Transcription Factor Activating Enhancer Binding Research Articles

USP15, activated by TFAP4 transcriptionally, stabilizes SHC1 via deubiquitination and deteriorates renal cell carcinoma.

Ubiquitin-specific peptidase 15 (USP15), a critical deubiquitinating enzyme, has been demonstrated to improve substrate stabilization by hydrolyzing the bond between the substrate and ubiquitin, and is implicated in multiple carcinogenic processes. Prompted by the information cited from The Cancer Genome Atlas (TCGA) database and the Cancer Proteogenomic Data Analysis Site (cProSite), USP15 is selectively overexpressed in clear cell renal cell carcinoma (ccRCC) samples. We aimed to investigate the function of USP15 on ccRCC malignant features, which was emphasized in its deubiquitination of SHC adaptor protein 1 (SHC1). The overexpression of USP15 promoted the capacity of proliferation, migration, and invasion in ccRCC CAKI1 and 769-P cells, and these malignant biological properties were diminished by USP15 deletion in 786-O cells. USP15 accelerated tumor growth and lung metastasis invivo. In addition, deubiquitinase USP15 was further identified as a new protector for SHC1 from degradation by the ubiquitination pathway, the post-translational modification. In sequence, transcription factor activating enhancer binding protein 4 (TFAP4) was shown to be partly responsible for USP15 expression at the level of transcription, as manifested by the chromatin immunoprecipitation and pull-down assay. Based on the invitro and invivo data, we postulate that USP15 regulated by TFAP4 transcriptionally deteriorates ccRCC malignant biological properties via stabilizing SHC1 by deubiquitination.

Inhibitory effect of CC chemokine ligand 23 (CCL23)/ transcription factor activating enhancer binding protein 4 (TFAP4) on cell proliferation, invasion and angiogenesis in hepatocellular carcinoma

ABSTRACT Hepatocellular carcinoma (HCC) is a highly vascularized solid tumor with a fast growth rate. According to bioinformatics analysis, CC chemokine ligand 23 (CCL23) has clinical significance for survival and prognosis in HCC. The online databases TCGA and CCLE were used to analyze the expression level of CCL23, and its expression was also measured in HCC cell lines by RT-qPCR and Western blotting. The STRING database and co-immunoprecipitation were employed to evaluate the association between CCL23 and transcription factor activating enhancer binding protein 4 (TFAP4). Overexpression plasmids for CCL23 (Ov-CCL23) and TFAP4 (Ov-TFAP4) were transfected into Huh-7 cells to detect TFAP4 expression. Huh-7 cells injected with OV-negative control (NC)/Ov-CCL23 or OV-NC/Ov-CCL23 plus Ov-TFAP4 were utilized to study the function of CCL23/TFAP4. Cell proliferation, invasion and human umbilical vein endothelial cell tube formation assays were conducted. The database revealed decreased expression of CCL23 in HCC and that it was commonly downregulated in HCC cell lines. TFAP4 expression was negatively correlated with CCL23. The overexpression of CCL23 inhibited the proliferation and invasion of Huh-7 cells, whereas TFAP4 blocked these effects. Similarly, the supernatant of CCL23-upregulated cells exhibited significantly lower tube formation potential, and low vascular endothelial growth factor A (VEGFA), VEGFRs expression compared with those of non-transfected Huh-7 cells, while TFAP4 plasmid co-transfected markedly increased these. Taken together, the present study suggests that CCL23 is expressed at low levels in HCC; it inhibits HCC cell proliferation, invasion and angiogenesis in vitro; and its action is negatively associated with and can be blocked by TFAP4.

TFAP4 promotes the growth of prostate cancer cells by upregulating FOXK1.

