrast, SOX9 lacking ei- for acidic transactivation domains in many transcription ther TAC or TAM-D was unable to do so, lending further factors, including GAL4 (yeast), P53, NFAT and NF-kB Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6923 Figure 4. Identification of subdomains of SOX9 TAM mediating transactivation. ( A) From top to bottom, schematic of the SOX9 protein; alignment of DBD the TAM sequences of the three human SOXE proteins; segments of TAM fused to GAL4 ; and TAM-A to TAM-D sequences deleted in the SOX9 DBD protein. (B) Reporter assay comparing the abilities of proteins made by fusing GAL4 with subdomains of SOX9 TAM to transactivate pG5Luc. Reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures per condition. Data were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the presence of an empty expre ssion plasmid. The western blot of cell lysates shows that all protein forms were efficiently made in the cells and thus that major differences in reporter act ivities among proteins genuinely reflect intrinsic differences in transactivation capabilities. These results were reproduced in multiple experiments. ( C) Reporter assay comparing the abilities of wild-type SOX9 and SOX9 proteins lacking the whole TAM or TAM segments to transactivate the Col2a1 reporter. Reporter activities are presented as described in panel B. The western blot of cell lysates shows that all protein forms were efficiently made in the cel ls and thus that major differences in reporter activities among proteins genuinely reflect intrinsic differences in transactivation capabilities. These results were reproduced in multiple experiments. proteins (mammals) and VP16 (human herpes virus) (28). ing protein structures directly from amino acid sequences, Accordingly, the Piskacek algorithm identified one such to predict the secondary and tertiary structures of TAM- motif in all SOXE TAM-C regions, one overlapping TAM- CD. The SWISS-MODEL model that reached the high- C and TAM-D in SOX9, and one in SOX9 and SOX10 est quality score (QMEAN, –1.36; sequence identity with TAM-D (Figure 5A). In many cases, 9-aa-TAD sequences the template, 23.53%) was built according to a region of contain a XX core motif (, hydrophobic residue; X, the CdiI Immunity protein from Yersinia kristensenii (PDB any residue) that interacts with basal transcription machin- ID: 4ZQV). The best I-TASSER model (C-score, –1.95; se- ery components, such as hTAF 31 (47). We found a par- quence identity with the template, 19%) was based on a II tially related, but distinct motif in TAM-D in the three hu- glycosylated calcitonin growth factor from Anguilla japon- man SOXE proteins (Figure 5A). This motif responds to ica (PDB ID: 1BYV). Of 10 models proposed by PEP- an E[D/E]QY consensus and is remarkably conserved FOLD3, we retained the best one (sOPEP score: –45.011). not only in all SOX8, SOX9, and SOX10 vertebrate se- All models concurred that TAM-CD could form two - quences (Supplementary Figure S1), but also in the lamprey helices, one using the TAM-C 9-aa-TAD motif and the SOXE3 protein and in the sole SOXE protein existing in in- other one using most of the TAM-D 9-aa-TAD sequence vertebrates (Figure 5B). Since the P53 XX motif was and E[D/E]QY motif (Figure 5A, C and D). These he- shown to transit from a random coil to an -helix upon lices would fold into a protein-binding pocket coated exter- binding to hTAF 31 (47), we used SWISS-MODEL and nally with polar residues and internally with hydrophobic II I-TASSER, which are template-based structure prediction and aromatic residues. software, and PEP-FOLD3, a de novo program predict- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6924 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 5. In silico analysis of transactivation domain features of the SOXE TAM-CD region. (A) Plots of the hydropathy scores of TAM-CD and flanking residues in human SOX8, SOX9 and SOX10. Amino acids are typed in colors according to the nature of their side chains, as indicated underneath the plots. Two predicted -helices are shaded in the plots; 9-aa-TAD motifs are delineated with brown brackets underneath the sequences; and the conserved E[D/E]QY motif is highlighted with a green box. (B) ClustalW sequence alignment showing a high degree of conservation of the E[D/E]QY motif in SOXE proteins from various vertebrate and invertebrate species. (C and D) Binding-pocket structure of the SOX9 TAM-CD domain predicted by SWISS- MODEL, I-TASSER and PEP-FOLD3. Top, ball-and-stick representation showing amino acid cores and side chains. Bottom, cartoon representations. The N- and C-termini of the domain are marked. The -helices are indicated as H1 and H2. The E[D/E]QY motif is highlighted with a green bubble. The color code is otherwise the same as for the sequences in the panel A. Specific residues in the E [D/E]QY motif are critical for cantly change the hydropathy index. These findings further transactivation supported the