Potential Binding Sites For Transcription Factors Research Articles

Estrogen regulates the transcription of guppy isotocin receptors.

Estrogen can regulate oxytocin receptor expression, which is mediated through estrogen receptors (ESRs) in mammals, initiating parturition. To further study the reproductive physiological process of ovoviviparous teleosts, guppies (Poecilia reticulata) were employed as the research model in the present study to identify the transcriptional regulation of ESRs on isotocin receptors (itrs). Since guppy embryos develop inside the ovary, in the present study, the levels of itrs in the ovarian stroma of pregnant female guppies treated with estradiol (E2) in vitro were tested. E2 increased only itr2 mRNA levels 3 h post-treatment, with no variation in itr1 mRNA expression levels. In vivo, pregnant guppies were immersed in different concentrations of E2, significantly increasing the relative expression levels of itr1 and itr2 in the ovary. Moreover, based on dual-fluorescence in situ hybridization (ISH), both esrs and itrs mRNAs were localized in the same cells around the embryos in the ovary. To further investigate the regulation of itr transcription by estrogen, a luciferase reporter assay was performed, and the results demonstrated that E2 treatment could induce E2-dependent repression of luciferase activity in cells transfected with ESR1. However, overexpression of ESR2a or ESR2b caused a robust ligand-independent increase in itr2 promoter activity. Deletion analysis of the itr2 promoter indicated that there were novel potential ESR transcription factor-binding sites at -360 bp upstream of the 5' end of the itr2 promoter. Overall, our study provided novel results regarding the ESRs mediating the onset of parturition in ovoviviparous teleosts.

Conservation of putative transcription factor binding sites of co-expressed Plasmopara halstedii genes in two Phytophthora species

Oomycetes, a large group of fungus-like organisms, include some destructive plant pathogens causing enormous economic damage. Phylogenetically, oomycetes belong to the kingdom Straminipila and have diverse lifestyles, including saprotrophs and both general and specialized pathogens of various eukaryotic supergroups. A rapid increase in genomic studies and next-generation sequencing technologies have led to significant progress in understanding oomycete lifestyles. However, their genetics, including transcriptional regulation, have been studied to a much lesser extent. Here, we provide a cross-species analysis of oomycete promoter for providing a first step towards elucidating gene regulation networks related to pathogenicity and life cycle stages. The clustered DNA sequences of Plasmopara halstedii transcriptome time-series expression level dataset from a preliminary study have been used as a core reference for cross-species comparisons. Using a computational pipeline, 46 potential transcription factor binding site (TFBS) motifs in 25 clusters with functionally conserved downstream genes of downy mildew and two Phytophthora species, regardless of the gene expression levels of Phytophthora transcriptomes, were found. This can now be followed up by knock-out experiments in oomycete species amenable for genetic modification.

Identification of putative enhancer-like elements predicts regulatory networks active in planarian adult stem cells.

Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms.

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Genome analysis of Pseudomonas chlororaphis subsp. aurantiaca mutant strains with increased production of phenazines.

Genomes of three strains-phenazine producers-Pseudomonas chlororaphis subsp. aurantiaca (B-162 (wild type), mutant strain B-162/255, and its derivative B-162/17) were sequenced and compared. Comparison of a wild-type strain and B-162/255 mutant genomes revealed 32 mutations. 19 new mutations were detected in the genome of B-162/17. Further bioinformatics analysis allowedus to predict mutant protein functions and secondary structures of five gene products, mutations which might potentially influence phenazine synthesis and secretion in Pseudomonas bacteria. These genes encode phenylalanine hydroxylase transcriptional activator PhhR, type I secretion system permease/ATPase, transcriptional regulator MvaT, GacA response regulator, and histidine kinase. Amino acid substitutions were found in domains of studied proteins. One deletion in an intergenic region could affect a potential transcription factor binding site that participates in the regulation of gene that encodes ABC transporter.

