SIX1 Research Articles

Characterization of the promoter region of the bovine SIX1 gene: Roles of MyoD, PAX7, CREB and MyoG

The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5′ regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5′ flanking region of bovine SIX1. However, a CpG island was predicted in the region −235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5′ flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region −689/−40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.

NRF1 and ZSCAN10 bind to the promoter region of the SIX1 gene and their effects body measurements in Qinchuan cattle

The SIX1 homeobox gene belongs to the six homeodomain family and is widely thought to play a principal role in mediating of skeletal muscle development. In the present study, we determined that the bovine SIX1 gene was highly expressed in the longissimus thoracis and physiologically immature individuals. DNA sequencing of 428 individual Qinchuan cattle identified nine single nucleotide polymorphisms (SNPs) in the promoter region of the SIX1 gene. Using a series of 5′ deletion promoter plasmid luciferase reporter assays and 5′-rapid amplification of cDNA end analysis (RACE), two of these SNPs were found to be located in the proximal minimal promoter region −216/−28 relative to the transcriptional start site (TSS). Correlation analysis showed the combined haplotypes H1-H2 (-GG-GA-) was significantly greater in the body measurement traits (BMTs) than the others, which was consistent with the results showing that the transcriptional activity of Hap2 was higher than the others in Qinchuan cattle myoblast cells. Furthermore, the electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assay (ChIP) demonstrated that NRF1 and ZSCAN10 binding occurred in the promoter region of diplotypes H1-H2 to regulate SIX1 transcriptional activity. This information may be useful for molecular marker-assisted selection (MAS) in cattle breeding.

SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B

BackgroundSIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family. It plays a vital role in the nervous system development. Our previous study showed that the SIX3 gene is hypermethylated, and its expression is decreased in astrocytoma, but the role of SIX3 remains unknown.MethodsChromatin-immunoprecipitation (ChIP) and luciferase reporter assay were used to confirm the binding of SIX3 to the promoter regions of aurora kinase A (AURKA) and aurora kinase B (AURKB). Confocal imaging and co-immunoprecipitation (Co-IP) were used to detect the interaction between AURKA and AURKB. Flow cytometry was performed to assess the effect of SIX3 on cell cycle distribution. Colony formation, EdU incorporation, transwell, and intracranial xenograft assays were performed to demonstrate the effect of SIX3 on the malignant phenotype of astrocytoma cells.ResultsSIX3 is identified as a novel negative transcriptional regulator of AURKA and AURKB, and it decreases the expression of AURKA and AURKB in a dose-dependent manner in astrocytoma cells. Importantly, interactions between AURKA and AURKB stabilize and protect AURKA/B from degradation, and overexpression of SIX3 does not affect these interactions; SIX3 also acts as a tumor suppressor, and it increases p53 activity and expression at the post-translational level by the negative regulation of AURKA or AURKB, reduces the events of numerical centrosomal aberrations and misaligned chromosomes, and significantly inhibits the proliferation, invasion, and tumorigenesis of astrocytoma in vitro and in vivo. Moreover, experiments using primary cultured astrocytoma cells indicate that astrocytoma patients with a low expression of SIX3 and mutant p53 are more sensitive to treatment with aurora kinase inhibitors.ConclusionSIX3 is a novel negative transcriptional regulator and acts as a tumor suppressor that directly represses the transcription of AURKA and AURKB in astrocytoma. For the first time, the functional interaction of AURKA and AURKB has been found, which aids in the protection of their stability, and partially explains their constant high expression and activity in cancers. SIX3 is a potential biomarker that could be used to predict the response of astrocytoma patients to aurora kinase inhibitors.

Associations of six SNPs of POU1F1-PROP1-PITX1-SIX3 pathway genes with growth traits in two Chinese indigenous goat breeds

