ATF1 rs11169552 (P = 4.36 × 10−10) and DUSP10 rs6687758 (P = 1.25 × 10−9), and positive association found with CASC8 rs10505477 (P = 8.2 × 10−5). Additional CRC marker haplotypes were also significantly associated with TA. Genotyping an independent dataset consisting of 52 cases with TA and 427 controls confirmed the association with CASC8. Atf1 and Dusp10 expression was detected in the mouse developing teeth from early bud stages to the formation of the complete tooth, suggesting a potential role for these genes and their encoded proteins in tooth development. While their individual contributions in tooth development remain to be elucidated, these genes may be considered candidates to be tested in additional populations.Previously reported co-occurrence of colorectal cancer (CRC) and tooth agenesis (TA) and the overlap in disease-associated gene variants suggest involvement of similar molecular pathways. Here, we took an unbiased approach and tested genome-wide significant CRC-associated variants for association with isolated TA. Thirty single nucleotide variants (SNVs) in CRC-predisposing genes/loci were genotyped in a discovery dataset composed of 440 individuals with and without isolated TA. Genome-wide significant associations were found between TA and  Its etiology is multifactorial and ~33% of all cases are attributed to genetic factors2. Tooth agenesis (TA), the congenital absence of one or more permanent teeth, results from disturbances occurring during tooth development, and represents one of the most common craniofacial anomalies in humans3. Over a decade ago, Lammi et al.4 reported that mutations in the tumor suppressor gene AXIN2 were found co-segregating with colorectal cancer and tooth agenesis in a large multiplex family. Moreover, these authors showed the expression of AXIN2 in developing mouse teeth and suggested that genes involved in colorectal cancer could also be involved in tooth development4.Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world and a leading cause of cancer-related deaths5. Any alteration of the epithelial-mesenchymal interactions can have deleterious effects on tooth development, and may affect growth, differentiation and pattern formation, or even have systemic effects6. TA can occur as part of a syndrome, although it is more frequently found as an isolated trait that may appear sporadically or segregating in families, for which the prevalence ranges between ~2–10% excluding third molars. Based on the number of missing teeth, TA is referred to as hypodontia (up to 5 teeth missing), oligodontia (≥6 teeth missing), or anodontia , the latter being mostly associated with syndromic TA3.Tooth development requires a sophisticated series of signaling interactions between the oral epithelium and mesenchyme under strict genetic control by a number of signaling molecules and their downstream signaling pathwaysMsx1, Pax9, Pitx2, and Lef1 genes in proper tooth formation and morphogenesis, since the absence of these genes results in arrest of tooth development at the bud stage9. In humans, the best characterized mutations for both syndromic and nonsyndromic TA involve MSX1 and PAX910, with concordance between mouse and human phenotypes. Additional mutations in genes belonging to the canonical Wnt pathway  have also been increasingly implicated in the susceptibility to nonsyndromic TA14, although mouse models have not yet been able to replicate the human phenotypes. Interestingly, Wnt pathway genes have also been shown to play critical roles in tumorigenesis, particularly in colorectal cancer17.Studies in mice have revealed more than 200 genes involved in tooth development and shown the functional importance of 11. Recently, genome-wide association studies (GWAS) have uncovered over 50 loci and single nucleotide polymorphisms (SNPs) that are significantly correlated with the susceptibility to CRC, most of which map to genes with established roles in tumorigenesis, or involved in developmental processes such as transcriptional regulation, genome maintenance, and cell growth and differentiation20. Of note, SNPs in CDH1, BMP2, BMP4, and GREM1 genes, known to have important roles in craniofacial and/or tooth development6, have also been reported in association with CRC18.In addition to epithelial-mesenchymal interactions, cell growth and cell differentiation, the signaling pathways involved in tooth development , also overlap with those implicated in colorectal cancerGiven the previously reported co-occurrence of CRC and TA and the overlap in disease-associated pathways, we assessed the association of genome-wide associated CRC variants with isolated TA. Here, we describe the association of CRC gene polymorphisms with isolated TA and reveal potentially novel candidate genes for TA in humans and mice.A total of 440 unrelated Caucasian individuals, 93 with nonsyndromic TA  and 347 unrelated control individuals without TA or family history of TA  were included in this study. All individuals were examined by a dentist, and the presence of tooth agenesis was determined through clinical and radiographic examinations, and considered if one or more permanent teeth were missing from the oral cavity, excluding third molars. Individuals showing signs of syndromic forms of tooth agenesis  were excluded. Of the 93 cases with TA, 61 presented with hypodontia while 32 presented with oligodontia. Positive family history of cancer was reported by 28 cases and 39 control individuals and included various cancer types including colorectal cancer (Supplementary Table −8) from previously published GWAS22, for genotyping in our case-control dataset (Table https://share.fhcrc.org/sites/gecco/), and prioritized additional SNPs for genotyping based on: (1) location in regulatory or enhancer regions of a given gene, (2) having an effect on motifs in the distal regulatory regions of respective genes, and/or (3) interactions with known genes of biological relevance. Genotypes were generated using Taqman chemistry23 and automatic call rate was >98%.We selected 30 CRC-predisposing SNVs with genome-wide significance 5 × 100−8 from DUSP10) gene, and allelic (P = 2 × 10−9) and genotypic (P = 1.25 × 10−9) associations were found between this SNP and TA, particularly under a recessive model (P = 1.27 × 10−9). At the 12q13.1 locus, rs11169552 is located 2Kb upstream of the activating transcription factor 1 (ATF1) gene promoter, for which genome-wide significant genotypic (P = 4.36 × 10−10) and positive allelic associations (P = 1.87 × 10−7) were also found (Table CASC8) gene on chromosome 8q24 and TA for SNP rs10505477 genotypes and alleles  and rs7014346 (P = 0.0005 for genotype under a recessive model)  and TA, particularly under a dominant model (P = 0.001).Genome-wide significant associations were found between TA and individual SNPs on 1q41 and 12q13 , followed by DUSP10 rs6687758 and rs6691170 , and ATF1 rs7136702 and rs11169552  (Supplementary Table ATF1 rs11169552 (P = 0.00004), and for carriers of a GG genotype in DUSP10 rs6687758 (P = 0.003)   .ATF1, DUSP10 and CASC8 during tooth development, as a second step into determining the relevance of these genes in tooth development, we sought to assess whether their transcripts and/or encoded proteins were expressed in the oral and dental tissues of developing mouse embryos.Since no information was available from public databases regarding the expression of Atf1 mRNA expression had been shown to be expressed in mouse embryos as early as E10.5, with strong expression noted in the mesenchyme of frontonasal prominences, branchial arches and limbs26 , a dental-derived stem cell line28, however, no expression was detected.et al.4 identified AXIN2 as a novel gene involved in tooth development after finding mutations in this gene to be co-segregating with CRC and TA in a large multiplex family. Moreover, these authors showed the expression of Axin2 in developing mouse teeth and showed for the first time that a gene involved in CRC could also be involved in tooth development. Here, we describe the findings of CRC-predisposing genes with potential roles in tooth development and as new candidate genes for isolated TA.Over a decade ago, Lammi DUSP10  and ATF1 (activating transcription factor 1) genes, respectively. Meanwhile, positive associations  were also found between CASC8 (cancer susceptibility candidate 8) and TA. These genes had not yet been described to have a role in tooth development and prompted investigations regarding their biological relevance to the TA phenotype. Intriguingly, no associations were found between TA and CDH1, BMP2, BMP4, or GREM1 genes, known to have important roles in craniofacial and/or tooth development6, and which have also been reported in association with CRC18.From the thirty CRC-predisposing variants assessed, our association analyses revealed genome-wide significant associations between TA and loci on 1q41 and 12q13, containing ATF1 belongs to the ATF/CREB family of transcription factors, which binds to the consensus ATF/CRE site ‘TGACGTCA’ and regulates the transcription of target genes to participate in various cellular processes29. Previous reports showed that the function of ATF1 appears to depend on the cellular and genetic context to play an important role in tumor progression in a tumor-specific manner30. ATF1 was found to be over-expressed in several cancer types including lymphoma, melanoma and nasopharyngeal carcinoma, and functioned as a tumor promoter both in vitro and in vivo34. In contrast, disruption of ATF1 activity suppressed its tumorigenicity and metastatic potential33. However, the exact role of ATF1 during craniofacial development is still largely unknown. Loss of Atf1 in mice did not cause any obvious