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IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion

Mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFβ3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfβ3-induced fusion defect. These findings indicate that IRF6 is involved in TGFβ3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFβ3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfβ3-induced fusion defect. These findings indicate that TGFβ3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFβ3, which regulates EMT and fusion process during the embryonic palate development.

Cleft lip and palate are common congenital craniofacial disorders that occur once in every 600 new births 34,35 . Orofacial cleft can be categorized into syndromic or non-syndromic cleft according to the presence or absence of associated anomalies. Van der Woude syndrome (VWS) is the most common form of syndromic cleft and is an autosomal dominant disorder. Mutations in the interferon regulatory factor 6 (IRF6) gene lead to VWS [36][37][38] . In addition to VWS, IRF6 mutations are also known to cause popliteal pterygium syndrome (PPS) and non-syndromic cleft lip/palate 37,[39][40][41] . IRF6 is a transcription factor that regulates cell proliferation, cell cycle, periderm formation, and keratinocyte differentiation [42][43][44][45] . Irf6 null and Irf6 R84C mutant mice have abnormal skin, limb, and craniofacial development 46,47 . In addition, Irf6 clft1 mutant mice, with an ENU-induced P39L mutation in Irf6, show abnormal adhesion between the palate and tongue resulting in cleft palate 48 . Recently, an interaction between TGFβ signaling and IRF6 activity has been reported. TGFβ increases Irf6 expression through both SMAD-dependent pathway and p38 MAPK pathway; during the palatal fusion this effect regulates MEE apoptosis through IRF6/Δ Np63/ p21 signaling cascade 16 . These studies suggest that IRF6 is important to MEE apoptosis and palate development. In addition to apoptosis, IRF6 regulates EMT and cellular migration. It was reported that IRF6 regulated N-cadherin, an EMT related gene, in human breast cancer cells 49 . Loss of Irf6 in mouse embryonic keratinocytes leads to a delay in cellular migration and wound healing via RhoA pathway 50 . These findings suggest that IRF6 may regulate EMT and cellular migration. However, whether IRF6 is involved in TGFβ 3-regulated EMT during palatal fusion remains poorly understood.
Irf6-null and Irf6-mutant homozygous embryos showed a phenotype involving intraoral adhesions that inhibited shelf elevation and eventually resulted in cleft palate. However, it is not known whether and how IRF6 is involved in palatal fusion. In this study, we investigate the role of IRF6 in TGFβ 3-regulated palatal fusion using palatal shelves organ culture, and find that IRF6 regulates EMT during palatal fusion via SNAI2.

Results
Knockdown of Irf6 delays TGFβ3 mediated palatal fusion. To determine whether Irf6 contributes to the TGFβ 3 regulated EMT pathway during palatal fusion, we first examined whether Irf6 knockdown affects palatal fusion in the organ culture system. To set up virus-mediated gene knockdown in mouse palatal shelves organ culture, a GFP reporter lentivirus was used to assess lentivirus infection efficiency in palatal shelves organ culture. Palatal shelves were infected with GFP reporter lentivirus for different time intervals, then changed to fresh media and cultured for a total of 48 hours. In palate pairs infected for 12 hours, weak GFP staining was detected in 66% of palatal epithelium cells. In palate pairs exposed to the virus for 18 hours, expression of GFP was detected in 100% palatal epithelium and 65% mesenchymal cells. In palate pairs infected for 24 hours, strong GFP was detected in 100% of the palatal epithelium cells and 100% of the mesenchymal cells ( Supplementary Fig. S1). The optimal lentivirus concentration for infection of palate organ cultures was evaluated. The results showed that infection with 3.3 × 10 6 R.I.U./ml lentivirus for 24 hours, followed by incubation for another 24 hours, resulted in the best GFP expression during palatal shelves tissue culture. Thus, the data show that the lentivirus vector can efficiently infect palatal shelves in vitro. Five mouse Irf6 shRNAs were introduced into cultured palatal shelves to knockdown Irf6 expression. Immunohistochemistry staining and Western blotting indicated that mouse Irf6 shRNA clone TRCN0000085329 had the best efficiency in terms of Irf6 knockdown in the culture ( Supplementary Fig. S2a, S3). The timing of Irf6 