Abstract In the process of anterior pituitary gland development and maturation, numerous transcription factors were involved in the pituitary organogenesis. To date, the lack of knowledge on POU1F1-PROP1-PITX1-SIX3 pathway genes affecting growth traits was reported in Chinese goat breeds. The objective of this work was to identify genetic variants of the POU1F1, PROP1, PITX1 and SIX3 genes, as well as to evaluate their associations with growth traits in goats. The results showed that one novel and five reported single nucleotide polymorphisms (SNP) were confirmed in above four genes. The novel SNP (NC_019460.2:g.1100T>A) was located in intron 1 of SIX3 gene. Among these loci, DQ826397.1:g.102T>G (SNP1), DQ826397.1:g.279T>C (SNP2) and NC_019460.2:g.1100T>A (SNP6) were associated with growth traits. In details, the SNP1 locus had significant association with hucklebone width (P<0.05) and hucklebone width index (P<0.05) in the Guanzhong dairy goat. The SNP2 locus was associated with body weight (P<0.05), chest circumference (P<0.01), chest width (P<0.05), hucklebone width (P<0.05), cannon circumference (P<0.01) and trunk index (P<0.01) in the Hainan black goat. The SNP6 locus was significantly associated with hucklebone width index (P<0.05) and chest width (P<0.05) in the Guanzhong dairy goat and Hainan black goat, respectively. These findings indicated that goat POU1F1-PROP1-PITX1-SIX3 pathway genes were important genetic factors, which could affect growth traits. Three SNPs detected in this study may be useful for potential marker-assisted selection programs in goat breeding and production.

Identification and characterization of variants and a novel 4 bp deletion in the regulatory region of SIX6, a risk factor for primary open‐angle glaucoma

BackgroundPrimary open‐angle glaucoma (POAG) is a complex disease of multigenic inheritance and the most common subtype of glaucoma. SIX6 encodes a transcription factor involved in retina, optic nerve, and pituitary development. Previous studies showed a genetic association between the SIX6 locus and POAG, identifying risk alleles. Whether these alleles are present also in the south Indian population is unclear.MethodsTo address this question, the SIX6 gene and an already characterized and highly conserved SIX6 enhancer (Ch14:60974427‐60974430) were sequenced in two south Indian cohorts, respectively, composed of 65/65 and 200/200 POAG cases/age‐matched controls. We next used Taqman‐based allelic discrimination assay to genotype a common variant (rs33912345: c.421A>C) and the rs1048372 SNP in two cohorts, respectively, composed of 557/387 and 590/448 POAG cases/age‐matched controls. An additional cohort of 153 POAG cases was subsequently recruited to assess the association of the rs33912345:c.421A>C and rs10483727 variants with more prominent changes in two POAG diagnostic parameters: retinal nerve fiber layer thickness and vertical cup/disc ratio, using spectral domain optical coherence tomography. The activity of the newly identified enhancer variants was assessed by transgenesis in zebrafish and luciferase assays.ResultsWe identified two known rare and two common variants in the SIX6 locus and a novel 4 bp deletion in the analyzed enhancer. Contrary to previous studies, we could not establish a significant association between the rs10483727 and rs33912345:c.421A>C variants and PAOG in the south Indian ethnicity but patients carrying the corresponding C or T risk alleles exhibited a dose‐dependent reduction of the thickness of the retinal nerve fiber layer and a significant increase in the vertical cup/disc ratio. Transgenesis in zebrafish and luciferase assays demonstrated that the newly identified 4 bp deletion significantly reduced reporter expression in cells of the retinal ganglion and amacrine layers, where human SIX6 is expressed.ConclusionAltogether, our data further support the implication of SIX6 variants as POAG risk factors and implicates SIX6 haploinsufficiency in POAG pathogenesis.

LncRNA-Six1 Encodes a Micropeptide to Activate Six1 in Cis and Is Involved in Cell Proliferation and Muscle Growth.

Long non-coding RNAs (lncRNAs) play important roles in epigenetic regulation of skeletal muscle development. In our previous RNA-seq study (accession number GSE58755), we found that lncRNA-Six1 is an lncRNA that is differentially expressed between White Recessive Rock (WRR) and Xinghua (XH) chicken. In this study, we have further demonstrated that lncRNA-Six1 is located 432 bp upstream of the gene encoding the protein Six homeobox 1 (Six1). A dual-luciferase reporter assay identified that lncRNA-Six1 overlaps the Six1 proximal promoter. In lncRNA-Six1, a micropeptide of about 7.26 kDa was found to play an important role in the lncRNA-Six1 in cis activity. Overexpression of lncRNA-Six1 promoted the mRNA and protein expression level of the Six1 gene, while knockdown of lncRNA-Six1 inhibited Six1 expression. Moreover, tissue expression profiles showed that both the lncRNA-Six1 and the Six1 mRNA were highly expressed in chicken breast tissue. LncRNA-Six1 overexpression promoted cell proliferation and induced cell division. Conversely, its loss of function inhibited cell proliferation and reduced cell viability. Similar effects were observed after overexpression or knockdown of the Six1 gene. In addition, overexpression or knockdown of Six1 promoted or inhibited, respectively, the expression levels of muscle-growth-related genes, such as MYOG, MYHC, MYOD, IGF1R, and INSR. Taken together, these data demonstrate that lncRNA-Six1 carries out cis-acting regulation of the protein-encoding Six1 gene, and encodes a micropeptide to activate Six1 gene, thus promoting cell proliferation and being involved in muscle growth.