Chromosome 10q26–driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

Identification and characterization of SlbHLH, SlDof and SlWRKY transcription factors interacting with SlDPD gene involved in costunolide biosynthesis in Saussurea lappa

The genes involved in costunolide biosynthesis in Saussurea lappa have been identified recently by our lab. However, the study of transcriptional regulators of these genes was lacking for better opportunities for engineering the pharmacologically important biosynthetic pathway. Therefore, we cloned the promoter region of diphosphomevalonate decarboxylase gene (DPD) and analyzed its cis-acting regulatory elements to reveal the potential transcription factor (TF) binding sites for Dof, bHLH and WRKY family proteins in the gene promoter. The transcriptome study approach followed by the hidden Markov model based search, digital gene expression, co-expression network analysis, conserved domain properties and evolutionary analyses were carried out to screen out seven putative TFs for the DPD-TF interaction studies. Yeast one-hybrid assays were performed and three TFs were reported, namely, SlDOF2, SlbHLH3 and SlWRKY2 from Dof, bHLH and WRKY families, respectively that interacted positively with the DPD gene of the costunolide biosynthetic pathway. The tissue specific relative gene expression studies also supported the linked co-expression of the gene and its interacting TFs The present report will improve the understanding of transcriptional regulation pattern of costunolide biosynthetic pathway.

Meta-Analysis of Transcriptome Data Detected New Potential Players in Response to Dioxin Exposure in Humans.

Dioxins are one of the most potent anthropogenic poisons, causing systemic disorders in embryonic development and pathologies in adults. The mechanism of dioxin action requires an aryl hydrocarbon receptor (AhR), but the downstream mechanisms are not yet precisely clear. Here, we performed a meta-analysis of all available transcriptome datasets taken from human cell cultures exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Differentially expressed genes from different experiments overlapped partially, but there were a number of those genes that were systematically affected by TCDD. Some of them have been linked to toxic dioxin effects, but we also identified other attractive targets. Among the genes that were affected by TCDD, there are functionally related gene groups that suggest an interplay between retinoic acid, AhR, and Wnt signaling pathways. Next, we analyzed the upstream regions of differentially expressed genes and identified potential transcription factor (TF) binding sites overrepresented in the genes responding to TCDD. Intriguingly, the dioxin-responsive element (DRE), the binding site of AhR, was not overrepresented as much as other cis-elements were. Bioinformatics analysis of the AhR binding profile unveils potential cooperation of AhR with E2F2, CTCFL, and ZBT14 TFs in the dioxin response. We discuss the potential implication of these predictions for further dioxin studies.

Genetic variability of the U5 and downstream sequence of major HIV-1 subtypes and circulating recombinant forms

The critical role of the regulatory elements at the 5′ end of the HIV-1 genome in controlling the life cycle of HIV-1 indicates that this region significantly influences virus fitness and its biological properties. In this study, we performed a detailed characterization of strain-specific variability of sequences from the U5 to upstream of the gag gene start codon of diverse HIV-1 strains by using next-generation sequencing (NGS) techniques. Overall, we found that this region of the HIV-1 genome displayed a low degree of intra-strain variability. On the other hand, inter-strain variability was found to be as high as that reported for gag and env genes (13–17%). We observed strain-specific single point and clustered mutations in the U5, PBS, and gag leader sequences (GLS), generating potential strain-specific transcription factor binding sites (TFBS). Using an infrared gel shift assay, we demonstrated the presence of potential TFBS such as E-box in CRF22_01A, and Stat 6 in subtypes A and G, as well as in their related CRFs. The strain-specific variation found in the sequence corresponding at the RNA level to functional domains of the 5ʹ UTR, could also potentially impact the secondary/tertiary structural rearrangement of this region. Thus, the variability observed in this 5′ end of the genomic region of divergent HIV-1 strains strongly suggests that functions of this region might be affected in a strain-specific manner. Our findings provide new insights into DNA–protein interactions that regulate HIV-1 replication and the influence of strain characterization on the biology of HIV-1 infection.

Global Survey and Expressions of the Phosphate Transporter Gene Families in Brassica napus and Their Roles in Phosphorus Response.

Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, currently little is known about these genes in oil crops. In this study, we aimed to characterize the five Pi transporter gene families (PHT1-5) in allotetraploid Brassica napus. We identified and characterized 81 putative PHT genes in B. napus (BnaPHTs), including 45 genes in PHT1 family (BnaPHT1s), four BnaPHT2s, 10 BnaPHT3s, 13 BnaPHT4s and nine BnaPHT5s. Phylogenetic analyses showed that the largest PHT1 family could be divided into two groups (Group I and II), while PHT4 may be classified into five, Groups I-V. Gene structure analysis revealed that the exon-intron pattern was conservative within the same family or group. The sequence characteristics of these five families were quite different, which may contribute to their functional divergence. Transcription factor (TF) binding network analyses identified many potential TF binding sites in the promoter regions of candidates, implying their possible regulating patterns. Collinearity analysis demonstrated that most BnaPHTs were derived from an allopolyploidization event (~40.7%) between Brassica rapa and Brassica oleracea ancestors, and small-scale segmental duplication events (~39.5%) in the descendant. RNA-Seq analyses proved that many BnaPHTs were preferentially expressed in leaf and flower tissues. The expression profiles of most colinearity-pairs in B. napus are highly correlated, implying functional redundancy, while a few pairs may have undergone neo-functionalization or sub-functionalization during evolution. The expression levels of many BnaPHTs tend to be up-regulated by different hormones inductions, especially for IAA, ABA and 6-BA treatments. qRT-PCR assay demonstrated that six BnaPHT1s (BnaPHT1.11, BnaPHT1.14, BnaPHT1.20, BnaPHT1.35, BnaPHT1.41, BnaPHT1.44) were significantly up-regulated under low- and/or rich- Pi conditions in B. napus roots. This work analyzes the evolution and expression of the PHT family in Brassica napus, which will help further research on their role in Pi transport.

3579 The role of CypD/mPTP during osteogenic differentiation - a potential target to prevent bone loss

OBJECTIVES/SPECIFIC AIMS: The study aims to further investigate how cyclophilin D (CypD), the key mPTP opening regulator, affects BMSCs fate and to determine potential regulatory mechanisms involved in CypD regulation during osteogenesis. METHODS/STUDY POPULATION: We evaluated CypD mRNA expression in mouse BMSCs and in osteogenic-like (OL) cells during the course of OB differentiation. CypD protein level was also probed. Moreover, BMSCs had their mPTP activity recorded during osteoinduction. We further analyzed the effect of CypD genetic deletion on osteogenesis in vitro and in vivo. For our in vivo model, we performed the ectopic bone formation assay to asses differences in ossicle formation when CypD KO BMSCs were transplanted compared to wild type littermate BMSCs. In our in vitro model, we transfected OL cells with either CypD gain of function or CypD loss of function vector and measured their osteogenic differentiation potential. Additionally, we treated BMSCs with CypD inhibitor and compare to non-treated BMSCs for mineralization level. To determine potential regulatory mechanisms involved in CypD regulation, we analyzed the CypD gene (Ppif) promoter for potential transcription factor (TF) binding sites and found multiple Smad-binding elements within this promoter. Smads (Smad1, 5, 8) are TFs downstream from Bone Morphogenic Protein (BMP) signaling pathway that transmit cell differentiation signaling, and exert either activating or inhibitory effects on a variety of genes. We also transfect OL cells with Smad1 vector and analyzed for CypD mRNA levels. RESULTS/ANTICIPATED RESULTS: - Our data showed that CypD mRNA levels decreased in both primary cells and OL cells at day 7 and day 14 in osteogenic media. - Osteogenic induction also decreased mPTP activity. - In vivo ectopic bone formation assay showed increased ossicle fo DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that downregulation of CypD increases OB differentiation due to improved OxPhos activity led by mPTP closure. Our results corroborate reports of CypD downregulation and mPTP closure during neuronal differentiation in developing rat brains as well as in cardiomyocyte differentiation in developing mouse hearts. Our studies also suggest a yet unknown mechanism linking differentiation signaling with mitochondrial function – BMP/Smad mediated downregulation of CypD transcription. As initially mentioned, in a previous study, our lab showed that CypD KO mice present higher mitochondrial function and osteogenicity in aged BMSCs and less osteoporosis burden. Taken together, these results suggest that CypD can be a potential target to prevent bone loss in aging.

Molecular Characterization, Expression Profiles, and Immunostimulation Responses of TRAF6 and TAK1 in Japanese Flounder (Paralichthys olivaceus)

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important adaptor molecules in Toll-like receptor (TLR) signaling pathway. In this study, TRAF6 (PoTRAF6) and TAK1 (PoTAK1) were cloned and characterized in Japanese flounder (Paralichthys olivaceus). The full-length cDNA sequence of PoTRAF6 is 1953 bp, with an open reading frame (ORF) of 1713 bp encoding a putative protein of 570 amino acids. PoTRAF6 contains one really interesting new gene (RING) domain, two zinc fingers, one coiled-coil region, and one meprin and TRAF homology (MATH) domain, which shows a high similarity to TRAF6s in other species. The full-length PoTAK1 cDNA sequence is 2086 bp, with an ORF of 1728 bp that encodes a putative protein of 575 amino acids. PoTAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The promoter regions of PoTRAF6 and PoTAK1 were also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of these two genes were examined in developmental stages, different tissues, and challenged samples. PoTRAF6 and PoTAK1 were expressed during the whole developmental stages, and the highest expressions were in intestine and heart, respectively. In challenged embryonic cells with LPS, CpG ODN, and poly I:C, the expressions of PoTRAF6 and PoTAK1 were both up-regulated significantly. These results suggest that PoTRAF6 and PoTAK1 play crucial roles in immune responses and may be involved in the developmental process of Japanese flounder.

