Ajuba transactivates N‐cadherin expression in colorectal cancer cells through interaction with Twist

Abstract Ajuba is a multiple LIM domain‐containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene N‐cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E‐box element within N‐cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist‐related tumour metastasis.


| INTRODUC TI ON
The Ajuba adaptor or scaffold protein belongs to the Zyxin/Ajuba family of LIM proteins. 1,2 The family contains Ajuba, Limd1, Wtip, Zyxin, Lpp and Trip6. These proteins have the same structural characteristics, which are the preLIM domain at their amino-terminus and two or three tandem LIM motifs at their carboxyl-terminal region. The preLIM domain is diverse in sequence and abundant in glycine and proline residues and constitutes an SH3 consensus recognition site. The LIM motif is a cysteine-rich, double zinc finger domain and primitively discovered in three critical transcriptional factors including Caenorhabditis elegans Lin-11, rat Isl-1 and Mec-3, which regulate cell fate determination and differentiation. 1,3 LIM domain is highly conservative and mediates protein-protein interaction as a diverse protein module. 4 In consistence with its feature to shuttle between cytoplasm and nucleus, Ajuba contains a nuclear localization sequence (NLS) in the LIM domain and nuclear exporting sequence (NES) in the pre-LIM domain. 5 Therefore, Ajuba can participate in assembling various protein complex and be implicated in a series of cellular processes such as cytoskeletal organization, cell-cell adhesion, regulation of gene transcription, mitosis, cell differentiation, proliferation and migration in cytoplasm and nucleus. 6 In nucleus, Ajuba can act as a transcriptional corepressor or coactivator to take part in transcriptional regulation. For example, Ajuba acts as a scaffold protein to recruit 14-3-3 and PRMT5 to Snail to repress E-cadherin expression. 7,8 Comparably, Ajuba can interact with PPARγ using its non-NR box element within the preLIM region to coactivate the transcriptional ability of PPARγ. 9 In recent years, a series of investigation showed that Ajuba exerts a critical role in epithelial-mesenchymal transition (EMT) and cancer metastasis. 10,11 Moreover, Ajuba can function as a Snail corepressor to modulate EMT and metastasis. 5 EMT is known to be critical for embryonic development, tissue regeneration and tumour metastasis. 12 Twist, a basic helix-loop-helix (bHLH) transcription factor, facilitates tumour invasion and metastasis by boosting EMT of cancer cells. During the EMT, Twist induces the transcription of genes encoding mesenchymal markers, such as fibronectin and N-cadherin. 13 In prostate carcinoma cells, Twist transactivates N-cadherin expression by directly binding to an E-box regulatory element within its promoter. 14 However, the underlying mechanism of transcriptional activation by Twist is little known. In this paper, we report that Ajuba functions as the coactivator of Twist to activate the transcription of N-cadherin.

| Cell lines and cell culture
HEK 293T, SW1116 and SW480 cells were obtained from ATCC and authenticated by DNA typing, and no signs of mycoplasma contamination were found. The cells were seeded in Dulbecco's modified Eagle's medium (Gibco, Cat#11005500T8) supplemented with 10% fetal bovine serum (Sigma, Cat#F2442) and incubated at 37°C with 5% CO 2 .

| Plasmids, shRNA and lentiviruses
Plasmids of pMEX-Myc-Ajuba and their truncations have been described previously. 8 The murine Twist cDNA was amplified from pBabe-Flag-Twist and digested with BamHI and XbaI, then inserted into the pcDNA3.1-N-Flag vector to create a Flag-tagged Twist fusion protein in the N terminus. The Twist truncation mutants were subcloned into pcDNA3.1-N-Flag vector by the PCR method. The sequence of N-cadherin gene promoter was obtained as described previously. 15 The first intron (+746-+3156 bp) from genomic DNA was amplified by PCR and subcloned into pGL3-basic luciferase reporter plasmids. The primer set is as follows: 5'-AAATTTG-  Forty-eight hours later, 2 μg/ml puromycin was added to the medium and the stable cells were selected.