conclusion that hydrophobic residues pro- jecting inside the binding pocket are critical for transacti- We introduced a series of missense mutations in TAM- vation and that even non-hydrophobic residues composing CD to test the importance of highly conserved residues in the E[D/E]QY motif are critical too. transactivation (Figure 6A). These mutations were selected to significantly alter the hydropathicity, polarity or size of amino acid side chains. Overall, mutations in residues par- The E[D/E]QY motif is conserved in both SOXE and ticipating in the -helix 1 had no drastic effect on SOX9 SOXF proteins activity (Figure 6B). In the GAL4 assay, where transacti- The discovery that the E[D/E]QY motif of TAM-CD vation is only driven by TAM-CD and is thus more sen- is fully evolved in invertebrates prompted us to determine sitive, mutations of residues with hydrophobic side chains whether an identical or similar motif and its associated fea- protruding inside the binding pocket (Leu278 and Val282) tures are also present in the SOXE TAC domain and in were deleterious, whereas mutations in residues with acidic other transactivating SOX proteins. We used three criteria: side chains projecting outwards (Glu277 and Asp281) were (i) sequence conservation between group members; (ii) pres- inconsequential (Figure 6C). ence of 9-aa-TAD domains and (iii) presence of XX or Replacing the first residue of the E [D/E]QY motif E[D/E]QY-like motifs. The SOXE TAC domains con- with the residues present in the XX motif of VP16 tained 9-aa-TAD motifs and a VYXXL sequence resem- or P53 (Glu293Met or Glu293Thr, respectively) impaired bling a XX, but no E[D/E]QY-like sequence (Sup- the activity of full-length SOX9 and one of the mutations plementary Figure S6A). SOXB1 (SOX1, SOX2 and SOX3) also affected TAM-CD activity, explaining that this acidic and SOXC (SOX4, SOX11 and SOX12) proteins featured residue (or Asp) is conserved in all SOXE proteins (Figure 9-aa-TAD, and XX/E[D/E]QY-like sequences in 6D and E). All mutations introduced in other residues of their transactivation domains, but these sequences were the E[D/E]QY motif dramatically reduced SOX9 and very different from those of the SOXE TAM-CD domains TAM-CD activities, except F294L, which resulted in an aro- (Supplementary Figure S6B). Interestingly, SOXF proteins matic to aliphatic side chain change, but did not signifi- (SOX7, SOX17 and SOX18) featured a 9-aa-TAD sequence Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6925 Figure 6. Test of the effects of amino acid substitutions in TAM-CD on transactivation. (A) Schematic showing the SOX9 TAM-CD residues substi- DBD TAM-CD tuted in GAL4 /SOX9 and SOX9 expression plasmids. (B–E) Reporter assays comparing the abilities of wild-type and variant SOX9 and DBD TAM-CD GAL4 /SOX9 proteins to transactivate their respective Col2a1 and pG5Luc target reporters upon transfection in HEK-293 cells. Normalized reporter activities are presented for one representative experiment as the mean ± standard deviation obtained for triplicate cultures and in percentage of the activities obtained with wild-type proteins. Western blots of cell lysates prove that major differences in reporter activities are not due to differences in relative amounts of the various proteins. These results were reproduced in multiple experiments. with an E[D/E]QYL motif fully matching the SOXE stitutively stabilized -catenin from activating TOP-Flash, E[D/E]QY consensus in their transactivation domains a reporter gene classically used as a readout of canoni- (Figure 7A). This motif is also remarkably conserved from cal WNT signaling (51). SOX9 was also shown to inhibit invertebrates to humans (Figure 7B). This finding pairs with -catenin transcriptional activity, but to use its TAC do- the fact that the HMG domains of the SOXE and SOXF main to bind to -catenin (52). We therefore decided to di- proteins are more closely related to one another than to rectly compare the contributions of the SOX9 and SOX17 those of other SOX proteins (48). Altogether, the data sug- E[D/E]QYL motifs to the protein activities. As expected, gest that the SOXE and SOXF groups emerged from a com- deletion of the motif significantly reduced the abilities of mon ancestor that was already featuring an E[D/E]QY SOX9 and SOX17 to activate reporter genes (Supplemen- motif. We looked for the presence of an E[D/E]QY mo- tary Figure S7A and B). When tested with TOP-Flash, tif in all other SOX proteins, but did not find any (data not wild-type SOX9 and SOX17 inhibited the activity of con- shown). stitutively stabilized -catenin in a dose-dependent manner The E[D/E]QYL motif was previously recognized in (Supplementary Figure S7C and D). Deletion of the whole SOXF proteins, and SOX17 was shown to require it to ac- TAM or only its EFDQYL motif slightly reversed SOX9 tivate endodermal genes on its own and in synergy with - inhibition of -catenin, whereas deletion of TAC totally re- catenin (49) as