The CtBP1-HDAC1/2-IRF1 transcriptional complex represses the expression of the long noncoding RNA GAS5 in human osteosarcoma cells.

Growth Arrest Specific 5 (GAS5), a long noncoding RNA (lncRNA), functions as a tumor suppressor in multiple cancers. However, its function, downstream targets and upstream regulatory mechanism are still obscure in osteosarcoma cells. Here, we discovered that GAS5 was downregulated in cancerous osteosarcoma tissues and cells. Using a microarray analysis, we identified that GAS5 can regulate the expression of TP53, Bax, Bim, DDB2, TGFB and ROS1 in osteosarcoma cells. Specifically, GAS5 overexpression in the U2OS osteosarcoma cell line induced TP53, Bax and Bim levels but inhibited DDB2, TGFB and ROS1 expression, resulting in the inhibition of cell proliferation, invasion, colony formation and in vivo tumor formation. By analyzing the GAS5 promoter region (-2000), we identified several potential transcription factor-binding sites including NF-ĸB, IK-1, AP-1, SP1 and IRF1. By individually knocking down these transcription factors, we found that only knockdown of IRF1 affected GAS5 expression. Using immunoprecipitation (IP), mass spectrometry assays, and co-IP assays, we identified that IRF1 formed a transcriptional complex with Histone Deacetylase 1 and 2 (HDAC1/2) and C-terminal binding protein 1 (CtBP1). Functional analyses indicated that the CtBP1-HDAC1/2-IRF1 complex specifically bound to the GAS5 promoter and regulated its expression and downstream events. Knockdown of CtBP1 or overexpression of IRF1 in osteosarcoma cells can significantly reverse their oncogenic phenotypes. Altogether, our results indicated that the CtBP1-HDAC1/2-IRF1 transcriptional complex inhibited GAS5-mediated signaling in osteosarcoma cells, and it might be a potential therapeutic target for osteosarcoma treatment.

Rapid Communication: Genome-wide association analyses identify loci associated with colostrum production in Jersey cattle1.

Consumption of an adequate volume of high-quality colostrum soon after birth is critical for a calf's health. Few studies have focused on the genetics associated with colostrum production, even though several dairy herds in the United States have reported incidents of low to no colostrum production during the fall and winter seasons. The objectives of this study were to identify loci associated with quantity and quality of colostrum production in a herd of Jersey cattle (n = 345) and to identify potential positional candidate genes and/or transcription factor binding site motifs located near associated loci. Cattle that freshened between the months of October and December of 2016 at a single dairy were enrolled in the study and produced on average 3.03 kg of colostrum at their first milking. This study included 112 cattle genotyped with the GeneSeek GGP50k BeadChip and another 233 cattle previously genotyped with various other arrays. The 233 cattle genotyped at lower densities were imputed to the GGP50k BeadChip density using BEAGLE 4.1.1, and 2 genome-wide association analyses (GWAA) were conducted using an additive efficient mixed-model association expedited method with a genomic relationship matrix (EMMAX-GRM). The first GWAA investigated loci associated with colostrum quantity and identified 7 loci: 6 that were moderately associated (5 × 10-07 > P < 1 × 10-05) and 1 that was strongly associated (P < 5 × 10-07). The second GWAA investigated colostrum quality and identified 1 moderately (5 × 10-07 > P < 1 × 10-05) associated locus. Five loci harbored positional candidate genes which had functional relevance to colostrum production, and 1 locus located on BTA10 contained a transcription factor binding site motif for TFAP2A which has previously been linked to mammary gland development. Pseudoheritability estimates were moderate for colostrum quality (0.19 ± 0.06) and high for colostrum quantity (0.76 ± 0.11), suggesting that genomic selection for these traits would be possible. Diminished colostrum quantity or quality can have a significant impact on herd health and herd economics. The identification of loci, positional candidate genes, and transcription factor binding site motifs associated with colostrum production could be used in genomic selection to allow producers to select for cattle with good colostrum production, improving calf health, and reducing economic losses to the herd.

