Cis Regulatory Elements

Hereditary transthyretin (TTR) amyloidosis (hATTR) is a rare life-threatening disorder caused by amyloidogenic coding mutations located in TTR gene. To understand the high phenotypic variability observed among carriers of TTR disease-causing mutations, we conducted an epigenome-wide association study (EWAS) assessing more than 700,000 methylation sites and testing epigenetic difference of TTR coding mutation carriers vs . non-carriers. We observed a significant methylation change at cg09097335 site located in Beta-secretase 2 ( BACE2 ) gene (standardized regression coefficient = −0.60, p  = 6.26 × 10 –8 ). This gene is involved in a protein interaction network enriched for biological processes and molecular pathways related to amyloid-beta metabolism (Gene Ontology: 0050435, q  = 0.007), amyloid fiber formation (Reactome HSA-977225, q  = 0.008), and Alzheimer’s disease (KEGG hsa05010, q  = 2.2 × 10 –4 ). Additionally, TTR and BACE2 share APP (amyloid-beta precursor protein) as a val...

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

AimsAmyloidosis is caused by deposition of abnormal protein fibrils, leading to damage of organ function. Hereditary amyloidosis represents a monogenic disease caused by germline mutations in 11 amyloidogenic precursor protein genes. One of the important but non-specific symptoms of amyloidosis is hypertrophic cardiomyopathy. Diagnostics of hereditary amyloidosis is complicated and the real cause can remain overlooked. We aimed to design hereditary amyloidosis gene panel and to introduce new next-generation sequencing (NGS) approach to investigate hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance.MethodsDesign of target enrichment DNA library preparation using Haloplex Custom Kit containing 11 amyloidogenic genes was followed by MiSeq Illumina sequencing and bioinformatics identification of germline variants using tool VarScan in a cohort of 40 patients.ResultsWe present design of NGS panel for 11 genes (TTR, FGA, APOA1, APOA2, L...

The identification of transcription factor binding sites, enhancers, and transcriptional target genes often relies on the integration of gene expression profiling and computational cis-regulatory sequence analysis. Methods for the prediction of cis-regulatory elements can take advantage of comparative genomics to increase signal-to-noise levels. However, gene expression data are usually derived from only one species. Here we investigate tissue-specific cross-species gene expression profiling by high-throughput sequencing, combined with cross-species motif discovery. First, we compared different methods for expression level quantification and cross-species integration using Tag-seq data. Using the optimal pipeline, we derived a set of genes with conserved expression during retinal determination across Drosophila melanogaster, Drosophila yakuba, and Drosophila virilis. These genes are enriched for binding sites of eye-related transcription factors including the zinc-finger Glass, a ma...

Images form a significant information source in published biomedical articles, which is under-utilized in biomedical document classification and retrieval. Much current work on biomedical image retrieval and classification uses simple, standard image representation employing features such as edge direction or gray scale histograms. In our earlier work we have used such features as well to classify images, where image-class-tags have been used to represent and classify complete articles. Here we focus on a different literature classification task: identifying articles discussing cis-regulatory elements and modules, motivated by the need to understand complex gene-networks. Curators attempting to identify such articles use as a major cue a certain type of image in which the conserved cis-regulatory region on the DNA is shown. Our experiments show that automatically identifying such images using common image features (such as gray scale) is highly error prone. However, using Optical Character Recognition (OCR) to extract alphabet characters from images, calculating character distribution and using the distribution parameters as image features, forms a novel image representation, which allows us to identify DNAcontent in images with high precision and recall (over 0.9). Utilizing the occurrence of DNA-rich images within articles, we train a classifier to identify articles pertaining to cis-regulatory elements with a similarly high precision and recall. Using OCR-based image features has much potential beyond the current task, to identify other types of biomedical sequence-based images showing DNA, RNA and proteins. Moreover, automatically identifying such images is applicable beyond the current use-case, in other important biomedical document classification tasks.

