The TEAD transcription factors are the final effectors of the Hippo pathway, and to exert their transcriptional activity they need to interact with other proteins. The three paralogous vestigial-like proteins VGLL1, VGLL2 and VGLL3 bind to TEAD via a conserved short linear sequence, the Tondu motif. The TEAD-binding domain of human VGLL2 contains in addition an Ω-loop, which is also present in Vg (vestigial) from arthropods and the YAP proteins, another family of TEAD interactors. In this report, using the available structural data, we study the amino acid sequence of the TEAD-binding domain of more than 2400 putative VGLL proteins from vertebrates. This analysis shows a strong link between sequence conservation and functional role for the residues from the Tondu motif. It also reveals that one protein sequence containing both a Tondu motif and an Ω-loop is present in most (if not all) vertebrate species. This suggests that there is a selective pressure to keep a VGLL paralog with a functional Ω-loop in vertebrates. Finally, this study identifies, particularly in mammals, variants of VGLL2 and VGLL3 with an altered TEAD-binding domain suggesting that they may have a different biological function than their homologs.

BACKGROUND: Jaceosidin (JA) is a natural flavone extracted from Artemisia that is used as a food and traditional medicinal herb. It has been reported to possess numerous biological activities. However, the regulatory mechanisms underlying amelioration of hepatic fibrosis remain unclear.
OBJECTIVE: We hypothesized that jaceosidin acid (JA) modulates hepatic fibrosis and inflammation.
METHODS: Thioacetamide (TAA) was used to establish an HF mouse model. In vitro, mouse primary hepatocytes and HSC-T6 cells were induced by TGF-β, whereas mouse peritoneal macrophages received a treatment lipopolysaccharide (LPS)/ATP.
RESULTS: JA decreased serum transaminase levels and improved hepatic histological pathology in TAA-treated mice stimulated by TAA. Moreover, the expression of pro-fibrogenic biomarkers associated with the activation of liver stellate cells was downregulated by JA. Likewise, JA down-regulated the expression of vestigial-like family member 3 (VGLL3), high mobility group protein B1 (HMGB1), toll-like receptors 4 (TLR4), and nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3), thereby inhibiting the inflammatory response and inhibiting the release of mature-IL-1β in TAA-stimulated mice. Additionally, JA suppressed HMGB1 release and NLRP3/ASC inflammasome activation in LPS/ATP-stimulated murine peritoneal macrophages. JA decreases the expression of pro-fibrogenic biomarkers related to liver stellate cell activation and inhibits inflammasome activation in mouse primary hepatocytes. It also down-regulated α-SMA and VGLL3 expressions and also suppressed inflammasome activation in HSC-T6 cells. VGLL3 and α-SMA expression levels were decreased in TGF-β-stimulated HSC-T6 cells following Vgll3 knockdown. In addition, the expression levels of NLRP3 and cleaved-caspase-1 were decreased in Vgll3-silenced HSC-T6 cells. JA enhanced the inhibitory effects on Vgll3-silenced HSC-T6 cells. Finally, Vgll3 overexpression in HSC-T6 cells affected the expression levels of α-SMA, NLRP3, and cleaved-caspase-1.
CONCLUSIONS: JA effectively modulates hepatic fibrosis by suppressing fibrogenesis and inflammation via the VGLL3/HMGB1/TLR4 axis. Therefore, JA may be a candidate therapeutic agent for the management of hepatic fibrosis. Understanding the mechanism of action of JA is a novel approach to hepatic fibrosis therapy.

UNASSIGNED: High-grade serous ovarian carcinoma (HGSOC) is the most common histologic type of epithelial ovarian cancer (EOC). Due to its poor survival outcomes, it is essential to identify novel biomarkers and therapeutic targets. The hippo pathway is crucial in various cancers, including gynaecological cancers. Herein, we examined the expression of the key genes of the hippo pathway and their relationship with clinicopathological significance, immune cells infiltration and the prognosis of HGSOC.
UNASSIGNED: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data were curated to analyse the mRNA expression as well as the clinicopathological association and correlation with immune cell infiltration in HGSOC. The protein levels of significant genes in the HGSOC tissue were analysed using Tissue Microarray (TMA)-based immunohistochemistry. Finally, DEGs pathway analysis was performed to find the signalling pathways associated with VGLL3.
UNASSIGNED: VGLL3 mRNA expression was significantly correlated with both advanced tumor stage and poor overall survival (OS) (p=0.046 and p=0.003, respectively). The result of IHC analysis also supported the association of VGLL3 protein with poor OS. Further, VGLL3 expression was significantly associated with tumor infiltrating macrophages. VGLL3 expression and macrophages infiltration were both found to be independent prognostic factors (p=0.003 and p=0.024, respectively) for HGSOC. VGLL3 was associated with four known and three novel cancer-related signalling pathways, thus implying that VGLL3 is involved in the deregulation of many genes and pathways.
UNASSIGNED: Our study revealed that VGLL3 may play a distinct role in clinical outcomes and immune cell infiltration in patients with HGSOC and that it could potentially be a prognostic marker of EOC.