phenotypic abnormalities although led to increased programmed cell death35.2-terminal kinase (JNK). Activation of MAPK by various stimuli including growth factors, cytokines, or stress conditions, regulates major cellular responses such as proliferation, differentiation, survival, migration or production of soluble factors36. Dusp10 has been regarded as an important regulator of tumorigenesis in animal models37, and loss of Dusp10 in mice caused enhanced immune and inflammatory responses although effects on skeletal phenotype have not been described38. Nonetheless, other DUSPs have been found to play critical roles in development. Loss of function studies revealed that Dusp4 is essential for early development and endoderm specification in the zebrafish39, whereas Dusp5 was described to control angioblast populations in the lateral plate mesoderm40.DUSP10 is a dual specificity phosphatase (DUSP) that negatively regulates the activation of the mitogen-activated protein kinase (MAPK) gene family, which includes the extracellular-regulated kinases (ERKs) and the stress-activated protein kinases p38 and c-Jun NHCASC8 is a long noncoding RNA (lncRNA), located in the gene desert region on 8q24.21. LncRNA is a new class of transcripts that are involved in multiple cellular functions including the regulation of expression of multiple genes. Studies have shown that SNPs in lncRNAs may affect the biological processes of messenger RNA conformation, and results in the modification of its interacting partners. Interestingly, various cancer types, including CRC, prostate, breast and gastric cancer, have been reported in association with CASC841, possibly through its function as a lncRNA.ATF1, DUSP10 and CASC8 have been associated with increased risk of CRC in different GWAS with multiple populations22, although functional studies addressing the potential effects of these variants on gene function and/or disease mechanisms are scarce. While the biological function of the associated ATF1, DUSP10, and CASC8 variants is yet to be determined, our bioinformatics analyses revealed that all three variants are predicted to be within DNAse I hypersensitivity clusters, or in enhancer histone marks, with likely functional effects on gene expression. Moreover, ATF1 rs11169552 is predicted to harbor a binding site for POLR2A, essential for RNA polymerase activity and DNA transcription. Another regulatory variant in ATF1 (rs11169571) was shown to increase breast/ovarian cancer risk through modifying miRNA binding43. Interestingly, this variant is in linkage disequilibrium with ATF1 rs11169552, associated in the present study, and could be transmitting the same genetic information as well as functional potential. DUSP10 rs6687758 is predicted to have a putative binding motif for TBP (TATA-box binding protein), which controls the transcription machinery. CASC8 rs10505477 has a putative binding motif to NK2-5, a homeobox-containing transcription factor with roles in embryonic development. These findings suggest that these genes are active in early cell processes being important in both embryogenesis and tumorigenesis.Variants in ATF1, DUSP10 and CASC8 in tooth development is unknown. Therefore, we assessed the expression of these genes or their encoded proteins in relevant tissues or cell lines. During mouse embryonic development, Atf1 mRNA was detected at embryonic day 10.5, with strong expression noted in the mesenchyme of the frontonasal prominences, branchial arches and limbs26. Our findings showed that Atf1 is expressed throughout mouse tooth development stages, in the oral epithelial cells and subjacent condensed ectomesenchymal cells during the early bud stage of tooth development, then shifting to the inner dental epithelium and dental papilla, and finally in the ameloblasts at the final stages of tooth development. Dusp10 mRNA expression was also observed in the mouse craniofacial region and oral cavity at E14.5, particularly in the mouth and tongue27. In our study, the expression of Dusp10, albeit weak, was also noted in the proliferating dental tissues at the early stages of tooth development. At later stages, Dusp10 expression was evident in the enamel organ, pre-ameloblasts and odontoblastic layer. Expression of CASC8 mRNA has been observed in the brain, skin, minor salivary gland, colon and esophagus. However, no expression of CASC8 was detected in our study with human dental stem cells of the apical papilla (SCAP). Since CASC8 has only recently been annotated in the human genome (and not yet annotated in mice), we were limited in the choice of relevant tissues to assess its expression thus hampering our interpretation of the role of this gene in tooth development. Of note, the observed association between TA and the CASC8 locus on chromosome 8q24.21 raises intriguing questions as this locus has been associated with increased risk of several malignancies including CRC41 and also cleft lip/palate45, for which an expanded phenotype including tooth agenesis has been proposed46. Furthermore, the associated CASC8 rs10505477, is located at approximately 250–300 Kb away from the transcription start site of cMYC, a gene well-known for its reprogramming capacity and as a proto-oncogene. Enhancer elements of cMYC are known to be up to 500 Mb away from the gene start site, therefore it is possible that the CASC8 SNP may be in a LD block associated with cMYC enhancer elements and drive the association findings47. The presence of Myc has been detected in dental pulp cells, dental follicle cells, ameloblasts and odontoblasts, and could be related to cell reprogramming or differentiation during tooth development48.The relevance of 49 was proposed for some families in which a single genetic variant could not explain the TA phenotypes13. In this context, the association observed with the CRC-predisposing genes may represent a direct or indirect role in the background of additional causal genes.Among the limitations of this study is the limited availability of individual cancer history or family history of cancer. Since most of our patients with TA are relatively young, our individual patient tumor/cancer history was low (3 patients). In addition, the majority of the TA patients’ parents and/or immediate family members are also younger than the ages in which preventive cancer screening begins. Regarding family history of cancer, in some instances, patients were unsure of cancer status in the family and thus their responses were not accounted for. We considered positive family history of cancer when the individual could name the relative that was affected, and despite various cancer types  were reported for relatives of TA patients, the number of cases was considered small to warrant conclusive genotype-phenotype comparisons between cases and controls. It is also possible that the genetic variants associated with TA in the present study may be in linkage disequilibrium with a more distant causal variant, and acting as surrogate markers for the condition. Additional fine-mapping around the associated loci may provide additional insights into the association signal. Lastly, we cannot rule out that additional variants in other genes may also be contributing to the TA phenotype observed. Similar to other genetic conditions, the hypothesis of multilocus genomic variationATF1, DUSP10 and CASC8 in TA remain to be further elucidated, our findings further support that genes involved in colorectal cancer may also be involved in tooth development and provide additional insights into deciphering the complex etiology of the condition.In summary, our findings provide evidence that novel genes and gene pathways may be yet unknown for a role in tooth development, and unbiased genetic studies have the potential to identify the full spectrum of candidate genes and variants associated with isolated TA. While the exact contributions of This study was approved by the University of Texas Health Science Center at Houston Committee for the Protection of Human Subjects and the University of Pittsburgh Institutional Review Board, and all methods were performed in accordance with the relevant guidelines and regulations. Unrelated individuals with and without tooth agenesis were invited to participate in the study when presenting for treatment at the University of Texas Health Science Center at Houston School of Dentistry clinics. Written informed consent was obtained from all participating individuals and/or from their parents or guardians. Clinical and demographic information, and saliva samples were collected from all individuals. When known, individual and familial history of cancer was also recorded. All individuals were examined by a dentist, and the presence of tooth agenesis was determined through clinical and radiographic examinations, and considered if one or more permanent teeth were missing from the oral cavity, excluding third molars. Differential diagnosis of tooth agenesis from other potential causes of tooth loss  was ensured through comprehensive evaluation of patients’ electronic dental records and conversation with the patient and/or legal guardian about previous tooth extractions and history of trauma. Individuals showing signs of syndromic forms of tooth agenesis  were excluded.To avoid potential effects of population stratification in genetic studies, only individuals with self-reported Caucasian ethnicity (up to 2 generations) were included in the study. A total of 440 unrelated individuals, 93 with nonsyndromic TA  and 347 ethnicity-matched, control individuals without TA or family history of TA  were included in this study. No selection regarding gender was used; the female to male ratio observed in this study is similar to the overall