Research ArticleCELL BIOLOGY

Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

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Science Advances  17 Feb 2021:
Vol. 7, no. 8, eabe0849
DOI: 10.1126/sciadv.abe0849
  • Fig. 1 SOX9 represses Wnt/β-catenin signaling in mammalian cells.

    (A and B) TopFlash reporter luciferase assays in HEK293T cells treated for 16 hours with purified Wnt3a (A) or GSK3 inhibitor CHIR99021 (CHIR) (B) and transfected with a SOX9 expression plasmid. (C) Same as (A) and (B) except Wnt signaling was activated by transfection of N-terminally mutated (S33Y) β-catenin (β-catenin*) and with an increasing dose of SOX9. (D) Same as (C) except an Axin2 enhancer reporter was used instead of TopFlash. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of SOX9 and two Wnt targets performed in pTRE-SOX9WT cells. Dox treatment was 13.5 hours. (F) TopFlash reporter assays of several cell lines transfected with β-catenin* with or without SOX9. See Results and Materials and Methods for details about each cell line. Each bar represents the means of biological triplicates ± SD. *P < 0.05; **P < 0.005; ***P < 0.001.

  • Fig. 2 SOX9 reduces β-catenin protein levels independent of TCFs and the proteasome.

    (A) Western blots of endogenous β-catenin and transfected FLAG-SOX9 from pTRE-SOX9WT cell lysates treated with 5 μM CHIR and Dox for the indicated times (hour). (B) Western blots of transfected FLAG–β-catenin* and FLAG-SOX9 from HEK293T cell lysates. (C) Western blots of parental HEK293T cells (left half) or HEK293T cells with CRISPR-induced deletions of all four TCF genes (TCFQKO cells; right half) transfected with β-catenin* and an increasing dose of SOX9. (D) TopFlash reporter assay performed on HEK293T cells transfected with or without FLAG-SOX9 and treated with the proteasome inhibitors MG132 and bortezomib for 8 hours before lysis. Each bar represents the means of biological triplicates ± SD. (E) Western blots of HEK293T cell lysates, transfected with FLAG–β-catenin* with or without SOX9 and treated with MG132 for indicated periods of time. Images cropped from the same blot for plus/minus Sox9. (F) Western blots of endogenous β-catenin from pTRE-SOX9WT cells supplemented with Dox (24 hours) and 5 μM CHIR (16 hours) and then treated with CHX for indicated period of time before harvest. (G) Western blots of transfected FLAG–β-catenin* from HEK293T cells transfected with β-catenin* with or without SOX9 and then treated with CHX for indicated period of time before harvest. More extract was loaded for the SOX9-expressing cells to provide a fair comparison of β-catenin decay. All images in (F) and (G) were cropped from the same blot. α-Tubulin was used as a loading control for all Western blots. In numerous experiments, Sox9 down-regulated β-catenin to levels shown in (A) to (C) and (E). In (three) separate experiments, Sox9 accelerated the turnover of β-catenin, as shown in (F) and (G). ***P < 0.001.

  • Fig. 3 SOX9 requires transcriptional activation activity to suppress Wnt/β-catenin signaling.

    (A) Schematic representations of wild-type (WT) full-length and transcriptionally disabled SOX9s. The location of the DNA binding HMG domain and the three known transcriptional activation domains (TAM, PQA, and TAC) are shown (12). All SOX9 constructs were tagged with two Flag epitopes at their N termini. SOX9HMG has three point mutations in the N terminus of the HMG domain (star) and contains an exogenous NLS to ensure nuclear localization. SOX9332 lacks the last 177 amino acids (aa) of the protein, and three variants of SOX9332 containing distinct transcriptional activation domains (KIX, GAL4, and VP16) were constructed (see fig. S2B for sequences of these domains). (B) SoxFlash reporter luciferase assays in HEK293T cells transfected with SOX9 constructs. The differences between SOX9 and the SOX9 mutants in activation of SoxFlash were significant (P > 0.001). (C) TopFlash reporter assay with SOX9 constructs. Each bar represents the means of biological triplicates ± SD. (D) Western blots on HEK293T cell lysates from transfections of FLAG–β-catenin* and various FLAG-SOX9 proteins. α-Tubulin was used as a loading control. (E) Western blots of HEK293T cell lysates transfected with different β-catenin* domain mutants (fig. S3B for details) with or without SOX9. In numerous experiments, SOX9 down-regulated β-catenin proteins to levels shown in (D) and (E). ***P < 0.001.