Transcription factor activating enhancer binding protein 4 (TFAP4) has been indicated to be correlated with the progression of various human malignancies. However, the effect and regulatory mechanism of TFAP4 in prostate cancer (PC) remain unclear. The protein and mRNA expression were detected by western blotting and RT-qPCR. TFAP4 was overexpressed or knocked down in PC cells. The viability, invasion and migration of PC cells were analyzed by CCK-8, Transwell and wound healing assays. The colony formation was also determined. TFAP4 expression was upregulated in PC patients and cells; high TFAP4 expression predicted poor prognosis, and was associated with a range of clinicopathological features, including metastasis, clinical stage and Gleason score. Moreover, overexpression of TFAP4 promoted cell viability, migration, and invasion in vitro, whereas knockdown of TFAP4 revealed the opposite results. TFAP4 also positively regulated forkhead box K1 (FOXK1) expression. In addition, overexpression of FOXK1 reversed the effects of TFAP4 knockdown on PC cells. These findings clarified the biologic significance of TFAP4 in PC progression and revealed an association between TFAP4 and FOXK1, thus providing a new potential target for clinical therapy of PC.

Silencing of the Long Non-Coding RNA TTN-AS1 Attenuates the Malignant Progression of Osteosarcoma Cells by Regulating the miR-16-1-3p/TFAP4 Axis.

ObjectivesOsteosarcoma (OS) is a type of bone malignancy. This study attempted to explore the effect of long non-coding RNA TTN-AS1 (TTN-AS1) on OS and to determine its molecular mechanisms.MethodsThe expression of TTN-AS1, microRNA-16-1-3p (miR-16-1-3p), and transcription factor activating enhancer binding protein 4 (TFAP4) in OS was assessed using qRT-PCR. The OS cell proliferation, migration, and invasion were measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and transwell assays. N-cadherin and MMP-2 protein level was determined with western blot. Interactions between TTN-AS1 and miR-16-1-3p or TFAP4 and miR-16-1-3p were confirmed using the dual-luciferase reporter assay. Additionally, an OS xenograft tumor model was constructed to assess the effect of TTN-AS1 on tumor growth.ResultsTTN-AS1 and TFAP4 expression was increased in OS, while miR-16-1-3p expression was decreased. TTN-AS1 silencing restrained OS cell proliferation, migration, invasion, N-cadherin and MMP-2 protein expression, and hindered tumor growth. MiR-16-1-3p overexpression retarded the malignant behavior of OS cells. TTN-AS1 played a carcinostatic role by down-regulating miR-16-1-3p in the OS cells. Moreover, miR-16-1-3p inhibition or TFAP4 elevation weakened the suppressive effect of TTN-AS1 silencing on OS cell tumor progression.ConclusionTTN-AS1 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OS cells via mediating the miR-16-1-3p/TFAP4 axis. TTN-AS1 may be a critical target for improving OS.

TFAP2A-induced SLC2A1-AS1 promotes cancer cell proliferation.

Long non-coding RNAs (lncRNAs) are involved in the occurrence and development of human cancers including lung adenocarcinoma (LUAD). SLC2A1-AS1 is a novel lncRNA that has been reported to be exceptionally expressed in several cancer types. However, the expression and role of SLC2A1-AS1 in cancer remains largely unclear. In this study, it was revealed that lncRNA SLC2A1-AS1 was notably over-expressed in LUAD and was closely correlated with patients' overall survival (OS). Knockdown of SLC2A1-AS1 could significantly restrain cell proliferation of LUAD in vitro, while over-expression of SLC2A1-AS1 had the accelerative effect. SLC2A1-AS1 enriched in the cytoplasm of LUAD cells could directly bind to miR-508-5p and negatively regulate its level. The inhibitory effect of miR-508-5p on LUAD cell proliferation was in part abrogated by SLC2A1-AS1 manipulation. Moreover, the transcription factor activating enhancer binding protein 2 α (TFAP2A) was highly expressed in LUAD and predicted worse patients' OS. TFAP2A could directly bind to the promoter region of SLC2A1-AS1 encoding gene and positively regulate the transcription of SLC2A1-AS1 in LUAD cells. Furthermore, TFAP2A-induced SLC2A1-AS1 promoted cell proliferation of lung squamous cell carcinoma (LUSC) and pancreatic adenocarcinoma (PAAD). Collectively, these findings suggest that TFAP2A-mediated lncRNA SLC2A1-AS1 works as an oncogene to drive cancer cell proliferation.