gender prevalence reported in the literature.Of the 93 cases with TA, 61 presented with hypodontia while 32 presented with oligodontia. Positive family history of cancer was reported by 28 cases and 39 control individuals. Details of known cancer types reported are available in Supplementary Table 22 to genotype in this study (Table https://share.fhcrc.org/sites/gecco/), and prioritized additional SNPs for genotyping based on: (1) location in regulatory or enhancer regions of a given gene, (2) having an effect on motifs in the distal regulatory regions of respective genes, and/or (3) interactions with known genes of biological relevance.We selected 30 CRC-risk-associated SNPs that reached genome-wide significance (5 × 10–8) in previously published GWAS23 in 5-μL final reaction volumes in a ViiA7 Sequence Detection System . Results were analyzed using EDS v.1.2.3 software (Applied Biosystems). For quality control of genotyping reactions, we used a non-template reaction as negative control and a DNA sample of known genotype as positive control. Genotyping was performed in duplicates and genotypes that did not have a 95% pass rate were repeated.Genotyping was performed using Taqman chemistryhttp://zzz.bwh.harvard.edu/gpc/) and indicate that the study sample size provided approximately 86% power to detect an association with an alpha of 0.05, if the markers selected are in linkage disequilibrium with the causal factor (D′ = 0.80) and their frequencies are around 80%. Data analysis was performed using PLINK version 1.0650. Hardy-Weinberg equilibrium was calculated for cases and controls, and SNPs showing evidence of deviation in controls were excluded from further analyses. Differences in allele and genotype frequencies for each polymorphism between cases and controls were compared using chi-square and Fisher Exact tests. We corrected for multiple testing using the Bonferroni method, and the significance level was set considering the number of tests (n = 30) to give a corrected P-value (α = 0.002). Haplotype analyses were performed using the ‘haplotype-based case-control association test’ as implemented in PLINK. Regression analyses were performed using EpiInfo v.7.1 (https://www.cdc.gov/epiinfo/) to identify potential preferential associations between specific SNP genotypes with tooth agenesis adjusted for family history of cancer and gender.Power calculations were performed using Genetic Power Calculator (24 and FuncPred (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html) databases. Information on the function of the associated SNPs regarding splicing regulation, stop codon, Polyphen predictions, transcription factor binding site predictions, miRNA binding site prediction regulatory potential score, conservation score, and nearby genes, were recorded. The presence of expression quantitative trait loci for each of the associated SNPs was assessed using the GTEx portal .Annotation of the newly identified loci was performed using the dbNSFPThe expression of Atf1 and Dusp10 proteins was evaluated using immunofluorescence and immunohistochemistry in developing mouse tooth sections, respectively. Wild type C57BL/6 mouse embryos at embryonic days (E) 12.5, 14.5, 16.5, 18.5 and postnatal day 0 (P0) were harvested, fixed in 4% paraformaldehyde and embedded in paraffin for sectioning at 7μm of thickness. Sections were deparaffinized in xylene solution, rehydrated in gradative alcohol baths and rinsed with deionized H2O at room temperature. For antigen retrieval, sections were immersed in sodium citrate buffer  at 100 °C for 30 minutes and allowed to cool down for 30 min at room temperature. To avoid nonspecific binding of the antibodies, sections were immersed in blocking solution  and incubated with AffiniPure Fab fragment goat anti-mouse , for 1 hour at room temperature. For Dusp10, an additional blocking step consisted of incubating sections in 2.5% normal horse serum  for 30 min at room temperature. Sections were then washed and incubated with either Atf1 , or Dusp10  primary antibodies at 4 °C overnight. For Atf1, sections were then washed and incubated with Alexa Fluor® 555  for 2 hours in a dark chamber at room temperature. Lastly, sections were washed (3 × 10′), counterstained with DAPI (Invitrogen) in a dark chamber for 10 min, and mounted with ProLong Gold AntifadeReagent (Invitrogen). For Dusp10, sections were incubated with biotinylated horse anti-rabbit IgG  for 30 minutes at room temperature, washed and incubated with Streptavidin/Peroxidase complex  for 5 minutes and washed. Sections were then incubated in 3,3′-Diaminobenzidine  chromogen for 2 minutes, washed, and counterstained with Mayer’s Hematoxylin. Lastly, sections were washed, dehydrated and mounted. Imaging was performed after 48 hours in a Nikon Eclipse Ni-U upright fluorescence microscope  equipped with a Zyla 5.5 sCMOS camera (Andor).CASC8 amplification were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) . RNA sample integrity was checked by analyzing 1 μg of total RNA on 2100 Bioanalyzer . After RNA extraction, complementary DNA was synthesized by using 3 μg of RNA through a reverse transcription reaction using QuantiTectRT kit . Four sets of primers for The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplemental Information


Pm6, a member of antimicrobial peptide in the antilipopolysaccharide factor (ALF) family from Penaeus monodon, plays important roles in shrimp immunity against pathogens. However, its antimicrobial activity and underlying mechanism have not been reported. The synthetic cyclic ALFPm6#29–52 peptide (cALFPm6#29–52) corresponding to the ALFPm6 LPS-binding domain can agglutinate and exhibited bacterial killing activity toward a Gram-negative bacterium, Escherichia coli 363 and Gram-positive bacteria, Bacillus megaterium, Aerococcus viridans, and Micrococcus luteus, with MIC values of 25–50 μM. Specifically, ALFPm6 and ALFPm3, the most abundant ALF isoforms, are different in terms of gene expression patterns upon pathogen infections. Herein, the regulation of ALFPm3 and ALFPm6 gene expression was studied. The 5′-upstream and promoter sequences were identified and the putative transcription factor (TF)-binding sites were predicted. The narrow down assay indicated that the ALFPm3 promoter and partial promoter of the ALFPm6 active regions were located at nucleotide positions (−814/+302) and (−282/+85), respectively. Mutagenesis of selected TF-binding sites revealed that Rel/NF-κB (−280/−270) of ALFPm3 and C/EBPβ (−88/−78) and Sp1 (−249/−238) sites of ALFPm6 were the activator-binding sites. Knockdown of the PmMyD88 and PmRelish genes in V. harveyi-infected shrimp suggested that the ALFPm3 gene was regulated by Toll and IMD pathways, while the ALFPm6 gene was regulated by the Toll pathway.ALF Vibrio species are considered major shrimp pathogens3. The major virulent strains of Vibrio in shrimp are Vibrio harveyi, Vibrio alginolyticus, and Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (AHPND)4. These virulent strains cause extreme losses of shrimp in hatcheries and grow-out farms, thus making the prevention and control of diseases a priority for shrimp production. To overcome these obstacles, knowledge regarding the innate immune system of shrimp is urgently required.Currently, white spot syndrome virus (WSSV) and bacteria of Pm isoforms to different microbial cell wall components5. The P. monodon ALF major isoform, ALFPm3, exhibits a broad antimicrobial activity spectrum against filamentous fungi, Gram-positive and Gram-negative bacteria including the natural shrimp bacterial pathogens, V. harveyi causing vibriosis and V. parahaemolyticus AHPND causing early mortality syndrome7. The recombinant protein of ALFPm3 (rALFPm3) induce membrane permeabilization and cell lysis of bacterial cells8. The antiviral properties of rALFPm3 against the major shrimp viral pathogen, WSSV, have been reported. ALFPm3 inhibits WSSV propagation in crayfish hematopoietic cell culture and in shrimp9. It has recently been shown that ALFPm3 performs its anti-WSSV activities by binding to the envelope proteins; WSSV189, WSSV395, and WSSV471; nucleocapsid proteins WSSV189; and tegument protein WSSV45811. Although ALFPm3 is the major ALF isoform responsible for fighting pathogen infection, other ALFPm isoforms such as the ALFPm6 gene have been shown to be up-regulated in P. monodon haemocytes in response to yellow head virus infection12 and are important for the shrimp immune response to V. harveyi- and WSSV infections13. However, no reports have described ALFPm6 activity. Moreover, knockdown of the ALFPm3 gene causes rapid mortality, while gene silencing of ALFPm6 has no effect on shrimp mortality but leads to a significant increase in cumulative mortality and a more rapid mortality rate following V. harveyi and WSSV infections, respectively13. Thus, the ALFPm3 and ALFPm6 genes might be controlled by different signalling pathways.Antimicrobial peptides (AMPs) in the antilipopolysaccharide factor (ALF) family are important effectors in shrimp. ALFs are mostly highly cationic polypeptides of approximately 100 residues with a hydrophobic N-terminal region. ALFs contain a signal peptide at the N-terminus and a LPS-binding domain (LPS-BD). Two conserved cysteine residues between LPS-BD are involved in an intramolecular disulphide bridge. The differences in the LPS-BD sequences contribute to the binding capability of different ALFDrosophila, the Toll and IMD pathways are clearly the two most important signaling pathways controlling antimicrobial peptide (AMP) genes16. The Toll pathway is mainly involved in defence against fungi, Gram-positive bacteria, and viruses19, while the IMD pathway plays key roles in controlling Gram-negative