Abstract A32: Upregulation of the Notch signaling pathway is associated with mortality and in vitro cell invasion in squamous cell carcinoma of the oral cavity

Abstract Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with low survival rates, which have not significantly improved despite advances in surgery and radiotherapy. Although human papillomavirus is a strong predictor of outcomes in oropharyngeal tumors, other HNSCC subtypes lack reliable prognostic and therapeutic biomarkers. The purpose of this study was to identify molecular targets to improve the management of oral cavity SCC (OSCC). We isolated RNA from 19 OSCCs treated surgically at UAB and evaluated gene expression using the NanoString nCounter system and PanCancer Pathways analysis module. Results were confirmed using data from the oral cavity anatomic subdivision of the Head and Neck Squamous Cell Carcinoma cancer study generated by the TCGA Research Network. Associated in vitro phenotype was assessed in HNSCC cell lines UM-SCC1, UM-SCC6 and FaDu treated with the activating Notch ligand DLL4 and included evaluation of gene expression, proliferation, cell cycle, migration, invasion, and radiosensitivity. We identified a significant association between upregulation of the Notch pathway and mortality in both the UAB and TCGA OSCC sample sets. In vitro activation of Notch signaling altered transcription in a pattern similar to that found in patient samples from the mortality cohorts. HNSCC cells with Notch activation had increased expression of pro-invasive genes FGF1, IL1B, SIX1, and CCNA1. In addition, Notch activated cells displayed increased cell migration and invasion. Proliferation, cell cycle distribution, and radiosensitivity were unchanged in ligand-treated cells. Our results identify a subset of OSCC patients who have increased Notch pathway activity and worsened outcomes. These clinical findings may reflect a more invasive cell phenotype driven by Notch-mediated changes in transcription. Additional studies are needed to fully elucidate the mechanism underlying this association and to investigate the efficacy of therapies targeting Notch signaling in OSCC. Citation Format: Alice N. Weaver, Tiffiny S. Cooper, Deborah Della Manna, Shi Wei, Eben L. Rosenthal, Eddy S. Yang. Upregulation of the Notch signaling pathway is associated with mortality and in vitro cell invasion in squamous cell carcinoma of the oral cavity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A32.

In-Silico Identification of Novel Resistant Genes for Fungal Pathogen Fusarium oxysporum f. sp. cubense Race 4: Causative Agent of Banana Vascular Wilt Disease

Cavendish, the most widely grown banana cultivar, is relatively resistant to Race 1 of Fusarium oxysporum f. sp. cubense (Foc1) that caused widespread Panama disease during the first half of the 20th century but is susceptible to Tropical Race 4 of the Foc (Foc TR4), a threat to world banana production. Foc TR4 can spread into the vascular system of banana roots during the early infection process. The genome of the diploid species Musa acuminata; the ancestor of majority of the triploid banana cultivars has recently been sequenced. The identification for the resistant gene in this cultivar is a challenge for the researchers as the resistant gene against Foc4 has not been identified, the major focus of this research work was to identify the pathogenic genes and some resistant genes against those pathogenic genes, to get some better results for the laboratory work. The four resistant genes I, I-1, I-2, and I-3 were identified which provides resistance against Foc4 in banana. About three pathogenic genes SIX1 (SIX1A, SIX1B, SIX1C), SIX2, and SIX8 of Foc4 were identified. Out of these pathogenic genes SIX8 showed the greatest number of interactions with the resistant proteins. So it is proposed that I-1, I-2 and I-3 genes can be used in the further research work for providing better improvements of the resistive cultivars in banana.