Trehalose-6-phosphate phosphatase (TPP) genes take part in trehalose metabolism and also in stress tolerance, which has been well documented in many species but poorly understood in wheat. The present research has identified a family of 31 TPP genes in Triticum aestivum L. through homology searches and classified them into five clades by phylogenetic tree analysis, providing evidence of an evolutionary status with Hordeum vulgare, Brachypodium distachyon and Oryza sativa. The exon-intron distribution revealed a discrete evolutionary history and projected possible gene duplication occurrences. Furthermore, different computational approaches were used to analyze the physical and chemical properties, conserved domains and motifs, subcellular and chromosomal localization, and three-dimensional (3-D) protein structures. Cis-regulatory elements (CREs) analysis predicted that TaTPP promoters consist of CREs related to plant growth and development, hormones, and stress. Transcriptional anal...

Amylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT-HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the appro...

Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control the majority of developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering against mutations in non-coding regulatory regions of genes implicated in human disease. Technological advances in genome editing and live imaging have shed light on how shadow enhancers establish precise gene expression patterns and confer phenotypic robustness. Shadow enhancers can interact in complex ways and may also help drive the formation of transcriptional hubs within the nucleus. Despite their apparent redundancy, the prevalence and evolutionary conservation of shadow enhancers underscore their key role in emerging metazoan gene regulatory networks.

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of subset of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

Scientists are only beginning to develop methods for whole genome comparisons. Computational methods which do not incorporate evolutionary relationships in their models are inadequate when there is high correlation between the DNA sequence data from closely related species. New genomes from various species are often sequenced specifically because of their relationships to other species as researchers wish to learn how species are differentiated at the DNA sequence level. Current methods do not provide a computational platform for efficiently analyzing data from many genomes simultaneously. Genomic scale analysis tends to result in hundreds of thousands of files across gigabytes of disk space with much redundancy. As the library of complete genomes grows, researchers will analyze larger sets of more complex genomes. A software system which scales to facilitate these multiple genome analyses will be necessary. This thesis aims to provide a computational system for identifying cis-regulatory elements in closely related genomes. As a demonstration, an analysis of cis-regulatory elements in E. coli and related bacterial genomes is provided. Previous computational methods for such an analysis are somewhat ineffective because the species are closely related. This closeness causes statistical problems due to the high correlation between the sequence data in the genomes. Methods for discovering potential cis-regulatory elements from DNA sequence data are reviewed, and a new Gibbs Sampling method is provided to address the correlation problem by accounting for evolutionary distances between species. A new software system is also shown to provide a computational platform necessary for more complex, collaborative analyses in the future. The software framework provides an object-oriented representation for the Gibbs Phylogenetic Sampler and generically lays the groundwork to accommodate other statistical bioinformatics applications. Many sampling strategies, biological data structures, and mathematical models may be shared between various analysis applications. On synthetic data, the new Gibbs Sampling method demonstrates greater positive predictive value and significantly greater specificity than other, comparable methods. On real data, the new method also produces encouraging results. A comparison of features between this and other methods is also provided. xii

Whole genome comparisons of distantly related species effectively predict biologically important sequences-core genes and cis-acting regulatory elements (REs)-but require experimentation to verify biological activity. To examine the efficacy of comparative genomics in identification of active REs from anonymous, non-coding (NC) sequences, we generated a novel alignment of the human and draft zebrafish genomes, and contrasted this set to existing human and fugu datasets. We tested the transcriptional regulatory potential of candidate sequences using two in vivo assays. Strict selection of nongenic elements which are deeply conserved in vertebrate evolution identifies 1744 core vertebrate REs in human and two fish genomes. We tested 16 elements in vivo for cis-acting gene regulatory properties using zebrafish transient transgenesis and found that 10 (63%) strongly modulate tissue-specific expression of a green fluorescent protein reporter vector. We also report a novel quantitative enhancer assay with potential for increased throughput based on normalized luciferase activity in vivo. This complementary system identified 11 (69%; including 9 of 10 GFP-confirmed elements) with cis-acting function. Together, these data support the utility of comparative genomics of distantly related vertebrate species to identify REs and provide a scaleable, in vivo quantitative assay to define functional activity of candidate REs.