bacteria and virus infection. In shrimp, Toll-like receptors (TLRs) and Toll signaling proteins such as Sptzle, myeloid differentiation factor 88 (MyD88), and Dorsal also play regulatory roles on AMP genes upon pathogen infection. LvToll2 from P. vannamei can significantly activate the promoters of the NF-κB pathway-controlled AMP genes, but not LvToll1 and LvToll320. The up-regulation of Spätzle proteins from Fenneropenaeus chinensis (FcSpz) and P. vannamei (LvSpz1–3) during Vibrio species and WSSV infections affect the expression of certain AMPs21. The FcMyD88 transcript from F. chinensis is up-regulated after bacterial infection22. Dual-luciferase reporter assays indicated that the Dorsal from P. vannamei  could regulate the transcription of the shrimp penaeidin-4 gene23. IMD and their signalling proteins in the IMD pathway have been found in shrimp. LvIMD and LvRelish from P. vannamei are involved in penaeidin-4 gene regulation24. Moreover, LvIMD can activate the expression of LvALF-AA-K and LvRelish transcripts in bacteria-challenged shrimp25. IMD from F. chinensis (FcIMD) and Procambarus clarkii (PcIMD) is involved in regulating the expression of several AMPs against Gram-negative bacteria in F. chinensis such as CrustinFc1, CrustinFc2, ALFFc1, ALFFc2 and LysFc1, and in P. clarkii such as CrustinPc1, CrustinPc3, ALFPc6, ALFPc8, and LysPc2. These results suggest that although the IMD distribution and expression patterns exhibit some differences, the IMD pathway may have a conserved function for AMP regulation in shrimp and crayfish immunity26. FcRelish is necessary for the expression of penaeidin-5 against Vibrio anguillarium infection27. In P. monodon, it has been demonstrated that PmRelish can transcriptionally regulate penaeidin-5, but not ALFPm3 and CrustinPm1, in YHV-infected shrimp28.In Pm6#29–52) corresponding to ALFPm6 LPS-BD was tested for antimicrobial activity and bacterial agglutination property. In addition, the difference in the expression levels of the ALFPm3 and ALFPm6 genes was explored by identifying of their promoter active regions and transcription factor binding sites that might play an important role in controlling gene expression. We also determined whether the Toll and IMD pathways were involved in regulating their gene expression.Although the control of AMP and its promoter has been reported in several shrimp, the crucial transcription factor binding sites and promoter active region involved in ALF regulation have not been studied. Moreover, the comparative regulation of each isoform of ALF is unclear. In this study, the synthetic cyclic peptide  in yeast Pichia pastoris. However, the rALFPm6 protein was successfully produced but the purification could not be accomplished (data not shown). To access the activity of ALFPm6, the synthetic cyclic peptide, cALFPm6#29–52, corresponding to the LPS-BD of ALFPm6 was examined for antimicrobial activity against various strains of Gram-negative bacteria and Gram-positive bacteria. The bactericidal activity of cALFPm6#29–52 peptide was observed in comparison to the positive control. The cALFPm6#29–52 peptide was able to kill a Gram-negative bacterium, E. coli 363, and some Gram-positive bacteria such as B. megaterium, A. viridans, and M. luteus with MBC values ranging from 25–50 μM  of the ALFPm3 and ALFPm6 genes were located at nucleotide positions −302 and −85, respectively, 5′-upstream from the start codon (ATG). The TATA box of ALFPm3 and ALFPm6 genes were located from −31 to −23 and from −29 to −24 5′-upstream from the transcription start site (+1), respectively , nuclear factor NF-κB binding sites, CCAAT-enhancer-binding proteins beta (C/EBPβ), CCAAT-enhancer-binding proteins alpha (C/EBPα), octamer-1 (OCT-1), hepatocyte nuclear factors-1 (HNF-1), hepatocyte nuclear factors-1C (HNF-1C), D1, cytoplasmic polyadenylation element binding protein (CPE bind) and interferon consensus sequence-binding protein (ICSBP), were predicted  was identified. Narrow down of the promoter fragment to −265/+302 showed the lowest activity /p(−1478/+302), was cloned and assayed for promoter activity. The promoter activity of del(−693/−358)/p(−1478/+302) was not significantly different when compared with the control p(−1478/+302) Fig. . These rκB transcription factor binding sites are involved in regulating the ALFPm3 gene. The −814/−266 ALFPm3 promoter region, was mutated at the transcription factor binding sites of Sp1 (−719/−701) and NF-κB (−280/−270) by rolling PCR using p(−1478/+302) as a template. The mutated plasmids, ΔSp1/p(−1478/+302) and ΔNF-κB/p(−1478/+302), were assayed for the promoter activity in comparison with the wild type plasmid p(−1478/+302). No significant change in promoter activity was observed with ΔSp1/p(−1478/+302). In constrast, mutation at the NF-κB binding site caused a decrease in promoter activity by 46.6% compared with the wild type plasmid  might have the activator-binding site at position −282 to −81. It contained many transcription factor binding sites such as Sp1 at position −249 to −238, ICSBP at position −146 to −135 and C/EBPβ at position −88 to −80.The promoter activity of each ALFity Fig. . The ALFPm6 promoter region, the mutated reporter plasmids at the transcription factor binding sites of Sp1 (−249/−238), ICSBP (−146/−135) and C/EBPβ (−88/−80) were constructed by rolling PCR using p(−419/+85) as a template. In comparison to the wild type plasmid p(−419/+85), the mutated plasmids, ΔICSBP/p(−419/+85), ΔC/EBPβ/p(−419/+85), and ΔSp1/p(−419/+85), showed an approximately 36.8%, 83.1%, and 98.4% decrease in promoter activity, respectively  using EF1α as an internal control. Normal saline- and GFP dsRNA-injected shrimp were used as negative controls. The result showed that MyD88 gene expression was suppressed at 0–72 h post-MyD88 dsRNA injection. In contrast, expression of the Relish gene was suppressed at 0–48 h post-Relish dsRNA injection and slightly recovered at 72 h post dsRNA-Relish injection  to 24 hpi (74.3%)  and 24 hpi (80.1%) when compared to that of dsGFP knockdown shrimp. In contrast, ALFPm6 gene expression increased by 34.3% at 6 hpi, and no difference was observed at 24 hpi between Relish knockdown shrimp and dsGFP-challenged shrimp  Fig. . It is limp Fig. . Our resP. monodon. ALFPm6 is the most recent and the second most abundant isoform identified in the P. monodon EST database (http://pmonodon.biotec.or.th)30. A previous report has indicated that the ALFPm6 gene is up-regulated upon YHV infection12. Moreover, silencing of the ALFPm6 gene revealed that it is essential for shrimp survival and plays protective roles in shrimp against V. harveyi and WSSV infections13. These findings suggest a potential role for ALFPm6 in the shrimp immunity. However, no reports have characterized ALFPm6. A previous study showed that the synthetic peptide of ALF LPS-BD could interact with LPS in vitro31. Additionally, the synthetic cycled loop peptide designed based on LPS-BD of P. monodon, Scylla paramamosain, and Scylla serrata showed inhibition effects on the growth of Gram-negative bacteria, such as V. harveyi, Pseudomonas aeruginosa and E. coli, and a Gram-positive bacterium, M. luteus34. In this research, the antimicrobial activity of the synthetic cyclic LPS-BD peptide, cALFPm6#29–52, exhibited antimicrobial activity against a Gram-negative bacterium, E. coli 363 with MBC ranging from 25 to 50 μM and Gram-positive bacteria, including B. megaterium, M. luteus and A. viridan, with MBC ranging from 25 to 50 μM. The cALFPm6#29–52, thus, showed less antimicrobial activity than the synthetic peptide of ALFPm3#35–51, which could act against a broader range of Gram-negative and Gram-positive bacteria6. It is noteworthy that the amino acid composition and the total numbers of positively charged amino acid residues (Arg and Lys) of LPS-BD of ALFPm3 and ALFPm6 were different. The LPS-BD of ALFPm3 had 4 Lys and 2 Arg, while ALFPm6 contained 3 Arg and 2 Lys with a net charge of LPS-BD of 6 and 5, respectively. Although the pI values of ALFPm3 (pI = 9.81) and ALFPm6 (pI = 9.96) affect their antimicrobial activities, it could not be responsible for the different antimicrobial activities in each isoform.ALF comprises a group of antimicrobial peptides that have been identified and characterized in shrimp and other crustaceans. Six isoforms of ALF have been discovered in Pm3 exhibits the antibacterial activity by binding to bacterial cell wall components and permeabilizing the bacterial membrane to cause a loss of membrane integrity and cell lysis8. Several AMPs not only exhibit antibacterial activity but also the agglutination activity. Bacterial agglutination activity has been reported for rcrustinPm1 and rcrustinPm7, a family of antimicrobial peptides from P. monodon35. In the mud crab, S. serrata, haemocyanin can bind to bacterial cells and mediated agglutination through the recognition of bacterial OmpA and Ompx proteins36. rEs-DWD1 from the Chinese mitten crab (Eriocheir sinensis) causes significant aggregation of B. subtilis and P. pastoris22. The synthetic peptide, GL13NH2, derived from the parotid secretory protein, agglutinates both Gram-negative and Gram-positive bacteria, including the oral pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii, but it does not showed bactericidal activity. Increasing the calculated net charge of GL13NH2 peptide from +1 to +5 resulted in the loss ability of bacterial agglutination but a gain in bactericidal activity against against Pseudomonas aeruginosa, S. gordonii and E. coli37. In the case of cALFPm6#29–52, it could agglutinate E. coli 363, B. megaterium, M. luteus and A. viridian and kill those bacteria. Taken together, we can conclude that different isoforms of ALFs