well as to reprogram somatic cells into in- versed this inhibition, and whereas deletion of the EFE- duced pluripotent stem cells (50). Further, this domain was QYL motif from SOX17 effectively reversed the inhibition. shown to help SOX17 bind to -catenin and prevent con- These data suggest that the E[D/E]QY motif might con- Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6926 Nucleic Acids Research, 2019, Vol. 47, No. 13 Figure 7. Identification of an E [D/E]QY motif in SOXF proteins. (A) Schematics showing the locations of the HMG and transactivation regions (TA, pale green boxes) of SOXF proteins and alignment of TA regions that show a high degree of conservation among group members, a 9-aa-TAD motif (brown bracket) and an E[D/E]QYL motif. The TA regions were previously delineated for SOX7 (63), SOX17 (64) and SOX18 (59,65). (B) ClustalW sequence alignment showing the high degree of conservation of the E[D/E]QYL motif in SOXF proteins from various vertebrate and invertebrate species. tribute to inhibition of canonical WNT signaling by both cancer and participates in tumorigenesis. The occurrence of SOXE and SOXF proteins, but that TAC has a dominant missense variants was lower in the TAM than in the HMG role in SOXE proteins and that SOXF proteins might fea- domain in COSMIC samples, but the difference did not ture specific sequences around EFEQYL that potentiate its reach statistical significance. We therefore closely examined inhibitory activity. The latter proposition is supported by the types of missense variants present in the SOXE and evidence that deletion of its entire C-terminus prevented SOXF E[D/E]QY motifs in gnomAD and COSMIC SOX17 from binding to -catenin, whereas the sole dele- samples. tion of the EFEQYL motif (located in the C-terminus) only Interestingly, gnomAD missense variants affected many had a partial effect (49). residues around the E[D/E]QY motifs of SOXE and SOXF proteins, but none occurred within the SOX9, SOX10 and SOX18 E[D/E]QY motifs and only a few occurred The SOXE/SOXF E[D/E]QY motif is highly conserved within the SOX8, SOX7 and SOX17 E[D/E]QY mo- in the human population tifs (Figure 8A and B). The SOX8 variants detected in The outstanding degree of conservation of the this motif represented conservative changes (D286E and SOXE/SOXF E[D/E]QY motif suggests that mu- Q287R) in the residues that occupy the X positions in tations in this motif would be incompatible with healthy the related XX sequence of other transcription fac- development and adult life. To test this hypothesis, we tors. The D286E change is unlikely to be consequential searched for literature reports of missense mutations or since both D and E are highly acidic and occupy the third other in-frame micro-alterations in this domain in SOXE position of the SOXE/SOXF E[D/E]QY motif. The and SOXF genes that were linked to a human disease, but other variant, Q287R, might be consequential since Q is did not find any. We then searched GnomAD, a database highly conserved in SOXE and SOXF proteins and since of genomic sequences from >140 000 unrelated control it is uncharged whereas R is positively charged. To test individuals, and COSMIC, a catalog of somatic mutations whether this variant could affect protein activity, we intro- in cancer. Detailed analysis of SOX9 revealed that synony- duced an equivalent mutation (Q296R) in the SOX9 and DBD TAM-CD mous and missense variants affected similar proportions GAL4 /SOX9 proteins. We observed that both of residues throughout all domains of the protein, except proteins still retained significant activity in their respective the HMG domain, where significantly fewer missense assays (Figure 8C), suggesting that the SOX8 Q287R vari- variants were detected in gnomAD individuals compared ant is not detrimental enough to cause a disease. to synonymous variants in the same cohort and compared Unlike the gnomAD database, the COSMIC database to missense variants in cancers (Supplementary Figure did contain missense variants in the SOX9 EFDQYL mo- S8A and B). This finding suggests that a particularly tight tif (Supplementary Figure S8C). We tested them along with sequence conservation constraint exists for this domain two variants in the -helix 1. The latter almost doubled the in healthy individuals and that this constraint is lifted in activity of SOX9, whereas the two variants affecting the E Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6927 Figure 8. Analysis of SOXE and SOXF missense variants in the human control population and model for the SOXE bipartite transactivation mechanism. (A) Missense variants listed in gnomAD in SOXE TAM-CD are presented underneath the domain sequences. The numbers of alleles detected in over 140 000 unrelated individuals are indicated for each variant. (B) Missense variants listed in gnomAD in the SOXF E[D/E]QYL motif and flanking residues. (C) Test of the effect of a Q296R variant detected in SOX8 in healthy human individuals on