In this review, the recent progress on genetic transformation of forest trees were discussed. Its described also, different applications of genetic engineering for improving forest trees or understanding the mechanisms governing genes expression in woody plants.

Pluripotent hematopoietic stem cells undergo maturation to regenerate blood cells. T-cell and B-cell maturation from common lymphoid progenitors (CLPs) is regulated by distinct transcription factors and signaling pathways. Notch signaling, GATA3, and TCF1 drive T-cell fate, while EBF1, PU.1, E2A, Pax5, TCF1, and Foxo1 regulate B-cell differentiation. Alterations in these factors can cause lineage deviations. Gene expression profiling of the chick thymus and bursa reveals dynamic expression patterns of transcription factors, cytokines, and signal molecules. Notch1 and Dll1 expression that increase during later stages indicate the ongoing role of Notch signaling in T-cell lineage maintenance. The current study aimed to identify differentially expressed genes during the development of the avian immune organs, focusing on the thymus and bursa using 24 Ross 308 avian breed. The mRNA libraries from these organs were analyzed using quantitative Real-time PCR analysis at six time points spanning the embryonic ages (days 15 and 18) and post-hatch age (days 3, 7, 14, and 28). The data for the gene expression indicated significant variations across different stages of immune organ development. Differential gene expression was observed between sorted T and B-cells, with GATA3, CD3e, CD4, and Ptprc showing higher expression in the T-cell population, and Pax5 and CD81 exhibiting higher expression in the B-cell population. Notably, ENO1 and IRF4 showed higher expression in T-cells at E15 and B-cells at E18. The study highlights the importance of regulatory factors and genes in maintaining cellular identity, furthers the understanding of avian immunology, and has the potential for improving poultry health and studying immune-related diseases in humans. These findings pave the way for further research on the role of biochemical components under important disease conditions in avian immunology and their potential applications for poultry health.

Respiratory failure associated with COVID-19 has placed focus on the lungs. Here, we present single-nucleus accessible chromatin profiles of 90,980 nuclei and matched single-nucleus transcriptomes of 46,500 nuclei in non-diseased lungs from donors of ~30 weeks gestation,~3 years and ~30 years. We mapped candidate cis-regulatory elements (cCREs) and linked them to putative target genes. We identified distal cCREs with age-increased activity linked to SARS-CoV-2 host entry gene TMPRSS2 in alveolar type 2 cells, which had immune regulatory signatures and harbored variants associated with respiratory traits. At the 3p21.31 COVID-19 risk locus, a candidate variant overlapped a distal cCRE linked to SLC6A20, a gene expressed in alveolar cells and with known functional association with the SARS-CoV-2 receptor ACE2. Our findings provide insight into regulatory logic underlying genes implicated in COVID-19 in individual lung cell types across age. More broadly, these datasets will facilitate...

Images form a rich information source, which remains underutilized in biomedical document classification. We present here work that uses both image-and text-based features in order to identify articles of interest, in this case, pertaining to cis-regulatory modules in the context of gene-networks. Extending on our new idea, which we have recently introduced, of using OCR-based features to identify DNA contents in images, we combine image and text based classifiers to categorize documents as relevant or irrelevant to cis-regulatory modules. Using a set of hundreds of articles, marked by experts as relevant or irrelevant to cisregulatory modules, we train/test image and text based classifiers, as well as classifiers integrating both. Our results indicate that the latter show the best performance with Recall, F-measure and Utility measures all above 0.9, demonstrating the significance of incorporating image data, and specifically OCR-based features, into the document categorization process. Moreover, the use of character distribution properties to represent images is directly relevant to other biomedical images containing text (e.g. RNA, proteins). Diagrams and other images containing text are also prevalent outside the biomedical domain, hence the work stands to be applicable and beneficial in other application areas.