The Expression of Pro-Platelet Basic Protein and Transcription Factor Activating Enhancer Binding Protein 4 Genes in Non-Small Cell Lung Cancer and Prognostic Value

To analyze the expression of pro-platelet basic protein and transcription factor activating enhancer binding protein 4 genes in non-small cell lung cancer and the prognostic value. 94 patients with non-small cell lung cancer undergoing lung cancer resection in our hospital from January 2015 to January 2016 were selected as study subjects and pro-platelet basic protein and transcription factor activating enhancer binding protein 4 messenger RNA expressions in cancer tissues and paracancerous tissues were detected by immunohistochemistry and other methods. The relationship between different levels of pro-platelet basic protein and transcription factor activating enhancer binding protein 4 messenger RNA expression levels and clinicopathological characteristics of patients was analyzed using Kaplan-Meier curves, used for survival analysis. Transcription factor activating enhancer binding protein 4 was lower in non-small cell lung cancer tissues than in paraneoplastic tissues (p<0.05) and the relative expression of pro-platelet basic protein was higher in all tissues than in paraneoplastic tissues (p<0.05). There were statistically significant differences in the maximum tumour diameter, tumor (T), nodes (N) and metastases (M) stage, proportion of lymph node metastasis and differentiation degree in the transcription factor activating enhancer binding protein 4 messenger RNA high expression group compared with the low expression group (p<0.05). The differences in maximum tumor diameter, tumor (T), nodes (N) and metastases (M) stage, proportion of lymph node metastasis and degree of differentiation were statistically significant in the pro-platelet basic protein messenger RNA high expression group compared with the low expression group (p<0.05). Survival analysis showed that the 5 y survival rate of the transcription factor activating enhancer binding protein 4 low expression group was higher than that of high expression group (p=0.028) and the 5 y survival rate of the pro-platelet basic protein high expression group was higher than that of low expression group (p=0.002). The expression of proplatelet basic protein and transcription factor activating enhancer binding protein 4 genes in cancer tissues of non-small cell lung cancer patients was significantly correlated with tumor (T), nodes (N) and metastases (M) stage, lymph node metastasis, maximum tumor diameter and differentiation degree, which could affect patients prognosis.

An integrated pan-cancer analysis of TFAP4 aberrations and the potential clinical implications for cancer immunity.

Studies have shown that transcription factor activating enhancer binding protein 4 (TFAP4) plays a vital role in multiple types of cancer; however, the TFAP4 expression profile is still unknown, as is its value within the human pan‐cancer analysis. The present study comprehensively analysed TFAP4 expression patterns from 33 types of malignancies, along with the significance of TFAP4 for prognosis prediction and cancer immunity. TFAP4 displayed inconsistent levels of gene expression across the diverse cancer cell lines, and displayed abnormal expression within most malignant tumours, which closely corresponded to overall survival. More importantly, the TFAP4 level was also significantly related to the degree of tumour infiltration. TFAP4 was correlated using gene markers in tumour‐infiltrating immune cells and immune scores. TFAP4 expression was correlated with tumour mutation burden and microsatellite instability in different cancer types, and enrichment analyses identified TFAP4‐associated terms and pathways. The present study comprehensively analysed the expression of TFAP4 across 33 distinct types of cancers, which revealed that TFAP4 may possibly play a vital role during cancer formation and development. TFAP4 is related to differing degrees of immune infiltration within cancers, which suggests the potential of TFAP4 as an immunotherapy target in cancers. Our study demonstrated that TFAP4 plays an important role in tumorigenesis as a prognostic biomarker, which highlights the possibility of developing new targeted treatments.

METTL3 contributes to renal ischemia-reperfusion injury by regulating Foxd1 methylation.

To investigate the mechanism of renal ischemia-reperfusion injury (IRI) via regulation of N6-methyl-adenosine (m6A) and relevant genes, IRI was induced in Sprague-Dawley rats, and urine and serum creatinine levels and tissue structure changes were observed. m6A and methyltransferase-like 3 (METTL3) protein levels were assessed via dot-blot and Western blot analyses, respectively. The hypoxia/reoxygenation (H/R) cell model was constructed using NRK-52E cells, and METTL3 protein levels were assessed. METTL3 was inhibited to observe its impact on NRK-52E cell apoptosis and m6A expression in H/R processes. Methylated RNA immunoprecipitation (MeRIP) sequencing was conducted followed by MeRIP-quantitative RT-PCR and quantitative RT-PCR validation. Our results indicated that urine and serum creatinine levels increased and that renal injury and cell apoptosis were both observed in the IRI model. In additon, m6A expression increased in the IRI model, and METTL3 protein levels significantly increased in the IRI and H/R models. When METTL3 was inhibited, m6A levels were accordingly decreased and cell apoptosis was suppressed in the H/R in vitro model. Based on MeRIP sequencing, transcription factor activating enhancer binding protein 2α (tfap2a), cytochrome P-450 1B1 (cyp1b1), and forkhead box D1 (foxd1) were significantly differentially expressed, as was m6A, which is involved in the negative regulation of cell proliferation and kidney development. We confirmed that foxd1 mRNA and its methylation levels contributed to IRI and H/R.