Effect of Six1,TGF-β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice

Objective:To research the effect of Six1,TGF-β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice. Method:Technology of RNA interference was used for silencing Six1 and TGF-β genes expression in laryngeal squamous cell, preparation Six1-targeting and TGF-β-targeting siRNA for transfecting into laryngeal squamous cell carcinoma, sieve out positive clone cell and amplify. Preparing bearing cancer mice, the mice were divided into five groups, group A (untransfected),group B(empty vector), group C(Six1-siRNA),group D(TGF-β-siRNA),group E(Six1+TGF-β-siRNA). 6-12mm when the tumor has grown, the mice were sacrificed by cervical. The size of each tumor and metastasis were observed and recorded.The protein expression of Six1,TGF-β and VEGF-C was determined by immunohistochemistry and Western blot.The mRNA of Six1,TGF-β and VEGF-C was determined by RT-PCR. Result:The average tumor volume and the number of metastasis cases in group B have no statistically significant compared with group A. The average tumor volume in group C, group D and group E has no significantly reduced, but there is a clear reduction of the number of metastasis cases compared with group A. The average tumor volume and number of metastasis cases in group E has no significantly reduced compared with group C and group D. Both protein and mRNA expression of Six1, TGF-β and VEGF-C in group B had no significant difference compared with group A. In group C, group D and group E,both protein and mRNA expression of VEGF-C was decreased,difference has statistically significant compared with group A. Both protein and mRNA expression of VEGF-C in group E had no significant difference compared with group C and group D. Conclusion:Both Six1 and TGF-β can mediate tumor lymphangiogenesis and lymph node metastasis by mediate the expression of VEGF-C. Suggest that Six1,TGF-β might be a potential therapeutic target for preventing lymph node metastasis of tumor.

Comparative analysis of the neurula transcriptomes of two species of flatfishes: Platichthys stellatus and Paralichthys olivaceus

Flatfish with left-right eye asymmetry are the most significant among vertebrates. However, the genetic basis for the control of this characteristic is still unclear. We propose that the gene(s) for eye asymmetry initially control minor differences in cell number in the tissues around the eyes during eye development. This minor difference is then amplified, causing eye migration during metamorphosis. Therefore, comparing the neurula transcriptomes between flatfish species with different eye-reversal mutants may provide very useful information to screen for genes involved in eye asymmetry. In this study, two cDNA libraries constructed from neurulas of P. stellatus (high ratio of eye reversal) and P. olivaceus (very low ratio of eye reversal) were sequenced and compared. There were 8121 and 8108 unigenes annotated to 32 categories in P. stellatus and P. olivaceus, respectively, and the highest KEGG pathways in both species were ‘signal transduction’, ‘immune system’, and ‘endocrine system’. In total, 62,692 and 18,938 putative single nucleotide polymorphisms (SNPs) were predicted in the P. stellatus and P. olivaceus transcriptomes, respectively. Furthermore, 8026 SNPs found in P. stellatus did not exist in P. olivaceus. Fifty-one SNPs were identified in nine genes (Fgf7, Wnt9, Sfrp2, Bmpr1B, Bmpr2, Pax3, Pax6, Six1 and Tgfβr2) related to eye development. In particular, Tgfβr2 with Asp77Glu found in P. stellatus but not in P. olivaceus will provide important information for screening genes associated with eye asymmetry.

Expanding the Phenotype of Treacher Collins Syndrome?

Treacher Collins Syndrome (TCS) is a congenital disorder of craniofacial development with variable phenotypic expression. Multiple genes have been implicated in TCS presentation, however most reported cases are caused by mutations in the TCOF1 gene. Here, we provide a description of a male infant with a familial mutation (c.4218dupG) in TCOF1 and concomitant holoprosencephaly (HPE), dysgenesis of the corpus callosum and cutis aplasia. Genetic analysis of common genes associated with HPE revealed a previously unreported variant of uncertain significance in the SIX3 gene but no definitive causal mutation. This is the first known case report of a CNS migrational malformation in TCS

Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets

Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

Association of EffectorSix6 with Vascular Wilt Symptoms Caused byFusarium oxysporumon Soybean