mRNA inhibition was determined. Irf6 mRNA level in palatal shelves was analyzed at 6, 12, and 18 hours by quantitative RT-PCR after lentivirus infection (Fig. 1). In the palatal shelves exposed for 6 hours, 58% of Irf6 mRNA expression Palatal shelves were dissected from E13.5 C57BL/6 mouse embryos and cultured for 3 hours. The palatal shelves were then infected with lentivirus, adenovirus, or treated with 20 ng/ml TGFβ 3 for indicated time interval. Then the palatal shelves were harvested for RNA extraction or fixed with 4% PFA or had a change of medium and then were cultured for another 6, 12, 18, or 24 hours, after which they were fixed with 4% PFA. was blocked. The expression level of Irf6 was reduced to 14% at 12 hours and 8% at 18 hours of virus infection ( Supplementary Fig. S2b). The results indicated that expression of shIrf6 started within 6 hours after infection. IRF6 was expressed in the cytoplasm of epithelium, including MEE cells, but not in the mesenchyme of the non-infected or shLuc lentivirus infected palatal shelves ( Supplementary Fig. S2c). IRF6 expression in epithelial and MEE cells was diminished by 93% in 24 hours shIrf6 lentivirus infected palatal shelves ( Supplementary Fig. S2c, d). However, shIrf6 lentivirus infection did not affect the protein expression level of the basal epithelial marker p63, the periderm cell marker K17, or the proliferation marker Ki67 (Supplementary Fig. S4). The results indicate that knockdown of Irf6 has no effect on the cell differentiation and proliferation of the palatal shelves. Culture of non-infected palate pairs for 48 hours led to complete fusion as marked by mesenchymal confluence (Supplementary Fig. S2c). At the end of 24 hours of control virus infection (24 hr), a two cell layer seam of epithelial cells was present in the midline of palate; this was also true for the non-infected controls. However, after the system was cultured for another 24 hours (a total of 48 hr), the MEE cells almost disappeared and the two palatal shelves completely fused together ( Supplementary Fig. S2c). These results show that lentivirus infection itself does not affect fusion between the palatal shelves.
We then examined if palatal fusion is affected by Irf6 knockdown. As shown in Supplementary Fig.  S5, palatal shelves infected with shLuc lentivirus and cultured for 30 hours formed a single cell layer seam and showed partial mesenchymal confluence (20%) in certain regions. When cultured for 36 hours, 66% mesenchymal confluence was detected. Furthermore, 86% and 94% mesenchymal confluence were detected after 42 hours and 48 hours culture respectively. In contrast, Irf6 knockdown palates failed to form a single cell layer seam until they had been cultured for 36 hours, which is 6 to 12 hours later than the control palates. The Irf6 knockdown palates reached 72% mesenchymal confluence at 42 hours culture (n = 4) ( Supplementary Fig. S5). Therefore, it appears that blocking of IRF6 expression is able to delay the fusion of cultured palatal shelves.
We tested if IRF6 expression is required in the TGFβ 3 induced palatal fusion. As in the shLuc controls, TGFβ 3 treatment significantly increased the expression of IRF6 protein in epithelial and MEE cells (Fig. 2). Consistent with other studies, TGFβ 3 treatment enhanced the disappearance of MEE cells and promoted palatal fusion at both anterior or posterior parts (Fig. 2a, Table 1) 32 . Interestingly, the TGFβ 3 induced palatal fusion process was delayed at least 24 hours when Irf6 was knocked down (Fig. 2a, Table 1). To investigate whether IRF6 is involved in the TGFβ 3-regulated EMT pathways, we examined the expression of SNAI2 and TWIST, two important TGFβ 3 regulated EMT regulators. Immunofluorescence staining revealed that SNAI2 and TWIST were expressed in the nuclei of epithelial cells of palatal shelves. Knockdown of Irf6 blocked 76% of SNAI2 expression but did not affect TWIST expression ( Fig. 2b and supplementary Fig. S6). TGFβ 3 treatment increased SNAI2 and TWIST expression within the epithelium in the oral, nasal, and medial edge of palatal shelves, however, induction of SNAI2 was blocked by Irf6 knockdown. TGFβ 3 diminished the expression of epithelial markers, such as ZO-1 and Plakophilin. Expression of these markers was restored by Irf6 knockdown (Fig. 2 and supplementary Fig. S6). Western blotting showed similar results (Fig. 3a). Additionally, Snai2 mRNA was significantly decreased by Irf6 knockdown (Fig. 3b). These findings suggest that IRF6 may regulate the expression of Snai2, which in turn regulates EMT during palatal fusion.