Dysregulated Transcription Factor TFAP2A After Peripheral Nerve Injury Modulated Schwann Cell Phenotype.

Transcription factors regulate the transcriptions and expressions of numerous target genes and direct a variety of physiological and pathological activities. To obtain a better understanding of the involvement of transcription factors during peripheral nerve repair and regeneration, significantly differentially expressed genes coding for transcription factors in rat sciatic nerves after sciatic nerve crush injury were identified. A total of 9 transcription factor genes, including GBX2, HIF3A, IRF8, LRRC63, SNAI3, SPIB, TBX21, TFAP2A, and ZBTB16 were identified to be commonly differentially expressed at 1, 4, 7, and 14 days after nerve injury. TFAP2A, a gene encoding transcription factor activating enhancer binding protein 2 alpha, was found to be critical in the regulatory network. PCR validation and immunohistochemistry staining of injured rat sciatic nerves showed that TFAP2A expression was significantly up-regulated in the Schwann cells after nerve injury for at least 2 weeks. Schwann cells transfected with TFAP2A-siRNA exhibited elevated proliferation rate and migration ability, suggesting that TFAP2A suppressed Schwann cell proliferation and migration. Collectively, our study provided a global overview of the dynamic changes of transcription factors after sciatic nerve injury, discovered key transcription factors for the regeneration process, and deepened the understanding of the molecular mechanisms underlying peripheral nerve repair and regeneration.

TFAP4 Promotes Hepatocellular Carcinoma Invasion and Metastasis via Activating the PI3K/AKT Signaling Pathway.

Transcription factor activating enhancer binding protein 4 (TFAP4) is established as a regulator of human cancer genesis and progression. Overexpression of TFAP4 indicates poor prognosis in various malignancies. The current study was performed to quantify TFAP4 expression as well as to further determine its potential prognostic value and functional role in patients with hepatocellular carcinoma (HCC). We identified that the expression of TFAP4 mRNA in 369 tumor tissues was higher than that in 160 normal liver tissues. Upregulated TFAP4 expressions were discovered in HCC cell lines compared to the healthy liver cell line, and similarly, the levels of TFAP4 were higher in tumor tissues than its expression in paratumor tissues. High mRNA and protein expression of TFAP4 was associated with worse overall survival (OS) and disease-free survival (DFS). Additionally, TFAP4 expression emerged as a risk factor independently affecting both OS and DFS of HCC patients. Functional studies demonstrated that TFAP4 increased HCC cell migration and invasion. Further investigations found that TFAP4 promotes invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) and regulating MMP-9 expression via activating the PI3K/AKT signaling pathway in HCC. In conclusion, our study demonstrated that TFAP4 is a valuable prognostic biomarker in determining the likelihood of tumor metastasis and recurrence, as well as the long-term survival rates of HCC patients. Exploring the regulatory mechanism of TFAP4 will also contribute to the development of new prevention and treatment strategies for HCC.

Overexpression of transcription factor activating enhancer binding protein 4 (TFAP4) predicts poor prognosis for colorectal cancer patients.