| Quantitative real-time PCR (qRT-PCR) and reverse transcription PCR (RT-PCR)
Total RNA from cells was prepared using TRIzol reagent (Life Sciences) following the manufacturer's protocol. Complementary DNA was synthesized with 2 µg of total RNA using SuperScript Ⅳ RT Reagent Kit

| Chromatin immunoprecipitation
SW480 cells were stably transfected with the indicated plasmids and subjected to ChIP assays. The method has been described previously. 16 The chromatin was sonicated to fragments ranging from 500 to 800 bp in size. Immunoprecipitation was performed using the specific anti-human antibodies against

| Statistical analysis
Experiments were performed at least three times, and results were shown as mean ± SD, Statistical analysis was performed using Student's two-tailed exact t test, and P values less than 0.05 were considered statistically significant.

| The LIM region of Ajuba binds to Twist
To further examine the mechanism of Ajuba promoting tumour   Figure 2B, the Twist mutants N180 and N102 did not pull down Ajuba, whereas the Twist C105 displayed the similar binding capability to Ajuba as the full-length Twist did. To further consolidate that the Twist box motif is pivotal for the interplay, we mutated the three conservative bases of Twist box 18 ( Figure 2C). Next, we cotransfected Myc-Ajuba and Flag-Twist wild type (WT) or mutant (MT) into 293T cells, and Co-IP experiment was performed with Flag antibody.

| The Twist box region of Twist binds to Ajuba
Interestingly, the wild-type Twist, not the mutant one, pulled down Ajuba. The other way round, we carried out the Co-IP assays with Myc antibody. The results showed that Ajuba intensely bound the wild Twist, but weakly bound the mutated one ( Figure 2D&E). These findings suggested that the Twist box region was essential for the interaction between Ajuba and Twist.

| Ajuba and Twist synergistically bind to Twist target promoter region to transactivate N-cadherin expression
The finding described above prompted us to wonder whether Ajuba affects Twist-mediated transcriptional activity. Next, SW1116 and SW480 cells were stably depleted using lentiviral shRNA ( Figure 3A).
The total RNAs were extracted and reverse-transcribed for qRT-PCR and RT-PCR analyses. During cancer metastasis, Twist is considered as a crucial transcription factor in regulating the gene expression of N-cadherin. 13 Here, N-cadherin was selected to assess the transcrip-

| Ajuba bridges Twist and CBP to regulate target gene transcription
It was reported that Ajuba can enhance the interaction between p300/CBP and PPARγ to activate the latter target gene transcription. 9 Here, we questioned whether Twist, Ajuba and p300/ To further verify the assembling of Ajuba, Twist and CBP ternary complex at endogenous chromatins, the ChIP assay was carried out in SW480-shNC and SW480-shAjuba cells using Twist, Ajuba, CBP and H3-Ac antibodies respectively. We used the primer sets flanking the E-box site to measure precipitated DNA fragments by qPCR.
In SW480-shNC cells, Twist, Ajuba and CBP were identified to bind to Twist target promoter in different degrees, and acetylated histone maintained at the middle level represented by H3-Ac antibody ( Figure 4C). Surprisingly, loss of Ajuba triggered strikingly decreased binding of Twist and CBP at the target chromatin region accompanying remarkably decreased histone acetylation at the same locus ( Figure 4D). Taken together, all data show that Ajuba is indispensable for Twist to efficiently recruit p300/CBP to its target chromatin region. triggers defection of EMT in Myc-Cap cells. 17 Our results demonstrated that the mutation of the three conserved residues in the Twist box strikingly inhibits the binding to Ajuba, suggesting that Ajuba might be required for transactivation. Collectively, these results supplied new mechanism for Ajuba-and Twist-mediated metastasis regulation.

| D ISCUSS I ON
Mechanistically, the previous investigation showed that Ajuba can function as a coactivator for PPARγ and ERα by recruiting CBP/ P300. 9,22 In this paper, we further verified that Ajuba acted as a scaffold protein to recruit Twist and CBP to target chromatin and collaboratively activate Twist target gene expression.
In summary, our results not only enriched the research on the tumour metastasis mechanism of Twist and Ajuba, but suggested that Ajuba/Twist/CBP/p300 pathway may be a new target for therapeutics to cancer metastasis.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.