have different abilities to kill bacteria via different mechanisms.Generally, AMPs exhibit antimicrobial activity via several mechanisms. ALFPm3 and ALFPm6 genes were characterized. Previously, approximately 600 bp of ALFPm3 5′-upstream sequences from the transcription start site were obtained by the genome walking technique. The putative promoter of ALFPm3 was identified at position −29 of the 5′-upstream sequences. Several transcription factor (TF)-binding sites, including octamer (OCT-1), GATA, CCAAT box and GAAA motifs, were predicted in the 5′-upstream sequences38. However, the promoter activity has not been tested. In ALFFc from F. chinensis, immune-related TF-binding sites such as one AP4, one NF-κB, one Sp-1, two GAAA, three OCT-1, and three GATA, were identified in the region from −702 to +1. Analysis of the ALFFc promoter activity in insect Sf9 cell lines showed that the putative promoter region from −702 to +33 was induced by lipopolysaccharide or -β-D-glucan, but the shorter promoter sequence pALF-318 (from −318 to +33) could only be induced only by -β-D-glucan39. However, the important TFs controlling ALFs transcription remain uncharacterized.In addition to the difference in activity, previous studies have also shown that different ALF isoforms are expressed at different levels in response to microbial infection, which could be due to the disparity in gene regulation. Herein, the promoter regions of the ALFPm3 and ALFPm6 gene expression were identified using the narrow down technique. From our analysis, we concluded that the ALFPm3 promoter region at the position (−814/−266) contained the activator-binding site. The genomic DNA sequence of the ALFPm3 promoter at position −814 to −266 contained many TF-binding sites such as Sp1, ICSBP, and NF-κB. Tandem repeats of DNA are prevalent and hypervariable in higher eukaryotic genomes. TR’s of DNA represent a section of the genome that may be highly evolvable, as repeat numbers can change with frequencies 100–10000 higher than point mutations40. The numbers of tandem repeats present in coding regions, promoters, and introns have demonstrated functional roles in modulating protein activity and gene expression and a correlation with disease43. However, the functional role of intergenic tandem repeats that are found far away from coding regions is unclear. A deletion assay for 21 units of GAAAGAGAGTAAGAG[T/C] tandem repeats and ICSBP suggested that this tandem repeat and ICSBP were not involved in ALFPm3 gene regulation. In contrast, Sp1 and NF-κB might be repressor- and activator-binding sites, respectively, on the ALFPm3 promoter. Rel/NF-κB family genes play a central role in the transcription of innate immune effectors44. The NF-κB binding sites were found in different antimicrobial peptide (AMP) genes, including penaeidin, crustin and ALFPm246. In this study, the NF-κB binding sites of ALFPm3 were also found at nucleotide position −280 to −270 from the transcription start sites. The NF-κB family genes, Relish and Dorsal, could regulate the transcription of shrimp AMP genes such as Pen4, Pen5, crustin, and ALF27. The transcription of other antimicrobial peptides in shrimp including crustin and ALF, was also regulated by Relish47. In this study, using site-directed mutagenesis, the NF-κB binding site at position (−280/−270) was identified as the activator-binding site that regulated the ALFPm3 gene regulation.In this study, the promoter active regions with designated the regulatory regions involved in regulating ALFPm3 promoter, the ALFPm6 promoter fragment obtained is relatively short and could probably only part of its regulatory region, although the important regulatory elements could be identified. After deletion of the promoter region at position (−282/−81), the promoter activity was decreased. Based on this result, we concluded that the ALFPm6 promoter region at the position (−282/−81) contained the activator-binding site. The predicted transcription factor-binding sites identified here were Sp1, C/EBPβ and ICSBP, which were shown to be involved in the positive regulation of ALFPm6 gene by site-directed mutagenesis. Previously, the C/EBPβ binding site was identified as one member of the transcription factor binding motif of the crustinPm7 gene45. C/EBPβ is a member of a family of transcription factors involved in important physiological processes, such as cellular proliferation and differentiation, regulation of energy homeostasis, inflammation, and haematopoiesis. Previously, LPS-induced C/EBPβ was shown to specifically bind to the C/EBP response element in the SerpinB2 proximal promoter in mice, and loss of C/EBPβ abrogates constitutive SerpinB2 gene transcription and the response to LPS48. C/EBPβ has been reported to physically interact with AP-1 and NF-κB to promote gene expression of inflammatory mediators49. Sp1 is a ubiquitous nuclear factor that plays a key role in maintaining the basal transcription of house-keeping genes50. According to previous reports, Sp1 and NF-κB are key transcription factors in the regulation of CD40 gene. Sp1 is a key transcription factor controlling the basal expression of CD40. In LPS-stimulated cells, the transcription factor NF-κB up-regulates CD40 expression. In contrast, Sp1 is phosphorylated and its DNA binding activity is reduced after LPS stimulation51. Therefore, it can be hypothesized that Sp1 is also involved in ALFPm3 and ALFPm6 gene regulation. However, further investigations are needed. In summary, our studies showed that NF-κB at nucleotide position (−280/−270), C/EBPβ at nucleotide position (−88/−80) and Sp1 at nucleotide position (−249/−238) in the ALFPm3 and ALFPm6 promoter active region, respectively, are necessary for positive regulation.Compared to the ALFκB signalling pathways controlling the expression of antimicrobial peptide genes16. Previously, the expression profiles of Toll and IMD in bacterial-challenged shrimp were detected and found to be up-regulated at an early stage in haemocytes and hepatopancreas after challenge with M. lysodeikticu and V. anguillarum. Furthermore, ALF expression was significantly increased in response to V. anguillarum and M. lysodeikticu challenge52. In this study, silencing of the adaptor protein, MyD88, and the transcription factor, Relish, which are key molecules in the Toll and IMD pathways, respectively, was performed to investigate the pathway responsible for controlling the gene expression of each ALF. Instead of down-regulation of the ALFPm6 gene, up-regulation of the ALFPm6 gene was observed at 6 and 24 hpi in Relish knockdown shrimp. These findings suggested that the IMD pathway was not involved in the regulation of ALFPm6 gene expression. However, the positive effect of Relish knockdown on ALFPm6 gene expression remains unclear and necessitates further investigation. It is noteworthy that the transcription levels of ALFPm3 in MyD88- and Relish-silenced shrimp were down-regulated at 6 h post-V. harveyi infection. Therefore, ALFPm3 gene expression was probably regulated by both Toll and IMD pathways. A recent study showed that the expression of ALFPm3 was not regulated by PmRelish upon yellow-head virus (YHV) infection. In V. harveyi-infected shrimp, it has been reported that the expression of ALFPm3 relies not only on the PmRelish-mediated pathway but also on another un-identified signalling pathway53.Toll and IMD pathways are the important NF-44, In P. vannamei, RNAi-mediated knockdown of Akirin significantly reduced the expression of NF-κB dependent LvALF-AA-K, Lvcrustin-P and LvPen3 and the transcription factors, Dorsal and Relish post-V. anguillarum and M. lysodeikticu challenge. These findings suggested that ALF, crustin, and Pen3 might be regulated by both Toll and IMD pathways54. Our result together with others implied that Toll and IMD pathways could interact synergistically to induce the independent activation of overlapping target genes. For instance, some target promoters might contain TF-binding sites for both Dif and Relish. Therefore, both pathways could activate them independently, and the response depends on the affinity and number of NF-κB sites55. Cooperation regulation is sometime mediated through an interaction of the NF-κB-related transcription factors in the two pathways. As stated previously, ALFPm3 was the most responsive ALFPm isoform during pathogen infections13. Our results supported this finding based on the observation that Toll and IMD pathways caused independent activation of ALFPm3 genes and, in turn, enhanced ALFPm3 gene expression.According to previous reports, some antimicrobial peptides can be activated by both Toll and IMD pathways, such as Defensin and Metchnikowin in DrosophillaPm against bacterial infection. ALFPm3 gene expression was regulated by Toll and IMD pathways upon bacterial infection. Promoter analysis implied that ALFPm3 transcription is positively modulated by the Rel/NF-κB transcription factor. Furthermore, ALFPm6 mRNA expression was regulated by the Toll pathway during bacterial challenge. C/EBPβ and Sp1 transcription factors may be the activator that controlling the expression of ALFPm6  with a flanking amino acid residue at both the N and C terminal ends, in which a disulphide bond was formed between two cysteine residues, was designed and synthesized service by BIO BASIC INC., Canada. The lyophilized cALFPm6#29–52 was resuspended in ultrapure-water and an aliquot was kept at −20 °C until used.The amino acid sequences (YCSFNVTPKFKRWQLYFRGRMWCP) corresponding to the cyclic LPS-BD of ALFE. coli 363, K. pneumoniae, E. cloacae, V. harveyi, non-virulent V. parahaemolyticus, V. parahaemolyticus (AHPND) and E. carotovora, and the Gram-positive bacteria B. megaterium, A. viridans, M. luteus, S. haemolyticus