In addition to increasing IRF6 expression, TGFβ 3 treatment resulted in nuclear accumulation of IRF6 (Fig. 4a). In TGFβ 3 treated MEE cells, the number of nuclear IRF6 positive cells was found to be six-fold higher than that of the MEE cells in the controls (16.9 ± 1.5% vs. 2.7 ± 0.9% ) (Fig. 4b). These findings suggest that TGFβ 3 regulates IRF6 translocation into nucleus, which affects expression of Snai2 and other downstream genes.

IRF6 regulates various EMT markers and palatal fusion.
To further investigate the importance of IRF6 in TGFβ 3-regulated palatal fusion, the palatal shelves were infected with adenovirus carrying IRF6-expressing cDNA (AdIRF6), and assessed whether ectopic expression of IRF6 was able to rescue shTgfβ 3 blocked palatal fusion. Palatal shelves infected with control adenovirus (AdIE) were found to fuse completely after they had been cultured for 48 hours, and this was also true for palatal shelves infected with adenovirus carrying IRF6 (AdIRF6). It could be clearly seen that the mesenchyme was confluent and the MEE cells disappeared in the midline with no epithelial triangle (Fig. 5a). At 24 hours after infection, MEE cells were still found in the control palatal shelves, whereas a partial disappearance of the MEE cells was observed in the AdIRF6 infected palatal shelves (n = 6) (Fig. 5a). These findings indicate that ectopic expression of IRF6 could promote palatal fusion. Furthermore, ectopic expression of IRF6 increased SNAI2 but not TWIST expression in the epithelium and MEE cells of palatal shelves (Fig. 5a,c). In addition, overexpression of IRF6 decreased the expression of Plakophilin and ZO-1 in the epithelial cells of palatal shelves (Fig. 5b). The findings indicate that IRF6 regulates SNAI2 expression and that this regulation affects EMT process during palatal fusion.

Discussion
In addition to VWS and PPS, mutations in IRF6 have been associated with non-syndromic cleft lip with or without cleft palate. Both Irf6 null and Irf6 mutant mice show the phenotype of cleft palate, indicating that IRF6 is important for palate development [46][47][48] . Although IRF6 is known to be a transcription factor, the downstream target genes and the signal pathways that regulate palatal fusion are not well understood. In this study, using palatal shelves organ culture, we showed that ectopic expression of IRF6 enhanced palatal fusion and rescued the fusion defect induced by shTgfβ 3. In addition, knockdown of Irf6 expression delayed palatal fusion for 12 hours, which in turn delayed TGFβ 3-mediated palatal fusion. These results indicate that IRF6 is important during palatal fusion and IRF6 is a mediator of TGFβ 3 during the regulation of palatal fusion. These findings agree with the results obtained using Tgfbr2 fl/fl ;K14-Cre;Irf6 Tg mice 16 . These findings showed that over-expression of IRF6 partially rescued palatal fusion in Tgfbr2 fl/fl ; K14-Cre mice. We have shown for the first time that IRF6 regulates the EMT regulator SNAI2 and that this can bring about a reduction in the expression of various epithelial markers, namely ZO-1, Plakophilin and E-cadherin. Snai2 knockdown delayed the palatal fusion and was able to block the rescue effect that IRF6 had on the shTgfβ 3-induced fusion defect. Therefore, Snai2 is a downstream target of IRF6 that is involved in the regulation of EMT, and IRF6 is involved in the EMT during palatal fusion. A previous study showed that Irf6 siRNA knockdown resulted in downregulation of Irf6 and Cdkn1a (P21) gene expression and upregulation of Trp63(P63) gene expression in organ culture. Overexpression of IRF6 rescued p21 expression and MEE disappearance in Tgfbr2 fl/fl ;K14-Cre mice. These data suggest that IRF6 regulates MEE apoptosis via the Δ Np63/p21 signaling cascade during the palatal fusion 16 . Taken together, IRF6 appears to be involved in both EMT and apoptosis during palatal fusion process (Fig. 7). These findings imply that loss function of IRF6 results in a delay in palatal shelves fusion. The delay subsequently causes separation of the shelves as the face continues to develop; this results in cleft palate in vivo.