transactivation. The variant was introduced in the DBD TAM-CD GAL /SOX9 and SOX9 proteins. The proteins were then tested in HEK-293 cells upon co-transfection with the pG5Luc or Col2a1 reporter. Other missense variants (as described in Figure 6) were tested in parallel for comparison. Data were calculated and are presented as in similar assays in previous figures. They were reproduced in multiple experiments. ( D) Model of the current view for the mechanism used by SOXE proteins to activate target genes. Previous studies demonstrated that SOXE proteins homodimerize upon binding to inverted recognition sites present in the enhancers or promoter of their target genes. These events involve their HMG and dimerization domains. The present study uncovered that they transactivate these genes through a bi-partite mechanism involving a centrally located domain (TAM) synergizing with the protein C-terminal region (TAC). The functional core of TAM is predicted to fold into binding pocket-like structure upon interaction with specific transcriptional co-activators or components of the basal tra nscription machinery. It contains a highly conserved E[D/Q]Y motif that is critical for transactivation and is thus likely involved in recognizing and firmly binding functional partners. Not shown here is a PQA-rich domain present solely in SOX9 that may facilitate transactivation in specific contexts. residue of the EFDQYL motif greatly decreased SOX9 ac- this motif could underlie congenital or acquired human dis- tivity and the variant affecting the D residue of the motif eases. modestly decreased SOX9 activity (Supplementary Figure S8D). Beside confirming the importance of EFDQYL for DISCUSSION SOX9 activity, these data add support to the notion that This study has brought unity and new information on the the SOXE/SOXF E[D/E]QY motif is critical for the mechanisms whereby the SOXE proteins achieve transacti- function of SOXE proteins and suggest that mutations in vation. It has provided evidence that each protein carries Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 6928 Nucleic Acids Research, 2019, Vol. 47, No. 13 two synergistic transactivation domains, TAM and TAC domain, and was mentioned to interact also with SOX8 (Figure 8D). By deploying a bipartite transactivation mech- TAM and SOX10 TAC (55,56). P53 and other proteins con- anism, SOXE proteins may engage exclusive sets of tran- tact CBP/p300 through 9-aa-TAD domains (57), but other scriptional partners to effectively activate target genes. The transcriptional partners have also been identified for 9-aa- C-terminal half of TAM is predicted to form a protein- TAD and XX domains, such hTAF 31 (47). All this II binding pocket. Its E[D/E]QY core motif is functionally suggests that SOXE proteins may use their two transacti- crucial and is highly conserved evolutionarily and in human vation domains to contact various partners and to do so healthy individuals not only in SOXE proteins but also in cooperatively or independently of one another. SOXF proteins. Dissection of functional segments revealed that the C- The unique PQA domain of SOX9 gained in length and terminal half of TAM (TAM-CD) is both needed and suffi- P-, Q- and A-enrichment upon mammalian evolution, but cient for transactivation. TAM-CD has characteristic fea- its role remains unclear. Glutamine-rich sequences exist in tures of acidic transactivation domains: enrichment for many types of proteins, especially in transactivation do- acidic residues that may promote an unstructured confor- mains, and there is evidence that they help stabilize pro- mation prone to encounter transcriptional partners, and en- teins and strengthen protein-protein interactions (53). The richment for aromatic and bulky hydrophobic residues that SOX9 PQA domain was not found in our study nor in could favor the formation of a specific structure upon inter- a previous study by Sudbeck ¨ et al. to have autonomous action with these partners (58). Protein modeling tools sup- transactivation function (22). McDowall et al. reported that ported this view by proposing that TAM-CD would indeed PQA enhanced the ability of SOX9 to transactivate an fold into a binding pocket. Functional assays added fur- artificial pS10E1bCat reporter, which likely bound SOX9 ther support to this view by showing that non-conservative monomers (23). We failed to replicate this finding using substitutions were most deleterious when occurring in hy- bona fide Col2a1 and Acan reporters that bind SOX9 drophobic residues predicted to line the inner surface of the DBD TAM>TAC homodimers, but observed that GAL4 /SOX9 binding pocket. was less abundant and less active in transactivating pG5Luc We found an E[D/E]QY sequence to be the most con- when it lacked PQA. This reporter is also likely to bind served segment of TAM-CD among all SOXE proteins from DBD GAL4 /SOX9 monomers. One can thus envision that invertebrates to humans and also to be the most critical PQA