Transcription factor activating enhancer binding protein 4 (TFAP4) is an important regulator in the genesis and progression of human cancers. Overexpression of TFAP4 has been found to be correlated with several malignancies. The present study assessed the clinical importance of TFAP4 in colorectal cancer (CRC). First, immunohistochemistry was used to analyze TFAP4 expression and the association of TFAP4 expression with clinicopathological features on a tissue microarray containing 208 CRC patients. The results revealed that TFAP4 protein expression was significantly upregulated in CRC tissues compared with that in normal colon tissues (P<0.001). Of note, statistical analysis revealed that TFAP4 expression was significantly correlated with a high pathological grade (P=0.034), advanced clinical stage (P=0.024), enhanced tumor invasion (P=0.002) and lymph node metastasis (P=0.041). In addition, the Cancer Genome Atlas dataset further validated that TFAP4 mRNA levels were increased in CRC with advanced clinical stage (P=0.026), lymph node metastasis (P=0.018) and vascular invasion (P=0.046). Kaplan-Meier survival analysis demonstrated that CRC patients with high TFAP4 expression had shorter overall survival compared with those with low TFAP4 expression (P=0.011). Importantly, overexpression of TFAP4 was a valuable independent prognostic factor for CRC patients (hazard ratio, 8.200; 95% confidence interval, 1.838–36.591; P=0.006). In summary, TFAP4 may have an important role in CRC progression and upregulation of TFAP4 may be a predictor of poor prognosis for CRC patients.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4.

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.

Abstract 2450: MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis

Abstract Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4), which plays important roles in cancer progression, has been reported to be a direct transcriptional target of MYC. In this study, we have shown that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome and furthermore that siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells impaired migration and colony formation, and led to an increased proportion of cells in G1/S phase of the cell cycle. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that TFAP4 expression is positively regulated by MYCN through direct promoter binding. In addition, when MYCN was overexpressed in neuroblastoma cells, TFAP4 was required for the observed increase in cell migration. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study unveils a complex regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying relevant therapeutic targets for aggressive forms of this disease. Citation Format: Chengyuan Xue, Denise M. Yu, Samuele Gherardi, Jessica Koach, Giorgio Milazzo, Laura Gamble, Bing Liu, Amanda Russell, Tao Liu, Belamy B. Cheung, Glenn M. Marshall, Giovanni Perini, Michelle Haber, Murray D. Norris. MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2450.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

IntroductionThe transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e−/−) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e−/− mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice.MethodsMurine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e−/− mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels.ResultsOnly minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e−/− mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e−/− cartilage explants. OA progression was significantly enhanced in the Tfap2e−/− mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e−/− articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT.ConclusionsWe reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

Expression and function of transcription factor AP-2? in early embryonic development of porcine parthenotes.

Transcription factor AP-2? (TFAP2C) is a member of the transcription factor activating enhancer binding protein (AP) family. In the present study we determined the temporal and spatial expression patterns of TFAP2C in porcine parthenotes during preimplantation development. Porcine TFAP2C transcripts were expressed at all stages of preimplantation development, with highest expression at the 8-cell stage. In contrast with the mouse, TFAP2C protein was not restricted to the trophectoderm and was also detected in the ICM in blastocyst stage porcine parthenotes. In knockdown (KD) experiments, most TFAP2C-depleted embryos were arrested before the compacted 8-cell stage. This developmental failure is attributed to abnormal expression of genes involved in cell adhesion, tight junction biogenesis and cell proliferation. Interestingly, although the conserved region 4 (CR4) of the porcine OCT4 5? upstream regionlacked the AP2C-binding motif, OCT4 transcript levels were elevated in porcine TFAP2C-KD 8-cell embryos, suggesting TFAP2C may be involved in the regulation of OCT4 in porcine embryos through other mechanisms. In summary, the results suggest that TFAP2C is necessary for the transition from de novo transcript synthesis by activation to compaction and further development, and the different expression patterns of TFAP2C in porcine embryos may reflect species-specific functions during preimplantation embryo development.