EMT is an important process associated with the disappearance of MEE cells during palatal fusion. TGFβ 3 regulates several EMT related transcription factors, such as SNAI1/2, TWIST, and LEF1 12,27,51 . It  has been found that down regulation of Twist or Snai1 expression in vitro using siRNA results in delayed palatal fusion 27,52 . Here we demonstrated that Irf6 knockdown is able to diminish SNAI2 expression in the epithelial cells. In addition, both Snai2 knockdown and Irf6 knockdown delay palatal fusion (Figs 2a,  6b), but do not prevent eventual palatal fusion in organ culture. This suggests SNAI2 is not the only factor regulating EMT, and explains why Irf6 knockdown delays TGFβ 3-mediated palatal fusion but not prevents the eventual palatal fusion in organ culture (Fig. 2a). Our observation that TGFβ 3 increases TWIST expression when Irf6 is knockdown may result in compensation for the loss of SNAI2 and subsequently induction of EMT. Conversely, knockdown of TGFβ 3 was found to block both SNAI2 and TWIST expression, and re-expression of IRF6 only rescued the expression of SNAI2 (Fig. 6a,b). Ectopic expression of IRF6 was able to rescue shTgfβ 3-blocked palatal fusion. This rescue effect was then blocked by the Snai2 knockdown. These findings support the idea that SNAI2 is important to IRF6 regulated palatal fusion. Specifically, it may either compensate the TWIST function, or cooperate with other EMT related transcription factors in the regulation of EMT during palatal fusion.
During this investigation, we demonstrated that R84C mutant lost SNAI2 induction activity (Fig. 6a). This finding indicates that DNA binding ability is important for IRF6 in regulation of Snai2 mRNA    expression. In normal human keratinocytes, one IRF6 binding peak was identified in the promoter region of SNAI2 gene by ChIP-seq analysis 43 . By promoter sequence analysis, several IRF6 binding consensus sequences have been found in the proximal promoter region of murine Snai2. Whether IRF6 directly regulates the expression of Snai2 in mice needs further investigation.
As a transcription factor, nuclear localization is essential for transcriptional activation. Similar to other IRF members, IRF6 is predominantly present in the cytoplasm 44,53 . Recently, receptor-interacting protein kinase 4 (RIPK4) was reported to phosphorylate IRF6; this triggered trans-activator activity and induced nuclear translocation 54 . We have shown for the first time that TGFβ 3 promotes IRF6 nuclear accumulation. It is possible that TGFβ 3 regulates IRF6 in a manner that allows an association with one or more nuclear proteins and results in nuclear retention. Alternatively, TGFβ 3 may induce IRF6 phosphorylation and lead to nuclear translocation. In addition to SMAD-dependent pathway, TGFβ 3 activates various SMAD-independent pathways, such as ERK, p38 MAPK, and MEK1/2 pathways 15 . Further study is required to investigate whether these pathways are involved in the regulation of IRF6 phosphorylation and nuclear localization.