and S. aureus, were used for the antibacterial activity assay.The bacterial strains including the Gram-negative bacteria 24 to test the antibacterial activity of cALFPm6#29–52 peptide. Briefly, various concentrations of cALFPm6#29–52 peptide  were mixed with the mid-logarithmic growth phase of the bacterial culture in a 96-well plate. Bacteria were grown overnight under vigorous shaking at 30 °C or 37 °C according to the strains. By observation, a bacterial-peptide mixture with high turbidity is considered a the negative result. Only positive results with clear solution as a control were subjected for confirmation of the bacterial killing activity of cALFPm6#29–52 peptide. A 20 μl-aliquot of the bacterial culture incubated with the synthetic cALFPm6#29–52 peptide was plated on an LB agar plate and incubated at 30 °C for 16 h. As a positive control, sterile water was used instead of the tested peptide. The number of bacterial colonies that grew on the LB agar plate was determined. The minimum bactericidal concentrations (MBCs) of the synthetic cALFPm6#29–52 peptide was defined as the range between the highest concentration of the peptide where bacterial growth was observed and the lowest concentration of the peptide that killed the bacteria. To further investigate how the peptide inhibited the growth of bacteria, one drop of the above peptide-bacteria mixture was placed on a microscope slide with the bacterial sample and then Gram-stained and observed under a 100X magnification light microscope (Olympus CX31). Images were obtained using a microscope camera (Nikon DS-Fi1).The liquid growth inhibition assay was performed as previously describedPm3 and ALFPm6 genes were determined using the BD GenomeWalker Universal Kit . The restriction enzyme-digested P. monodon genomic DNA libraries  were used as template. The primary PCR using the outer adaptor primer (AP1) and the first gene-specific primer (GSP1) was amplified. The secondary PCR was performed with the nested adaptor primer (AP2) and a nested gene-specific primer (GSP2). The GSP1 and GSP2 primers designed from the known genomic sequence of ALFPm3 (accession no. EF523562) and ALFPm6 (accession no. JN562340) are shown in Supplementary Table www.cbs.dtu.dk) and BDGP  (www.fruitfly.org). The putative cis-regulatory elements in the 5′-upstream sequences were predicted by TF search Version 1.3, Match 1.0 and the Alibaba 2 analysis programme (TRANSFAC® Public database) (www.gene-regulation.com). The results were compared with the JASPAR database (jasper.genereg.net) to confirm the putative transcription factor binding sites.The 5′-upstream sequences of ALFPm3 and ALFPm6 promoters were randomly narrowed down. ALFPm3 promoter fragments included the nucleotide positions of (−1478/+324), (−814/+324), (−719/+324), (−265/+324), and (−71/+324). For the ALFPm6 promoter, various fragments at nucleotide positions (−419/+85), (−282/+85), (−162/+85), and (−80/+85) were constructed. Primer sequences containing the restriction sites NheI and BglII used for promoter fragment amplification are shown in Supplementary Table To construct the luciferase reporter plasmid harbouring various fragments of promoter sequences, the ALFDrosophila Schneider 2 (S2) cells were seeded in a 24-well plate and cultured in complete Schneider’s Drosophila Medium containing 10% heat-inactivated FBS and antibiotics  at 27 °C for overnight. The 200 ng of pGL3-Basic (control plasmid) or pGL3 plasmid containing either ALFPm3 or ALFPm6 promoter fragments were co-transfected with 50 ng of the pRL-TK plasmid containing the Renilla luciferase gene into the S2 cells using Effectene® transfection reagent (Qiagen) according to manufacturer’s protocol. Cell lysates were collected at 48 h after transfection. Firefly luciferase and Renilla luciferase activities were assayed using the Dual-Luciferase® Reporter Assay System (Promega). Luminescence was detected using a SpectraMax M5 Multi-Mode Microplate Reader (Molecular device). The relative light unit (RLU) was calculated by normalizing the firefly luciferase activity to the Renilla luciferase activity to correct the transfection efficiency. The data were reported as the relative luciferase activity. Independent triplicate experiments were performed for each construct, and the standard deviation was calculated.Pm gene regulation. To determine whether the 21 units of GAAAGAGAGTAAGAG[T/C] tandem repeat was involved in regulating ALFPm3 gene expression, the tandem repeat deletion construct, del(−693/−358)/p(−1478/+302), was prepared by rolling PCR using p(−1478/+324) as a template and the primer pair del(−693/−358)/p(−1478/+324)F and del(−693/−358)/p(−1478/+324)R  and NF-κB (−280/−270) were mutated using p(−1478/+324) as a template. For the ALFPm6 promoter region, mutation of the transcription factor binding sites of Sp1 (−249/−238), ICSBP (−147/−136) and C/EBPβ (−78/−88) were performed using p(−419/+85) as a template. The mutated constructs of these transcription factor-binding sites were prepared by rolling PCR. The conserved nucleotides of the interested TF-binding site were identified by JASPAR (an open-access database for eukaryotic TF-binding profiles) and their specific primers with mutated nucleotides were designed (Supplementary Table E. coli strain XL-1 blue by electroporation. The recominant plasmids were extracted using the High-speed plasmid mini kit (Geneaid). The mutated-transcription factor-binding sites were confirmed by DNA sequencing . The promoter activity of the mutant was measured and compared with wild type using the Dual-Luciferase® Reporter Assay (Promega).The obtained promoter active region was further analysed for the specific transcription factor binding site involved in ALF)R Table . The PCRThe dsRNA specific to MyD88, Relish and GFP were prepared according the T7 RiboMAX™ Express RNAi System (Promega) kit’s instructions. DNA template containing the T7 promoter sequence at the 5′-end was generated by PCR using the specific primer, T7-MyD88-F, MyD88-R, MyD88-F, T7-MyD88-R, T7-Relish-F, Relish-R, Relish-F, T7-Relish-R, and T7-GFP-F, GFP-R, GFP-F, T7-GFP-R (Supplementary Table μg/g shrimp), while the control groups were injected with GFP dsRNA and 0.85% NaCl, respectively. To test the silencing efficiency, the haemolymph of 9 individuals from each treatment was collected at 0, 24, 48, and 72 h post-dsRNA injection (hpi), and total RNA was extracted. Three sets of pooled samples were prepared, each comprising 3 different individuals. The first-strand cDNA was synthesized from 1 μg of total RNA using the RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific). Either MyD88 or Relish gene expression was analysed by quantitative real-time RT-PCR using the gene-specific primer pair, PmMyD88-RT_F, PmMyD88-RT_R, PmRelish-RT_F and PmRelish-RT_F, respectively (Supplementary Table α (EF1α) gene was used as an internal control. The relative expression of MyD88 and Relish was calculated using a comparative method described by Pfaffl56. The data are shown as the means ± standard deviations. Statistical analysis was performed using the one-way ANOVA followed by Duncan’s new multiple range test. Data differences were considered statistically significant at P < 0.01.For the dsRNA-mediated gene silencing experiments, 144 shrimps were randomly divided into four groups (n = 36 in each group). The experimental group (3–5 g shrimp) was treated with MyD88 dsRNA or Relish dsRNA (10 Pm3 and ALFPm6 transcript levels upon V. harveyi infection, shrimp were injected with 10 μg/g shrimp of the MyD88 dsRNA, Relish dsRNA or GFP dsRNA. After the first dsRNA injection for 12 h, shrimp were challenged with 106  CFU/ml of V. harveyi mixed with 10 μg/g shrimp dsRNA. At 0, 6 and 24 h post-V. harveyi infection, shrimp haemolymph was collected and used for total RNA extraction and cDNA synthesis. Transcript levels of ALFPm3 and ALFPm6 genes in each sample were analysed by quantitative real-time RT-PCR using the specific primers, qRT-ALFPm3_F/R, qRT-ALFPm6_F/R, respectively. The relative expression of ALFPm3 and ALFPm6 was calculated using a comparative method described by Pfaffl56. The data are shown as the means ± standard deviations. The statistical analysis was performed using the one-way ANOVA followed by Duncan’s new multiple range test. Data differences were considered statistically significant at P < 0.01.To investigate the effect of MyD88 and Relish gene knockdown on the ALFSupplementary Table S1