GTRD: a database on gene transcription regulation-2019 update.

The current version of the Gene Transcription Regulation Database (GTRD; http://gtrd.biouml.org) contains information about: (i) transcription factor binding sites (TFBSs) and transcription coactivators identified by ChIP-seq experiments for Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Arabidopsis thaliana; (ii) regions of open chromatin and TFBSs (DNase footprints) identified by DNase-seq; (iii) unmappable regions where TFBSs cannot be identified due to repeats; (iv) potential TFBSs for both human and mouse using position weight matrices from the HOCOMOCO database. Raw ChIP-seq and DNase-seq data were obtained from ENCODE and SRA, and uniformly processed. ChIP-seq peaks were called using four different methods: MACS, SISSRs, GEM and PICS. Moreover, peaks for the same factor and peak calling method, albeit using different experiment conditions (cell line, treatment, etc.), were merged into clusters. To reduce noise, such clusters for different peak calling methods were merged into meta-clusters; these were considered to be non-redundant TFBS sets. Moreover, extended quality control was applied to all ChIP-seq data. Web interface to access GTRD was developed using the BioUML platform. It provides browsing and displaying information, advanced search possibilities and an integrated genome browser.

Identification of suh gene and evidence for involvement of notch signaling pathway on gonadal differentiation of Yellow River carp (Cyprinus carpio).

The suh gene is crucial in Notch pathway and regulates mammalian gonad development. In this study, the sequences of suh1 and suh2 genes in Yellow River carp (Cyprinus carpio) were verified. The partial 5'-flanking regions of suh1 and suh2 were analyzed and several potential transcription factor-binding sites were identified. Phylogenetic, gene structure, and chromosome synteny analyses revealed that carp suh1 and suh2 were orthologs and homologous to vertebrate suh. Investigation of the expression profiles of suh1 and suh2 with qPCR showed that these genes were abundant in the brain and gonad of carp, with suh1 exhibiting sexual dimorphism expression pattern in gonad. To study the relationship between gonad differentiation and Notch signaling, primordial gonads were exposed to DAPT, an inhibitor of Notch signaling, in vitro and in vivo. The results revealed a significant downregulation of suh1 and other Notch genes in vitro. In addition, expression of male-biased genes, such as amh, dmrt1, etc., was downregulated, whereas that of female-biased genes, such as foxl2, gdf9, etc., was upregulated. When the primordial gonads were subjected to long-term DAPT exposure, an increased proportion of ovary and delay in testis development were observed. These results suggest that suh gene may have a conservative function between teleosts and mammals. Furthermore, Notch signaling was found to be involved in gonad differentiation in Yellow River carp, and DAPT was noted to inhibit and enhance the expression of male- and female-biased genes, respectively, and induce the increase in number of females.

Sequence-based prediction of putative transcription factor binding sites in DNA sequences of any length.

A transcription factor (TF) is a protein that regulates gene expression by binding to specific DNA sequences. Despite the recent advances in experimental techniques for identifying transcription factor binding sites (TFBS) in DNA sequences, a large number of TFBS are to be unveiled in many species. Several computational methods developed for predicting TFBS in DNA are tissue- or species-specific methods, so cannot be used without prior knowledge of tissue or species. Some computational methods are applicable to finding TFBS in short DNA sequences only. In this paper we propose a new learning method for predicting TFBS in DNA of any length using the composition, transition and distribution of nucleotides and amino acids in DNA and TF sequences. In independent testing of the method on datasets that were not used in training the method, its accuracy and MCC were as high as 81.84% and 0.634, respectively. The proposed method can be a useful aid for selecting potential TFBS in a large amount of DNA sequences before conducting biochemical experiments to empirically determine TFBS. The program and data sets are available at http://bclab.inha.ac.kr/TFbinding.

Antineoplastic effects of histone deacetylase inhibitors in neuroendocrine cancer cells are mediated through transcriptional regulation of Notch1 by activator protein 1.