The Fusarium oxysporum species complex (FOSC) is a widely distributed group of fungi that includes both pathogenic and nonpathogenic isolates. In a previous study, isolates within the FOSC collected primarily from soybean were assessed for the presence of 12 fungal effector genes. Although none of the assayed genes was significantly associated with wilt symptoms on soybean, the secreted in xylem 6 (Six6) gene was present only in three isolates, which all produced high levels of vascular wilt on soybean. In the current study, a collection of F. oxysporum isolates from soybean roots and F. oxysporum f. sp. phaseoli isolates from common bean was screened for the presence of the Six6 gene. Interestingly, all isolates for which the Six6 amplicon was generated caused wilt symptoms on soybean, and two-thirds of the isolates showed high levels of aggressiveness, indicating a positive association between the presence of the effector gene Six6 and induction of wilt symptoms. The expression profile of the Six6 gene analyzed by quantitative reverse-transcription polymerase chain reaction revealed an enhanced expression for the isolates that caused more severe wilt symptoms on soybean, as established by the greenhouse assay. These findings suggest the suitability of the Six6 gene as a possible locus for pathogenicity-based molecular diagnostics across the various formae speciales.

SIX2 haploinsufficiency causes conductive hearing loss with ptosis in humans.

The ossicles represent one of the most fundamental morphological features in evolutionary biology of the mammalians. The mobile ossicular morphology abnormalities result in the severe conductive hearing loss. Development and patterning of the middle ear malformation depend on genetic and environmental causes. However, the genetic basis for the risk of congenital ossicle malformation is poorly understood. We show here nine affected individuals in a Chinese pedigree who had bilateral conductive hearing loss with ptosis. We performed whole-genome sequencing and array comparative genomic hybridization (CGH) analysis on DNA samples from the Chinese pedigree. We confirmed the presence of a novel 60 kb heterozygous deletion in size, encompassing SIX2 in our family. Mutation screening in 169 sporadic cases with external ear and middle ear malformations identified no pathogenic variant or polymorphism. We suggest SIX2 haploinsufficiency as a potential congenital factor could be attributed to developmental malformation of the middle ear ossicles and upper eyelid. To the best of our knowledge, this is the first report to provide a description of copy number variation in the SIX2 gene resulting in syndromic conductive hearing loss.

Association of Putative Fungal Effectors in Fusarium oxysporum with Wilt Symptoms in Soybean.

Fungi within the Fusarium oxysporum species complex can cause root rot, seedling blight, and wilt of soybean. Isolates recovered from soybean vary in aggressiveness and also the type of symptoms they produce. The aim of this study was to identify genetic markers to detect aggressive soybean wilt isolates. Eighty isolates collected primarily from soybean were tested in the greenhouse for their ability to produce wilt symptoms using susceptible 'Jack' soybean. The same 80 isolates were assessed for the presence of fungal effector genes Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, and Snf1. All polymerase chain reaction amplicons were sequenced, phylogenies were inferred, and analysis of molecular variance (AMOVA) was performed for 10 of the 12 genes. High incidence of vascular discoloration of roots or stems was observed with 3 isolates, while moderate to low levels of incidence were observed for 25 isolates. Fungal effector genes Fmk1, Fow1, PelA, Rho1, Sge1, and Snf1 were present in all isolates screened, while Pda1, PelD, Pep1, Prt1, Six1, and Six6 were dispersed among isolates. The Bayesian and AMOVA analyses found that the genes Fmk1, Fow1, Pda1, PelA, Rho1, Sge1, and Snf1 corresponded to previously designated clades based on tef1α and mitochondrial small subunit sequences. None of the genes had a significant association with wilt symptoms on soybean. Interestingly, the Six6 gene was only present in three previously known wilt isolates from soybean, common bean, and tomato; of these, the soybean and common bean isolates produced high levels of vascular wilt in our study.

Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.

Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells invitro. However, their roles in adult muscle regeneration invivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD.

A SIX1 Homolog in Fusarium oxysporum f. sp. conglutinans Is Required for Full Virulence on Cabbage.