Here, we further explored the mechanism how the number of repeats of the STR regulates gene transcription. An inverse correlation between the number of repeats and the extent of transactivation was found in a haplotype consisting of three promoter SNPs (C-STR-T-T). We showed that these adjacent SNPs located outside the STR were required for the STR to function as eSTR. The C allele of rs35767 provides a binding site for CCAAT/enhancer-binding-protein δ (C/EBPD), which is essential for the gradational transactivation property of eSTR and FOXA3 may also be involved. Therefore, we propose a mechanism in which the gradational transactivation by the eSTR is caused by the interaction of one or more transcriptional complexes located outside the STR, rather than by direct binding to a repeat motif of the STR.Functional short tandem repeats (STR) are polymorphic in the population, and the number of repeats regulates the expression of nearby genes . STR in IGF1 promoter has been extensively studied for its association with IGF1 concentration in blood and various clinical traits and represents an important eSTR. We previously used an  Genetic variations in gene promoters play key roles in the determination of gene expression and phenotypes, including disease predisposition. Single nucleotide polymorphisms (SNPs) are the most commonly studied genetic variations and have been considered as the primary functional element in phenotype determination. The alternate alleles of a SNP in a gene promoter may result in either the formation or abolition of a binding site for transcription factors (TFs) and are therefore believed to play piloting roles in the transactivation of gene expression and quantitative trait loci1235789et al. studied STRs in both human and nonhuman great ape genomes and showed that STRs contribute significantly to the diversity and divergence of gene expression among species131819615Recently, a renewed interest in the functional role of STRs has developed. First, STRs are abundantly located in promoters across eukaryotic organisms (ranging from yeast to rodents and humans)12131415et al.522524et al. confirmed the presence of more than 2000 functional STR loci that are correlated with gene expression, which are termed expression STRs (eSTRs)26The renewed interest in STRs, particularly their functional potential and role in human diseases, has led to the development of tools and catalogs of STRs. For example, Willems Although evidence to support STRs as functional elements continues to accumulate, little information is known about how they operate. The only difference between any two STR alleles is the number of repeats; there is no change in the nucleotide base as in the case of SNPs, which could be responsible for alterations in the binding sites for nuclear proteins and transcriptional factors. The most abundant STRs are dinucleotide repeats, and the difference between alleles could be as little as lengthening (or shortening) by 2 bps. Furthermore, these changes in length (i.e. number of repeats) occur within a sequence of identical 2-bp repeating motifs; given such minute alterations, it is difficult to envision how eSTRs may regulate gene function.in-vitro luciferase model. Interestingly, this gradational transactivation property was only found in one of the two prevalent SNP haplotypes. In this study, we used an in-vitro reporter assay to study and localize the functional elements within this promoter and to investigate the mechanism of the gradational effect of microsatellite length on transactivation. We approached this question at two levels. First, at the genomic DNA level, we used serial deletion fragments of the promoter to localize functional units in the promoter. Second, we identified which transcriptional factors (TFs) were involved through a series of experiments by removing each TF from the expression plasmid mix. The results of this two-tiered experimental approach provided new insights into the gradational transactivation property of these microsatellites, which may represent a common mechanism of transactivation regulation shared with other eSTRs.We have studied the transactivation mechanism of STRs using IGF1 as a model for other dinucleotide repeats. Our early results were consistent with genetic epidemiological findings and supported a biological basis for the association between the blood concentration of IGF1 and the alleles of the STR in IGF1 promoter28FST) values were calculated according to the equation described by Weir and CockerhamGenotype data for these three SNPs and their neighboring SNPs were obtained from the HapMap database, and their fixation index (BglII restriction site (underlined) (5′-AGCAGATCTGCCCCAGGATAACACAAAGA-3′) and a reverse primer containing a HindIII restriction site (underlined) (5′-AGCAAGCTTGCTTCTGAAGTACAAAGTCT-3′). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System  according to the manufacturer’s instructions and digested by BglII  and HindIII . The gel-extracted products were purified as described above and cloned into the BglII and HindIII sites of a promoter-less firefly luciferase vector, pGL4.10 . Reporter constructs with uncommon promoter haplotypes   . Express2. This cell line was selected due to the previous finding that it was a suitable cell model for manipulation of TFs related to IGF1 promoterThe human lung epithelial cell line Beas-2B was obtained from American Type Culture Collection . The cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM)/F12 , supplemented with 10% heat-inactivated fetal bovine serum . Cells were incubated at 37 °C with 5% CO4 24 hours before transfection. Cell transfection was performed using X-tremeGene HP , according to the manufacturer’s protocol. For each well, 0.5 μg of the reporter construct was transfected along with 1ng of cytomegalovirus (CMV)-controlled Renilla luciferase vector pGL4.75 , which was used to adjust for transfection efficiency, and 0.5 μg of pcDNA3-hGHR, 0.25 μg of pcDNA3-hFOXA3 and 0.25 μg of pSX-hStat5B, which were used to activate the promoter fragments. The transfected cells were incubated for 48 hours before they were harvested for luciferase assay. The activity of firefly and Renilla luciferase was measured by a Dual Luciferase Reporter Assay System . For each reaction, 50 μl of LARII and 50 μl of Stop & Glo Reagent were added to 10 μl of cell lysate. The output signal was detected with a Victor X3 Multilabel Plate Counter . The firefly luciferase activity encoded by the promoter-reporter construct was normalized to the Renilla luciferase activity encoded by CMV promoter to control for variation in transfection efficiency. All assays were performed in four independent experiments, each of which consisted of four replicates for each haplotype (n = 16). Coefficient of variation (CV) was less than 25% for each group.The cells were seeded in 24-well plates at a density of 5 × 10FST analysisPopulation differentiation was determined by classic The allelic distribution of these three studied SNPs  differed significantly between Caucasians and Asians . The genFST), measures the differentiation of a subpopulation relative to the total population and is directly related to the variance in allele frequency among subpopulations. A high FST implies a high level of differentiation. Genotype data for these three SNPs and their neighboring SNPs were obtained from HapMap database and FST was calculated according to equation described by Weir and CockerhamFST of among the three SNPs, with FST = 0.025. For rs5742612 and rs2288377, the FST was 0.346 and 0.387, respectively. The low FST of rs35767 suggests that this is a universal allele, distributed across the world’s population, and may have an important biological function.This analysis suggested that significant differences in haplotype structure were present among the different populations. Fixation index  had significantly different transcription activitySerial deletion mutants were prepared from −1491 to −925 on the construct with haplotype C-T-T . The lucSacI digestion and compared the transcriptional activity of the full-length and digested (without the 125 bp) promoter fragments.In our previous study, we identified a gradational effect of the microsatellite in the haplotype C-T-T, in which promoters with a longer microsatellite had a lower transcriptional activityAs shown in It has been reported that a C/EBPD transcription activation complex binds exclusively to the C allele of rs35767 to activate IGF1 promoter activityOn the background of haplotype T-T-T, the gradational transactivation effect of different microsatellite lengths was abolished , indicatBecause rs35767 is located more than 1000 bp upstream of IGF1, it may interact with another TF close to the transcription start site of IGF1 to recruit transcription machinery and activate gene expressionIn the absence of FOXA3, gradational transactivation as a function of microsatellite with different lengths in haplotype C-T-T was abolished, and thus there was no relationship between microsatellite length and promoter activity . PromoteThe association between genetic variations in IGF1 promoter and inter-individual variation in circulating IGF1 levels and disease susceptibility has long been identified in epidemiology studies, but the underlying mechanism has not been elucidated373839287in-vitro luciferase reporter model used here had been developed earlier, and the optimization procedure was described in more detail in our previous publication  conditions, which is the essential limitation of all in-vitro studies.The To investigate the underlying mechanism of the differential transcriptional activities between haplotypes C-T-T and T-C-A, a 5′-serial deletion experiment was performed to analyze the effect of various segments of IGF1 promoter on transcriptional activity. We found that a 125-bp segment, in which rs35767 was located, and a 925-bp segment, in which rs5742612 and rs2288366 were located, contributed to the differential transcriptional activities between the two haplotypes. This indicates that functional elements are present inside these two promoter segments which enable the eSTR property.SNP rs35767 located in the 125-bp segment, is a recognized functional SNP41424344Furthermore, our results suggested that the importance of FOXA3 for gradational transactivation. FOXA3 is a liver-enriched TF and a known interaction partner of C/EBPD complex in the liver33FST among the neighboring SNPs. The mean genome-wide value of Fst across the population for all 2.8 million Phase II HapMap SNPs is 0.11. Assuming neutrality, all variants were similarly affected by only human demographic history. However, the significantly low population differentiation indicated by its Fst suggests a potentially strong natural selection pressure upon this IGF1 promoter SNP. On the basis of population genetic studies, it has been suggested that value of FST < 0.05 can be regarded as low, and some examples of SNPs with low FST are associated