Inhibition of neural crest formation by Kctd15 involves regulation of transcription factor AP-2

The neural crest develops in vertebrate embryos within a discrete domain at the neural plate boundary and eventually gives rise to a migrating population of cells that differentiate into a multitude of derivatives. We have shown that the broad-complex, tramtrack and bric a brac (BTB) domain-containing factor potassium channel tetramerization domain containing 15 (Kctd15) inhibits neural crest formation, and we proposed that its function is to delimit the neural crest domain. Here we report that Kctd15 is a highly effective inhibitor of transcription factor activating enhancer binding protein 2 (AP-2) in zebrafish embryos and in human cells; AP-2 is known to be critical for several steps of neural crest development. Kctd15 interacts with AP-2α but does not interfere with its nuclear localization or binding to cognate sites in the genome. Kctd15 binds specifically to the activation domain of AP-2α and efficiently inhibits transcriptional activation by a hybrid protein composed of the regulatory protein Gal4 DNA binding and AP-2α activation domains. Mutation of one proline residue in the activation domain to an alanine (P59A) yields a protein that is highly active but largely insensitive to Kctd15. These results indicate that Kctd15 acts in the embryo at least in part by specifically binding to the activation domain of AP-2α, thereby blocking the function of this critical factor in the neural crest induction hierarchy.

Genetic Polymorphisms of the Main Transcription Factors for Adiponectin Gene Promoter in Regulation of Adiponectin Levels: Association Analysis in Three European Cohorts

Adiponectin serum concentrations are an important biomarker in cardiovascular epidemiology with heritability etimates of 30–70%. However, known genetic variants in the adiponectin gene locus (ADIPOQ) account for only 2%–8% of its variance. As transcription factors are thought to play an under-acknowledged role in carrying functional variants, we hypothesized that genetic polymorphisms in genes coding for the main transcription factors for the ADIPOQ promoter influence adiponectin levels. Single nucleotide polymorphisms (SNPs) at these genes were selected based on the haplotype block structure and previously published evidence to be associated with adiponectin levels. We performed association analyses of the 24 selected SNPs at forkhead box O1 (FOXO1), sterol-regulatory-element-binding transcription factor 1 (SREBF1), sirtuin 1 (SIRT1), peroxisome-proliferator-activated receptor gamma (PPARG) and transcription factor activating enhancer binding protein 2 beta (TFAP2B) gene loci with adiponectin levels in three different European cohorts: SAPHIR (n = 1742), KORA F3 (n = 1636) and CoLaus (n = 5355). In each study population, the association of SNPs with adiponectin levels on log-scale was tested using linear regression adjusted for age, sex and body mass index, applying both an additive and a recessive genetic model. A pooled effect size was obtained by meta-analysis assuming a fixed effects model. We applied a significance threshold of 0.0033 accounting for the multiple testing situation. A significant association was only found for variants within SREBF1 applying an additive genetic model (smallest p-value for rs1889018 on log(adiponectin) = 0.002, β on original scale = −0.217 µg/ml), explaining ∼0.4% of variation of adiponectin levels. Recessive genetic models or haplotype analyses of the FOXO1, SREBF1, SIRT1, TFAPB2B genes or sex-stratified analyses did not reveal additional information on the regulation of adiponectin levels. The role of genetic variations at the SREBF1 gene in regulating adiponectin needs further investigation by functional studies.

Intronic Polymorphisms within TFAP2B Regulate Transcriptional Activity and Affect Adipocytokine Gene Expression in Differentiated Adipocytes

We have identified a gene encoding transcription factor activating enhancer binding protein-2beta (TFAP2B) as a candidate for conferring susceptibility to type 2 diabetes. Although we have also found that TFAP2B was preferentially expressed in adipose cells in a differentiation-dependent manner, the mechanisms by which the gene and gene polymorphisms contribute to conferring susceptibility to the disease have not yet been elucidated. The aim of this study was to evaluate the impact of the polymorphisms within the TFAP2B gene on conferring susceptibility to type 2 diabetes. We identified that a 300-bp DNA fragment in intron 1 of TFAP2B had significant enhancer activity, and the variations of this region affected this enhancer activity in differentiated adipocytes. In an experiment using adenovirus vectors encoding TFAP2B, the expression of TNF-alpha gene was shown to be elevated in the TFAP2B overexpressing cells compared with those in control cells. Furthermore, we demonstrated that the expression of TFAP2B was increased in the adipose tissues of subjects with the disease-susceptibility allele, and the plasma levels of TNF-alpha and high sensitivity C-reactive peptide were significantly elevated in the patients with the disease-susceptibility allele. These results suggest that TFAP2B may contribute to the pathogenesis of type 2 diabetes through regulation of adipocytokine gene expression, and that TFAP2B may be a promising target for treatment or prevention of this disease.