IRF6 is a transcription factor that contains a highly conserved helix-turn-helix DNA-binding domain and a less well conserved protein-binding domain. The missense mutations associated with VWS are evenly located in both the DNA-binding and protein-binding domains 37 . Most DNA-binding domain mutations lose their DNA binding ability 42 . However, there is little information in the literature on how mutations in IRF6 express functional aberrance during palatal development. Using adenovirus mediated overexpression and lentivirus based gene knockdown in palatal organ culture system, it is possible to elucidate the molecular mechanisms of IRF6 and determine how the disease causing mutation affect IRF6 function. TGFβ 3 upregulates the expression of IRF6 and enhances its nuclear translocation, which then seems to alter the expression of SNAI2. This change in SNAI2 expression represses Plakophilin and ZO-1 expression and induces the EMT, which is essential to the process of palatal fusion. In addition, IRF6 has been reported to regulate Δ Np63 protein degradation, which will result in an induction of p21 expression and MEE apoptosis; this is also crucial to the palatal fusion process.
Scientific RepoRts | 5:12791 | DOi: 10.1038/srep12791 Methods Lentivirus production. Lentivirus was produced according the protocol provided by the National RNAi Core Facility 55 . Briefly, lentiviral production was carried out by transfecting HEK293T cells with pLKO.1-shRNA plasmid and two packaging plasmids: pCMV-∆R8.91 and pMD.G. The virus supernatant was collected at 24 hrs and 36 hrs after transfection, and filtered through a 0.22 μ m filter (Merck Millipore). For lentivirus titration, HEK293T cells were infected for 24 hours with lentivirus after serial dilution and subsequently selected with 2 μ g/ml puromycin (Sigma Aldrich) for 3 days. Plasmids pCMV-∆R8.91, pMD.G, pPGK-GFP, and the shRNA expression plasmid (pLKO.1) were obtained from the National RNAi Core Facility, Taiwan. The shRNA clone used to knockdown mouse Tgfβ 3 was TRCN0000066147. The shRNA clone used to knockdown mouse Snai2 was TRCN0000319552. The five shRNA clones for mouse Irf6 were TRCN0000085328, TRCN0000085329, TRCN0000085330, TRCN0000085331, and TRCN0000085332.
Adenovirus production. Adenovirus production followed the AdEasy protocol 56 . Briefly, human IRF6 cDNA from pCMV-SPORT6-IRF6 plasmid was cloned into pAdTrack-CMV vector (Agilent Technologies) at Xho I and Xba I restriction enzyme sites. The AdIRF6 adenovirus expression plasmid was generated from recombination between pAdEasy-1 vector and pAdTrack-CMV-IRF6 in BJ5183 competent cells (Agilent Technologies). AdIRF6 plasmid (40 μ g) was linearized with Pac I restriction enzyme and subsequently transfected into AD293 cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol for adenovirus packaging. Adenoviruses were harvested at 14 to 20 days after transfection.

Organ culture and virus infection.
Palatal shelves were cultured using a submerged system as previous described 10,32 . Briefly, palatal shelves were dissected from E13.5 C57BL/6 mouse embryos using microscissors, placed on a 0.8 μ m pore size filter (Merck Millipore) in 35 mm culture dish, and cultured in 0.5 ml serum-free DMEM/F12 (Gibco) supplemented with 100 U/ml penicillin-streptomycin (Gibco), 2 mM L-glutamine (Gibco), and 0.1 mM non-essential amino acid (Biological Industries) in a 37 o C incubator with 5% CO 2 for 3 hours to let palatal shelves attach to the filters. After 3 hours incubation, the filters with palatal shelves attached were transferred to a 48-well culture plate and incubated with 20 ng/ml TGFβ 3 (R&D system), 3.3 × 10 6 Relative Infection Unit/ml (R.I.U./ml) lentivirus containing 8 μ g/ml polybrene (Sigma Aldrich), or 5.4 × 10 7 R.I.U./ml adenovirus for indicated time period (Fig. 1). Subsequently, the palatal shelves were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) in phosphate buffered saline (PBS) or had their medium changed and then they were cultured for another 24 hours. Fixed tissues were processed in STP 120 tissue processor (MICROM) and subjected to sectioning. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of National Yang-Ming University.
Histological examination and scoring of palatal fusion. Serial sections (5 μ m) were collected and numbered in sequence from the anterior to the posterior. The degree of fusion (DOF) was individually analyzed for the anterior, middle, and posterior sections of each sample. The degree of fusion (%) for each section was calculated as the length of mesenchymal confluence/total length of adherence × 100%.