with disease susceptibilityWe confirmed the population differentiation in variations of IGF1 promoter, e.g. rs35767, using population genetics and molecular evolution analysis. Our results suggested that rs35767 had the lowest Despite the recent genomic scale discovery of more than 2000 eSTRs, little is known about the mechanism on how the length of the microsatellite affects gene expression. There are two potential models for the operation of eSTR see . Model 1In conclusion, this study provides support for a model of the mechanism of eSTR based on cooperation between the STR and adjacent TF binding complexes. Our data, together with the results of our previous epidemiological and functional studies, demonstrate that the eSTR effect relies on both the microsatellite and the SNPs. In addition, our results suggest a novel regulatory mechanism for microsatellites in humans.in vitro reporter assays to compare the transcriptional activity of IGF1 promoter fragments with various STR lengths and allelic compositions. We identified two regions outside the STR that contribute to the distinct regulatory mechanisms of haplotype C-T-T and T-C-A: a 125-bp segment with a functional SNP, rs35767, and a 925-bp segment with two common SNPs, rs5742612 and rs2288377. In the haplotype C-T-T, an eSTR effect was found in which higher transactivation was correlated with shorter STRs. The eSTR property is dependent on the C/EBPD complex, which binds upstream to the microsatellite. This led us to suggest a model for eSTR function involving the binding of one or more TF in the vicinity of the microsatellite but not directly onto the repeat motifs.To investigate the regulatory mechanism of eSTR, we performed How to cite this article: Chen, H. Y. et al. The mechanism of transactivation regulation due to polymorphic short tandem repeats (STRs) using IGF1 promoter as a model. Sci. Rep.6, 38225; doi: 10.1038/srep38225 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Visfatin is an adipokine associated with glucose and lipid metabolism. We previously reported two visfatin upstream single nucleotide polymorphisms (SNPs), c.-3187G > A (rs11977021) and c.-1537C > T (rs61330082), which were in perfect linkage disequilibrium, in a Singaporean cohort of severely obese children and are associated with visfatin level and adverse cardiometabolic parameters. We aim to functionally characterize the effect of c.-3187G > A and c.-1537C > T SNPs on basal transcriptional activity.A 1.6 kb and 3.7 kb upstream promoter region of the visfatin gene was amplified by polymerase chain reaction and separately cloned into luciferase reporter vectors. Successful clones were transfected into human embryonic kidney (HEK293T) and human breast carcinoma (MCF7) cells and in-vitro dual-luciferase assay was performed. Electrophoretic mobility shift assay (EMSA) was also conducted to examine the binding affinity between transcription factors and visfatin promoter sequences.p < 0.01) in transcriptional activity. The longer 3.7kbp visfatin promoter sequence was also shown to have significantly higher transcriptional activity (p < 0.05) as compared to the shorter 1.6kbp visfatin promoter. Both c.-3187G > A and c.-1537C > T variants showed an increased binding with nuclear protein.Variant promoter with only c.-1537C > T SNP did not show a change in transcriptional activity as compared to the wild type. However, variant promoter with both c.-3187G > A and c.-1537C > T SNPs showed a statistically significant increase of 1.41 fold  contains supplementary material, which is available to authorized users. Visfatin is an adipokine secreted by adipose tissues  and plasPolymerase chain reaction (PCR) was performed to amplify the upstream promoter region of visfatin. The DNA samples of unrelated obese subjects identified to be homozygous for the respective wild-type and variant alleles of the 2 SNPs (rs61330082 and rs11977021) at perfect linkage disequilibrium were used for PCR . The pri4 cells/well and they were pre-cultured in 24-well tissue culture plate for 24 h.Human embryonic kidney cells, HEK293 and human breast cancer cells (MCF-7) were grown to 90% confluency. The seeding density of the cells was ~5 × 102 overnight for 24 h before they were used for luciferase assay study. The functionality of the visfatin insert was determined by dual-luciferase reporter assay  according to manufacturer’s instructions. The assay for each visfatin insert was performed in triplicates and the experiment was performed thrice. All the 3 transfection experiments showed similar fold-change. However, due to the variable transfection efficiency, we presented the transfection results as the mean and SD of the triplicates in one experiment setting. The results for other 2 biological replicates are shown in Additional file Cells were co-transfected with 1 ng/μl of visfatin pGL4.10 luciferase plasmid and 0.025 ng/μl of Renilla luciferase reporter vector using 0.03 μl /μl of Lipofectamine 2000 . The transfected cells were incubated at 37 °C with 5% COhttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3, last accessed on 30th May 2014) . The experiment was performed twice  of the visfatin promoter sequence and were designed to include 5 bases before and after the respective SNP. The probe sequences are c.-3187G: 5’-ACTGAt-test. Paired sample t-test was used to analyze the difference in EMSA band intensity between wild-type and variant promoter sequence .Data are expressed as mean ± standard deviation (SD). The difference in luciferase activity between different visfatin promoter sequences was analyzed by the Student’s We used the transcription factor binding site prediction program PROMO to predict any alteration in transcription factor binding affinity in visfatin promoter sequence previously found to contain single nucleotide polymorphisms (SNPs), rs61330082 (c.-3187G > A) and rs11977021 (c.-1537C > T). PROMO predicted the alteration of the transcription factor estrogen receptor-alpha  binding site by c.-3187G > A variant while c.-1537C > T variant was predicted to alter activating protein-2alphaA  transcription factor binding site as shown in Fig. p = 0.014) higher than that of wild-type 1.6kbp promoter in MCF7 and HEK293T cells respectively. The transcriptional activity of the variant 3.7kbp promoter sequence was 1.27 and 1.48 fold higher than that of variant 1.6kbp promoter in MCF7 and HEK293T cells respectively. The luciferase assay was performed in triplicates (n = 3) for each promoter sequence and a minimal amount of luciferase activity (0.21 ± 0.03 and 0.27 ± 0.02) was detected in HEK293T and MCF cells transfected with control pGL4.10 luciferase plasmid.The basal transcriptional activity was measured as the in-vitro luciferase activity of the different visfatin promoter sequences in transiently transfected MCF7 and HEK293T cells as shown in Fig. EMSA data showed that the variant alleles in the respective c.-3187G > A and c.-1537C > T SNPs have a significant increased binding with nuclear protein Fig. , b. The In this study, we functionally characterized two upstream visfatin promoter SNPs, c.-3187G > A rs11977021) and c.-1537C > T (rs61330082), that were previously detected in the upstream promoter region of visfatin gene in our cohort of severely obese children . Through77021 andThe 3.7kbp visfatin variant promoter sequence containing both c.-3187G > A and c.-1537C > T SNPs was shown to have a significantly higher luciferase activity than that of the wild-type 3.7kbp promoter Fig. . This inIn addition, we examined the transcriptional activity of the 1.6kbp variant visfatin promoter containing only c.-1537C > T SNP and found that there is no significant difference in transcriptional activity as compared to wild-type 1.6kbp promoter Fig. . Our finThe longer 3.7kbp wild-type visfatin promoter was found to have a significantly higher transcriptional activity as compared to the shorter 1.6kbp wild-type promoter. Moreover, the longer 3.7kbp variant promoter was also found to exhibit a higher transcriptional activity as compared to the shorter 1.6kbp variant promoter Fig. . Though Both c.-3187G > A and c.-1537C > T SNPs have been reported as expression quantitative trait loci (eQTLs) in left ventricular heart tissue with trends of decreasing expression as the genotype changes from homozygous wild-type to heterozygous to homozygous variant . HoweverData from our EMSA experiments indicated an increased binding of nuclear protein to the variant A and T alleles of c.-3187G > A and c.-1537C > T SNPs respectively Fig. . AlthougSince c.-3187G > A does not exist in solitary without c.-1537C > T in our obese children cohort, we did not examine the effect of c.-3187G > A SNP alone on the transcriptional activity. Hence, we could not determine if both SNPs are needed to work cooperatively in order to exert an effect on transcriptional activity. In addition, we did not examine the specific transcription factor that is affected by the c.-3187G > A and c.-1537C > T SNPs respectively. Hence, we are not able to determine if the SNPs have affected binding with ER-alpha and AP-2alphaA transcription factors as predicted by PROMO.In conclusion, we have demonstrated for the first time that visfatin variant promoter with both c.-3187G > A and c.-1537C > T SNPs result in increased in-vitro transcriptional activity. This supports our previous finding and postulation that these SNPs contribute to elevated visfatin levels which mediate higher triglyceride levels, severe systolic blood pressure and severe hypertension in obese children. This study supports the role of these upstream visfatin SNPs which could potentially affect phenotypic outcome in obese children through alteration of circulating visfatin level.