Notch signaling is minimally active in neuroendocrine (NE) cancer cells. While histone deacetylase inhibitors (HDACi) suppress NE cancer growth by inducing Notch, the molecular mechanism underlying this interplay has not yet been defined. NE cancer cell lines BON, H727, and MZ‐CRC‐1 were treated with known HDACi Thailadepsin‐A (TDP‐A) and valproic acid (VPA), and Notch1 mRNA expression was measured with RT‐PCR. Truncated genomic fragments of the Notch1 promotor region fused with luciferase reporter were used to identify the potential transcription factor (TF) binding site. The key regulatory TF was identified with the electrophoretic mobility shift assay (EMSA). The effect of HDACi on Notch1 level was determined before and after silencing the TF. TDP‐A and VPA induced Notch1 mRNA in a dose‐dependent manner. A functional DNA motif at −80 to −52 from the Notch1 start codon responsible for the HDACi‐dependent Notch1 induction was identified. Mutation of this core sequence failed to induce luciferase activity despite HDACi treatment. EMSA showed the greatest gel shift with AP‐1 in nuclear extracts. Knockdown of AP‐1 significantly attenuated the effect of HDACi on Notch1 induction. Interestingly, AP‐1 transfection did not alter Notch1 level, suggesting that AP‐1 is necessary but insufficient for HDACi activation of Notch1. Therefore, AP‐1 is the TF that binds to a specific transcription‐binding site within the Notch1 promotor region to trigger Notch1 transcription. Elucidating the HDACi activation mechanism may lead to the development of novel therapeutic options against NE cancers and facilitate the identification of clinical responders and prevent adverse effects.

Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface-binding.

Several Frankia strains have been shown to be lead-resistant. The mechanism of lead resistance was investigated for Frankia sp. strain EAN1pec. Analysis of the cultures by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDAX) and Fourier transforming infrared spectroscopy (FTIR) demonstrated that Frankia sp. strain EAN1pec undergoes surface modifications and binds high quantities of Pb+2. Both labelled and unlabelled shotgun proteomics approaches were used to determine changes in Frankia sp. strain EAN1pec protein expression in response to lead and zinc. Pb2+ specifically induced changes in exopolysaccharides, the stringent response, and the phosphate (pho) regulon. Two metal transporters (a Cu2+-ATPase and cation diffusion facilitator), as well as several hypothetical transporters, were also upregulated and may be involved in metal export. The exported Pb2+ may be precipitated at the cell surface by an upregulated polyphosphate kinase, undecaprenyl diphosphate synthase and inorganic diphosphatase. A variety of metal chaperones for ensuring correct cofactor placement were also upregulated with both Pb+2 and Zn+2 stress. Thus, this Pb+2 resistance mechanism is similar to other characterized systems. The cumulative interplay of these many mechanisms may explain the extraordinary resilience of Frankia sp. strain EAN1pec to Pb+2. A potential transcription factor (DUF156) binding site was identified in association with several proteins identified as upregulated with heavy metals. This site was also discovered, for the first time, in thousands of other organisms across two kingdoms.

Specific expression and promoter analysis of the albumin gene promoter of the duck (Anas platyrhynchos domesticus)

ABSTRACT1. Albumin (ALB) is a serum protein most highly expressed in liver and regarded as an effective indicator for liver pathologies. The objectives of this study were to determine the expression of duck ALB gene (duALB) in various non-hepatic tissues and identify the potential cis-regulatory elements in the promoter.2. A model was established to assess duALB promoter activity in different cell lines by construction of a duALB promoter-driven GFP (Green Fluorescent Protein)-expressing vector, which exhibited high expression activity in liver-derived cells and lower expression in other cells. Through the firefly luciferase reporter gene driven by a series of constructs carrying progressive deletions, the core transcriptional regulatory region within the duALB promoter was identified. Mutations in candidate-binding sites were made by site-directed mutagenesis.3. The core transcriptional regulatory region was located in the −190/−51 bp region. This region contains three potential transcription factor-binding sites, one each for hepatocyte nuclear factor (HNF-3β) (−158/−149), CCAAT/Enhancer-binding protein element (C/EBPα) (−119/−107) and nuclear factor-1 (HNF-1) (−67/−57). Site-directed mutagenesis of HNF-1 and C/EBPα-binding sites resulted in a significant reduction in duALB promoter activity. Two potential cis-regulatory elements (C/EBPα and HNF-1) were responsible for its transcriptional activity in liver-derived cells.4. These findings contribute to the further understanding of the fundamental mechanisms of ALB gene regulation and the use of tissue-specific gene promoters to regulate tissue-specific expression of exogenous genes in vivo.