In Atlantic salmon, age at maturation is a life history trait governed by a sex-specific trade-off between reproductive success and survival. Following environmental changes across large areas of the Northeast Atlantic, many populations currently display smaller size at age and higher age at maturation. However, whether these changes reflect rapid evolution or plasticity is unknown. Approximately 1500 historical and contemporary salmon from the river Etne in Western Norway, genotyped at 50,000 SNPs, revealed three loci associated with age at maturation. These included vgll3 and six6 which collectively explained 36%-50% of the age at maturation variation in the 1983-1984 period. These two loci also displayed sex-specific epistasis, as the effect of six6 was only detected in males bearing two copies of the late maturation allele for vgll3. Strikingly, despite allelic frequencies at vgll3 remaining unchanged, the combined influence of these genes was nearly absent in all samples from 2013 to 2016, and genome-wide heritability strongly declined between the two time-points. The difference in age at maturation between males and females was upheld in the population despite the loss of effect from the candidate loci, which strongly points towards additional causative mechanisms resolving the sexual conflict. Finally, because admixture with farmed escaped salmon was excluded as the origin of the observed disconnection between gene(s) and maturation age, we conclude that the environmental changes observed in the North Atlantic during the past decades have led to bypassing of the influence of vgll3 and six6 on maturation through growth-driven plasticity.

Age at maturity is a key life history trait and a significant contributor to life history strategy variation. The maturation process is influenced by genetic and environmental factors, but specific causes of variation in maturation timing remain elusive. In many species, the increase in the regulatory gonadotropin-releasing hormone 1 (GnRH1) marks the onset of puberty. Atlantic salmon, however, lacks the gnrh1 gene, suggesting gnrh3 and/or other regulatory factors are involved in the maturation process. Earlier research in Atlantic salmon has found a strong association between alternative alleles of vgll3 and maturation timing. Recently we reported strong induction of gonadotropin genes (fshb and lhb) in the pituitary of Atlantic salmon homozygous for the early maturation allele (E) of vgll3. The induction of gonadotropins was accompanied by increased expression of their direct upstream regulators, c-jun and sf1 (nr5a1b) but the regulatory connection between vgll3 and these regulators has never been investigated in any organism. In this study, we investigated the potential regulatory connection between vgll3 genotypes and these regulators through a stepwise approach of identifying a gene regulatory network (GRN) containing c-jun and sf1, and transcription factor motif enrichment analysis. We found a GRN containing c-jun with predicted upstream regulators, e2f1, egr1, foxj1 and klf4, to be differentially expressed in the pituitary. Finally, we suggest a vgll3 and Hippo pathway -dependent model for transcriptional regulation of c-jun and sf1 in the pituitary, which may have broader implications across vertebrates.

Understanding the potential of natural populations to adapt to altered environments is becoming increasingly relevant in evolutionary research. Currently, our understanding of adaptation to human alteration of the environment is hampered by lack of knowledge on the genetic basis of traits, lack of time series, and little or no information on changes in optimal trait values. Here, we used time series data spanning nearly a century to investigate how the body mass of Atlantic salmon (Salmo salar) adapts to river regulation. We found that the change in body mass followed the change in waterflow, both decreasing to ∼1/3 of their original values. Allele frequency changes at two loci in the regions of vgll3 and six6 predicted more than 80% of the observed body mass reduction. Modeling the adaptive dynamics revealed that the population mean lagged behind its optimum before catching up approximately six salmon generations after the initial waterflow reduction. Our results demonstrate rapid adaptation mediated by large-effect loci and provide insight into the temporal dynamics of evolutionary rescue following human disturbance.

One of the most well-known life-history continuums is the fast-slow axis, where \'fast\' individuals mature earlier than \'slow\' individuals. \'Fast\' individuals are predicted to be more active than \'slow\' individuals because high activity is required to maintain a fast life-history strategy. Recent meta-analyses revealed mixed evidence for such integration. Here, we test whether known life-history genotypes differ in activity expression by using Atlantic salmon (Salmo salar) as a model. In salmon, variation in Vgll3, a transcription cofactor, explains approximately 40% of variation in maturation timing. We predicted that the allele related to early maturation (vgll3*E) would be associated with higher activity. We used an automated surveillance system to follow approximately 1900 juveniles including both migrants and non-migrants (i.e. smolt and parr fish, respectively) in semi-natural conditions over 31 days (approx. 580 000 activity measurements). In migrants, but not in non-migrants, vgll3 explained variation in activity according to our prediction in a sex-dependent manner. Specifically, in females the vgll3*E allele was related to increasing activity, whereas in males the vgll3*L allele (later maturation allele) was related to increasing activity. These sex-dependent effects might be a mechanism maintaining within-population genetic life-history variation.

The mammalian Vestigial-like (VGLL) transcriptional cofactor family of proteins VGLL1-4 has recently emerged as an important player in the tumorigenesis of diverse neoplasms. The role of VGLL3 in soft tissue tumors is exemplified by its amplification in myxoinflammatory fibroblastic sarcoma and its rearrangement (fused to CHD7, CHD9, or MAMLD1) in hybrid schwannoma-perineurioma. This study characterizes a distinctive low-grade myogenic neoplasm with a striking predilection for the head and neck, characterized by VGLL3 fusions. The study includes five males and one female patient, aged 30-71 years (median, 56). Three tumors originated in the tongue, with one case each in the nasopharynx, oral cavity, and oropharynx. The VGLL3 fusion partners included TCF12 (n = 3), EP300 (n = 2), and PPARGC1A (n = 1). The tumor size range was 0.8-1.6 cm (all, but one, was <1 cm). Histologically, all tumors displayed bland spindle to ovoid cells arranged into vague fascicular and diffuse patterns. Mitotic activity ranged from 1 to 7 per 10 HPFs. Five tumors were muscle-centered and infiltrative, and one was centered beneath nasopharyngeal mucosa. Immunohistochemistry revealed consistent expression of desmin (diffuse in four and patchy in two cases) associated with patchy smooth muscle actin expression (4/6), and focal reactivity for myogenin (5/6) and myoD1 (1/3). All patients were managed surgically; one patient each received adjuvant radio- or chemotherapy. Three patients with follow-up were without disease at 8, 19, and 60 months and one was alive with unknown disease status at 24 months. All VGLL3 fusions were in-frame and involved exon 2, fused with either TCF12 exon 16, EP300 exon 31, or PPARGC1A exon 5, respectively. This series characterizes a distinctive subset of spindle cell rhabdomyosarcoma (RMS) with a predilection for the head and neck in adults, defined by VGLL3 fusions, likely indolent behavior and limited rhabdomyoblastic differentiation. Further delineation of this entity and differentiation from more aggressive molecular subtypes of spindle cell RMS is mandatory to define the most appropriate therapeutic strategy and avoid overtreatment.

Age at maturity is a major contributor to the diversity of life history strategies in organisms. The process of maturation is influenced by both genetics and the environment, and includes changes in levels of sex hormones and behavior, but the specific factors leading to variation in maturation timing are poorly understood. gnrh1 regulates the transcription of gonadotropin genes at pubertal onset in many species, but this gene is lacking in certain teleost species including Atlantic salmon (Salmo salar), which raises the possibility of the involvement of other important regulatory factors during this process. Earlier research has reported a strong association of alternative alleles of the vgll3 gene with maturation timing in Atlantic salmon, suggesting it as a potential candidate regulating reproductive axis genes. Here, we investigated the expression of reproductive axis genes in one-year-old Atlantic salmon males with immature gonads and different vgll3 genotypes during the spawning period. We detected strong vgll3 genotype-dependent differential expression of reproductive axis genes (such as fshb, lhb, amh and igf3) tested in the pituitary, and testis. In addition, we observed differential expression of jun (ap1) and nr5a1b (sf1), potential upstream regulators of gonadotropins in the pituitary, as well as axin2, id3, insl3, itch, ptgs2a and ptger4b, the downstream targets of amh and igf3 in the testis. Hereby, we provide evidence of strong vgll3 genotype-dependent transcriptional regulation of reproductive axis genes prior to sexual maturation and suggest alternative models for distinct actions of vgll3 genotypes on the related molecular processes.

Vestigial-like family member 3 (VGLL3) is a cofactor for TEA domain transcription factors (TEADs). Although VGLL3 is known to be highly expressed and stimulate cell proliferation in mesenchymal cancer cells, its involvement in mesenchymal phenotypes is largely unknown. In this study, we found that VGLL3 promotes epithelial-to-mesenchymal transition (EMT)-like phenotypic changes. We found that A549 human lung cancer cells stably expressing VGLL3 exhibit spindle-like morphological changes, reduction in the epithelial marker E-cadherin and induction of the mesenchymal marker Snail. Notably, VGLL3-expressing cells exhibited enhanced motility. The DNA-binding protein high-mobility group AT-hook 2 (HMGA2) was found to be a target of the VGLL3-TEAD4 complex, and HMGA2 knockdown repressed EMT-like phenotypic changes in VGLL3-expressing cells. VGLL3-dependent phenotypic changes are involved in transforming growth factor-β (TGF-β)-induced EMT progression. VGLL3 or HMGA2 knockdown repressed the motility of the mesenchymal breast cancer MDA-MB-231 cells. Importantly, high levels of VGLL3 expression were shown to have a positive correlation with poor prognosis in various human cancers, such as breast, colon, ovarian, head and neck, pancreatic, renal, gastric and cervical cancers. These results suggest that VGLL3 promotes EMT-like cell motility by inducing HMGA2 expression and accelerates cancer progression.