enhances SOX9 stability and transactivation efficiency segment for transactivation. Its conservation in SOXF pro- when the protein binds target genes as a monomer, as in Ser- teins unsettles a longstanding concept that proteins from toli cells, but may not be necessary when the protein binds different SOX groups share no significant identity out- targets as a homodimer, as in other cell types. side the HMG domain and strongly suggests that the two Different transactivation mechanisms were previously SOX groups recently evolved from a common ancestor. suggested for the three SOXE proteins, mostly because most It is intriguing, however, that the two groups diverged in DBD studies only used the GAL4 assay to map transactiva- their expression patterns and gene targetome to control dif- tion domains and did not test the consequences of dele- ferent cell types, but nevertheless conserved a core motif tions or point mutations in the protein sequences. The use for transactivation. Missense mutations introduced in the of both assays led us to find two functional domains in each E[D/E]QY sequence of SOX18 were previously shown protein. This finding was not unexpected considering the to impair transactivation, showing that this motif is critical high degree of sequence conservation and redundancy of in SOX18 too (59). Previous studies also showed that this se- SOXE proteins in many processes. What was unexpected quence facilitates interaction of SOX17 with -catenin and was to find that the two domains synergize with one an- may thereby allow SOX17 to cooperate with -catenin in other. Schreiner et al. indeed showed that deletion of SOX10 the activation of endodermal genes in Xenopus embryos TAM in the mouse led to a milder disease than total inac- (49) and to reprogram fibroblasts into pluripotent stem cells tivation of Sox10 (26). Melanogenesis and enteric nervous with higher efficiency than SOX2 ( 50). These data, how- system development were significantly impaired, but neural ever, contrast with evidence that SOX17 can also repress crest and oligodendrocyte development were only slightly -catenin/TCF activity and interact with -catenin to pro- affected. This led the authors to suggest that SOX10 TAM mote its degradation (60). Similarly, SOX9 and -catenin contributes to SOX10 activity in a cell type-specific con- were shown to physically interact with one another and text rather than being mandatory in all processes to syner- to have reciprocal antagonistic activities, including induc- gize with TAC. It remains possible, however, that TAM con- tion of mutual degradation (52). TAC was required for in- tributes to SOX10 activity in all cell types, but that its dele- teraction of SOX9 with -catenin. In direct comparisons, tion is pathogenic only in cells critically dependent on its we found that the EF[D/E]QYL motif is more critical for dosage. Supporting this model is the fact that humans with transactivation by SOX9 than by SOX17, but is more po- heterozygous mutations inactivating one SOX10 allele have tent in SOX17 than SOX9 to inhibit -catenin. Altogether, deficiencies primarily in melanogenesis and enteric nervous conservation, structural and functional data point to the system development. Synergy implies that TAC and TAM E[D/E]QY motif as being a pivotal sequence that com- domains may cooperatively contact the same protein or bines with other sequences, likely distinct in SOXE and may contact different components of transcriptional com- SOXF proteins, to engage in protein interactions with ei- plexes. CBP/p300, a major transcriptional co-activator, was ther transcriptional or non-transcriptional consequences. shown to interact with SOX9 via TAC, but apparently not SOXE and SOXF mutations cause severe developmen- through TAM (54). TRAP230, a member of the transcrip- tal diseases, namely Campomelic Dysplasia and XY sex re- tional mediator, was shown to interact with the SOX9 TAC versal for SOX9, Waardenburg-Shah syndrome for SOX10, Downloaded from https://academic.oup.com/nar/article-abstract/47/13/6917/5518312 by Ed 'DeepDyve' Gillespie user on 23 July 2019 Nucleic Acids Research, 2019, Vol. 47, No. 13 6929 and hypotrichosis-lymphedema-telangiectasia-renal defect FUNDING syndrome for SOX18 (61). The mutations vary from en- National Institute of Arthritis and Musculoskeletal and tire gene deletions to point mutations. Among the latter, Skin Diseases (NIAMS) [AR046249, AR072649 to V.L.]. nonsense mutations within the SOXE TAC domain have Funding for open access charge: NIAMS [AR072649 to demonstrated the critical importance of this domain. Mis- V.L.]. sense mutations are almost always located in the HMG do- Conflict of interest statement. None declared. main or SOXE dimerization domain. 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Tsuchiya,T., Suzuki,E., Miyado,M., Hata,K., Nakabayashi,K. et al. (2015) Testicular dysgenesis/regression without campomelic

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