  • Fig. 4 Transcriptional profiling identified MAML2 as a conserved inhibitor of Wg/Wnt signaling.

    (A and B) Venn diagrams depicting the genes that are activated (A) or repressed (B) after Dox treatment (13.5 hours) of pTRE-SOX9WT and pTRE-SOX9EFD-AAA cells. (C and D) Volcano plots summarizing the differently expressed genes pTRE-SOX9WT (A) and pTRE-SOX9EFD-AAA (B) cells treated with Dox for 13.5 hours. Location of MAML2 indicated by red dot in both plots. (E) Schematic representation of human MAMLs and Drosophila Mam proteins. The location of the polyglutamines (>12 sequential Qs), conserved acidic domains, and the N-terminal CSL/Nintra binding domains are indicated. (F to K) Micrographs of adult eyes of Drosophila containing P[GMR-wg] (F and I), P[GMR-arm*] (G and J), or P[GMR-hid] (H and K) transgenes without (F and G) or with (I to K) a P[EP] line expressing Mam. (L) Quantification of the adult eye phenotypes using ImageJ (n = 10 eyes per genotype). Each bar represents the means of biological triplicates ± SD. See Materials and Methods for details. ***P < 0.001; **P < 0.005. Photo credit: Ken M. Cadigan, University of Michigan.

  • Fig. 5 MAML2 is required for SOX9-mediated inhibition of Wnt signaling.

    (A) Table showing RPKM (Reads Per Kilobase per Million reads mapped) values with and without SOX9WT and SOX9EFD-AAA induction (13.5 hours) for all three MAML family members. (B) qRT-PCR analysis showing MAML2 expression upon 13.5-hour Dox treatment in pTRE-SOX9WT and pTRE-SOX9EFD-AAA cells. (C) Western blots of pTRE-SOX9WT cells transfected with either scrambled or two distinct MAML2A siRNAs (total length of time, 80 hours) and treated with or without Dox and 5 μM CHIR for 24 hours before harvest. (D) TopFlash reporter assays of pTRE-SOX9WT cells transfected with scrambled or siRNA targeting MAML2 and treated with Dox and CHIR as in (C). (E) TopFlash assay in parental pTRE-SOX9WT cells and two CRISPR-derived lines with small deletions in MAML2 (see fig. S4D for details) treated with Dox and CHIR as in (C). Each bar represents the means of biological triplicates ± SD. Depletion or mutation of MAML2 resulted in significant (P > 0.001) differences in SOX9 repression compared to controls. ***P < 0.001.

  • Fig. 6 MAML2 promotes β-catenin degradation through a mechanism distinct of the β-catenin destruction complex.

    (A) TopFlash reporter assays in parental and MAML2 KO lines in response to CHIR (1 to 4 μM). Each point represents the means of biological triplicates ± SD. (B and C) Western blots of parental and MAML2 KO line (#1) of endogenous β-catenin stabilized with overnight treatment of 5 μM CHIR (B) or transfected with β-catenin* (C) and treated with CHX for the indicated times. α-Tubulin was used as a loading control. Images cropped from the same blots. Blots shown were representative of three independent experiments. (D) Schematic representation of the N-terminal (MAML2-N) and C-terminal (MAML2-C) fragments used in the β-catenin binding assays shown in (E). The locations of the CSL/N binding domain (gray), acidic domains (stripped), and polyQ regions (black) are indicated. MAML2-N and MAML2-C are HIS-tagged at their C termini. (E) Western blots of in vitro pull downs using GST or GST–β-catenin with HIS-tagged MAML2-N (left) and MAML2-C (right). Input lanes contain 10% of the total binding reaction. Data are representative of three independent pull-down assays. (F and G) Cartoon depicting a SOX9-MAML2 axis that restricts the Wnt pathway independently of the β-catenin destruction complex, proteasome, and lysosomal degradation pathways. *P < 0.05.

Supplementary Materials

  • Supplementary Materials

    Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

    Abhishek Sinha, Vinson B. Fan, Aravinda-Bharathi Ramakrishnan, Nicole Engelhardt, Jennifer Kennell, Ken M. Cadigan

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