Fusarium oxysporum is a soil-born fungus that induces wilt and root rot on a variety of plants. F. oxysporum f. sp. conglutinans (Foc) can cause wilt disease on cabbage. This study showed that a homolog of SIX1 protein in the Arabidopsis infecting isolate Fo5176 (Fo5176-SIX1) had four isoforms in the conidia of Foc by proteomic analysis. Thus, we analyzed the roles of protein Foc-SIX1. Gene expression analysis showed that, compared to the expression in mycelia, dramatically altered expression of Foc-SIX1 could be detected after infecting cabbages, and Foc-SIX1 was highly expressed in conidia under axenic culture condition. Furthermore, we knocked out the Foc-SIX1 gene and found that Foc-ΔSIX1 mutants had significantly reduced virulence compared with wild type isolate, and full virulence was restored by complementation of Foc-ΔSIX1 mutants with Foc-SIX1. Thus, we concluded that SIX1 in Foc was required for full virulence on cabbage. We also complemented Foc-ΔSIX1 with SIX1 gene in F. oxysporum f. sp. lycopersici (Fol) and found Foc-ΔSIX1::Fol-SIX1 mutants did not affect the virulence of Foc-ΔSIX1. The results confirmed that Fol-SIX1 was not capable of replacing the role of Foc-SIX1 in Foc on the disease symptom development of cabbage. The roles of Fol-SIX1 on virulence might rely on host specificity.

Development of a TaqMan Real‐Time Polymerase Chain Reaction Assay for Detection and Quantification of Fusarium oxysporum f. sp. lycopersici in Soil

AbstractFusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is an important disease of tomato. Pathogenicity and vegetative compatibility tests, although reliable, are laborious for the identification of FOL isolates and cannot efficiently quantify population densities of FOL in the soil. The objective of this study was to develop a rapid, sensitive and quantitative real‐time polymerase chain reaction (PCR) assay for detecting and quantifying FOL in soil. An inexpensive and relatively simple method for soil DNA extraction and purification was developed based on bead‐beating and a silica‐based DNA‐binding method. A TaqMan probe and PCR primers were designed using the DNA sequence of the species‐specific virulence gene SIX1, which is only present in isolates of FOL, not in isolates of other formae speciales or non‐pathogenic isolates of F. oxysporum. The real‐time PCR assay successfully amplified isolates of three races of FOL used in this study and quantified FOL DNA in soils, with a detection limit of 0.44 pg of genomic DNA of FOL in 20 μl of the real‐time PCR. A spiking test performed by adding different concentrations of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by the real‐time PCR assay and the concentration of conidia added. In addition, the real‐time PCR assay revealed a significant quadratic regression for a glasshouse experiment between disease severity and DNA concentration of FOL. The soil DNA extraction method and real‐time PCR assay developed in this study could be used to determine population densities of FOL in soil, develop threshold models to predict Fusarium wilt severity, identify high‐risk fields and measure the impact of cultural practices on FOL populations in soils.

Chromosomal anomalies at 1q, 3, 16q, and mutations of SIX1 and DROSHA genes underlie Wilms tumor recurrences.

Approximately half of children suffering from recurrent Wilms tumor (WT) develop resistance to salvage therapies. Hence the importance to disclose events driving tumor progression/recurrence. Future therapeutic trials, conducted in the setting of relapsing patients, will need to prioritize targets present in the recurrent lesions. Different studies identified primary tumor-specific signatures associated with poor prognosis. However, given the difficulty in recruiting specimens from recurrent WTs, little work has been done to compare the molecular profile of paired primary/recurrent diseases. We studied the genomic profile of a cohort of eight pairs of primary/recurrent WTs through whole-genome SNP arrays, and investigated known WT-associated genes, including SIX1, SIX2 and micro RNA processor genes, whose mutations have been recently proposed as associated with worse outcome. Through this approach, we sought to uncover anomalies characterizing tumor recurrence, either acquired de novo or already present in the primary disease, and to investigate whether they overlapped with known molecular prognostic signatures.Among the aberrations that we disclosed as potentially acquired de novo in recurrences, some had been already recognized in primary tumors as associated with a higher risk of relapse. These included allelic imbalances of chromosome 1q and of chromosome 3, and CN losses on chromosome 16q. In addition, we found that SIX1 and DROSHA mutations can be heterogeneous events (both spatially and temporally) within primary tumors, and that their co-occurrence might be positively selected in the progression to recurrent disease. Overall, these results provide new insights into genomic and genetic events underlying WT progression/recurrence.

Transcriptional Reactivation of OTX2, RX1 and SIX3 during Reprogramming Contributes to the Generation of RPE Cells from Human iPSCs.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation.