View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Genomics 103 (2014) 147–153 Contents lists available at ScienceDirect Genomics journal homepage: www.elsevier.com/locate/ygeno Identification and characterization of transforming growth factor β induced gene (TGFBIG) from Branchiostoma belcheri:Insightsinto evolution of TGFBI family Xiaojun Song a,b,LuCaia,b,YafangLia,b, Jiu Zhu a,b,PingJina,b,LimingChenc,FeiMaa,b,⁎ a Laboratory for Comparative Genomics and Bioinformatics, College of Life Science, Nanjing Normal University, Nanjing 210046, PR China b Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210046, PR China c The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Science, Southeast University, Nanjing 210009, PR China article info abstract Article history: The transforming growth factor β induced gene (TGFBIG)encodesaprotein(TGFBI)whichplaysimportantrolesin Received 5 August 2013 many biological processes. However, no TGFBIG homolog has been reported in B. belcheri. Here, we identified a Accepted 7 October 2013 TGFBI-like gene from B. belcheri and extensively studied the evolutionary history of TGFBI family. We found that Available online 16 October 2013 the amphioxus genome contains a TGFBIG homolog designated as AmphiTGFBI which encodes a protein with 5 Fas1 domains. The TGFBIGs were present in a common ancestor with Amphimedon queenslandica.Wealso Keywords: demonstrated expression patterns of AmphiTGFBI in five amphioxus tissues. Interestingly, the gene structures and Amphioxus Domain loss conserved motifs of invertebrate TGFBIGs were found to present regular changes in the evolution. Positive selection Evolution and Fas1 domain loss might cause the regular changes of gene structures and conserved motifs in invertebrate Gene duplication TGFBIGs during evolution. Together, our findings provided an insight into the evolution of the TGFBI family. TGFBIG © 2013 Elsevier Inc. All rights reserved. TGFBI family Persiostin 1. Introduction [12,13]. In addition, TGFBI was required for embryogenesis through regulation of canonical Wnt signaling and suppression of mesothelioma The transforming growth factor β induced gene (TGFBIG) encoding an progression through the Akt/mTOR pathway [8,14]. extracellular matrix (ECM) protein (TGFBI), was identified more than two TGFBI family contains two genes: TGFBI gene and POSTN (periostin, decades ago from a TGFβ1 treated lung adenocarcinoma cell line[1].The OSF-2) gene. Interestingly, the TGFBI paralog POSTN which is a secreted TGFBI contains a secretary signal sequence, an N-terminal cysteine-rich extracellular matrix glycoprotein has a similar domain structure to (EMI) domain, four homologous internal domains, and a cell attachment TGFBI with the following exceptions: 1) TGFBI has 17 exons compared (Arg-Gly-Asp, RGD) site recognizing integrins [2].TheTGFBIplaysan to 23 exons in POSTN in human; 2) TGFBI is notably shorter and lacks important role in cell adhesion, migration, proliferation, apoptosis, and completely the C-terminal region that is subject to alternative splicing angiogenesis, and mutations of the TGFBIG have been shown to be in POSTN [15]. The fundamentally divergence of TGFBI and POSTN involved in several corneal dystrophies [3–5]. Recent studies showed sequences are after the fourth fasciclin domain. The POSTN protein as that the TGFBI can serve as a tumor suppressor in several cancers, such a secreted protein also has an N-terminal signal sequence, in addition as mesothelioma, breast cancer, and radiation carcinogenesis [6–8].The to an EMI domain encoded by exons 2 and 3, and four Fasciclin (Fas1) expression levels of TGFBI in tumor cells were significantly lower than domains [16]. Periostin were initially characterized as an adhesion that in normal cells [6,7]. And TGFBI can sensitize ovarian cancer cells to protein on the basis of its apparent homology to insect fasciclin and paclitaxel by inducing microtubule stabilization and that the loss of this characterization subsequently were refined by expanding the TGFBI induces drug resistance and mitotic spindle abnormalities in collection of known ECM binding partners and illuminating functional ovarian cancer cells [9]. TGFBI can also participate in mesoderm aspects consistent with a role in contact-signaling [17]. Periostin, differentiation and tissue branching morphogenesis [10,11].TGFBI- whose expression has been found to be promoted by several factors knockdown disrupts zebrafish somitogenesis and homozygous null including TGFβ1, 2, 3 and BMP-2 in multiple studies [18–20], can bind mutations in mice may retard postnatal development and has been to certain integrin receptors, which subsequently activate the Akt/PKB reported to predispose the animals to spontaneous and induced cancers pathway via FAK and PI3K, leading to an enhanced migratory or invasive phenotype [21]. Otherwise, periostin may be also involved in the development of FVMs (fibrovascular membranes) [22]. ⁎ Corresponding author at: Laboratory for Comparative Genomics and Bioinformatics, College of Life Science, Nanjing Normal University, Nanjing 210046, PR China. Although TGFBI and periostin have important roles in cell adhesion, E-mail address: [email protected] (F. Ma). migration, proliferation, apoptosis and cancer, there are also mounting 0888-7543/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygeno.2013.10.002 148 X. Song et al. / Genomics 103 (2014) 147–153 evidence that periostin and TGFBI can have complementary or opposite with tBLASTn against the genome sequence and EST libraries of the roles in cancer and development. In cancers, reports highlighted tumor- relevant organism. These data are shown in Table S1. suppressive roles of TGFBI, and complementary expression patterns of POSTN and TGFBI in the developing heart were explicitly studied 2.3. Cloning of full-length AmphiTGFBI cDNA by 3′-and5′-RACE [13,23,24]. TGFBI is dedicated to certain relatively common mutations causing a variety for corneal dystrophies [25], a functional aspect for Total RNA of the whole organism was extracted using Trizol reagent which there is no known counterpart for periostin. Like periostin, (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's TGFBI is a TGFβ-induced, secreted, integrin-binding ECM protein instruction. The first strand of cDNA was synthesized with Moloney expressed during cardiac development and differentially expressed in Murine Leukemia Virus reverse transcriptase (M-MLV; TaKaRa, Dalian, certain epithelial cancers [26]. Although an RGD binding motif for a China). According to the expressed sequence tag (EST) sequence, a subgroup of integrin adhesion receptors is found in TGFBI, TGFBI putative B. floridae TGFBI gene partial sequence was obtained from α3β1-integrin binding has been shown to be mediated not via the AmphiEST database [28]. Two primers, forward primer: 5′-TCGGATG canonical RGD binding motif, which via two pentapeptides containing ATCCGTTATGAGTGC -3′ and reverse primer: 5′-TAGCGCGAATCATC a central Asp-Ile (DI) dimer found in the second and fourth to be TCCAACTT-3′, were designed to amplify the Branchiostoma belcheri conformationary similar to RGD peptides [27]. While an RGD motif is TGFBI gene sequence by RT-PCR. To obtain a complete cDNA sequence, not present in periostin, the DI dimers in Fas1 domains 2 and 4 are 5′-and3′-RACE were performed using a SMARTer RACE cDNA conserved between the two proteins strongly suggesting a mechanism Amplification Kit (Clontech, CA, USA) according to the manufacturer's for periostin integrin binding [15]. instruction. The specific primers 5′-CCTGGTGATGAAGGCGGAGCA-3′ The amphioxus is the modern survivors of an ancient chordate and 5′-TTGCTGGGTGCGAAGACGGTG-3′ for 5′-RACE, and 5′-CACCG lineage, with a fossil record dating back to the Cambrian period. The ACACGGCGTTCCAGAAGG-3′ and 5′-GCCAGATGTGGCACCGCAAGG-3′ unique evolutionary position indicates that amphioxus is the basal for 3′-RACE, were designed according to the RT-PCR product. Primers chordate and a crucial organism for the study of chordate evolution. 5′-GACAGACGATTATCCGTTATGAGTGC-3′ (forward) and 5′-GCTAGA Thus, identification and characterization of TGFBIGs in amphioxus and TACTAACACGGAACAAGGGA-3′ (reverse) were used to amplify the studying the origin and evolution of TGFBI family will not only improve full-length cDNA sequence. The amplified fragments were cloned into our understanding of the process of development and disease of a pMD_19-T Simple Vector (TaKaRa, Dalian, China) and sequenced. amphioxus, but also provide the proximate evolution of TGFBIGs from invertebrates to vertebrates. Here we identified and characterized a 2.4. The spatial express patterns of B. belcheri TGFBI gene detected by TGFBI-like gene from B. belcheri, and also studied extensively the real-time PCR evolutionary history of TGFBI family genes. The amphioxus genome contains an AmphiTGFBI gene encoding a TGFBI homolog with 5 fas1 In the study of spatial distribution of the AmphiTGFBI in different domains. We also found positive selection and lost of Fas1 domain in tissues, total RNA was extracted from muscles, gills, intestine, notochord the evolution history of invertebrate. and hepatic cecum of fifteen healthy amphioxuses. The forward primer 5′-GCCAAGGACGAGGAGAGTAACG-3′ and reverse primer 5′-CCGCC 2. Material and methods GCCGACGATGACG-3′