BackgroundFrog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions.ResultsUsing a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, b...

Cis-regulatory elements (CREs) are non-coding DNA regions involved in the spatiotemporal regulation of gene expression. Gene regulatory changes drive animal development and play major roles during evolution of animal body plans. Therefore, we believe that determining CREs at different developmental stages and across animal lineages is critical to understand how evolution operates through development. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) is a powerful technique for the study of CREs that takes advantage of Tn5 transposase activity. Starting from fewer than 10 5 cells, in a 1-day procedure, it is possible to detect, at a genome-wide level, CREs located in open chromatin regions with high resolution. Here, we describe a detailed step-by-step ATAC-seq protocol for invertebrate chordate marine embryos. We have successfully applied this technique to amphioxus and two species of tunicate embryos. We also show an easy workflow to analyze data generated with this technique. Moreover, we point out that this method and our bioinformatic pipeline are efficient to detect CREs associated with Wnt signaling pathway by simply using embryos treated with a drug that perturbs this pathway. This approach can be extended to other signaling pathways and also to embryo mutants for critical genes. Our results therefore demonstrate the power of ATAC-seq for the identification of CREs that play essential functions during animal development in a wide range of invertebrate or vertebrate animals.

Background: Positional weight matrix (PWM) is a de facto standard model to describe transcription factor (TF) DNA binding specificities. PWMs inferred from in vivo or in vitro data are stored in many databases and used in a plethora of biological applications. This calls for comprehensive benchmarking of public PWM models with large experimental reference sets. Results: Here we report results from all-against-all benchmarking of PWM models for DNA binding sites of human TFs on a large compilation of in vitro (HT-SELEX, PBM) and in vivo (ChIP-seq) binding data. We observe that the best performing PWM for a given TF often belongs to another TF, usually from the same family. Occasionally, binding specificity is correlated with the structural class of the DNA binding domain, indicated by good cross-family performance measures. Benchmarking-based selection of family-representative motifs is more effective than motif clustering-based approaches. Overall, there is good agreement between in vitro and in vivo performance measures. However, for some in vivo experiments, the best performing PWM is assigned to an unrelated TF, indicating a binding mode involving protein-protein cooperativity. Conclusions: In an all-against-all setting, we compute more than 18 million performance measure values for different PWM-experiment combinations and offer these results as a public resource to the research community. The benchmarking protocols are provided via a web interface and as docker images. The methods and results from this study may help others make better use of public TF specificity models, as well as public TF binding data sets.

Fifty years ago, Francois Jacob and Jacques Monod published a seminal study in the Journal of Molecular Biology on the role of "regulator genes" in controlling the expression of "structural genes" in bacteria. This turned out to accurately define the transcription factors that act to determine the subset of genes to be expressed in each cell, in interaction with the external milieu. If these findings were rapidly recognized as a milestone, the contributions of this paper to collective scientific thought are even more impressive with regard to recent functional genomics. The authors already proposed in 1961 "that the genome contains not only a series of blueprints, but a coordinated program of protein synthesis and the means of controlling its execution." This represents an extraordinary vision of the way biological systems are hardwired by genomic information, a central concept in our current view of physiology, development, and evolution. Genetic analyses in model systems later provided an unbiased portal into the molecular control of embryonic development, showing the importance of transcription factors and signaling pathways that regulate their outputs. For example, Eric Wieschaus and Christiane Nüsslein-Volhard showed that most mutations affecting the embryonic pattern in Drosophila alter regulator genes. The discovery of so-called homeotic genes by Ed Lewis and their products (which contain the homeodomain DNA-binding motif) by Walter Gehring and colleagues further demonstrate that transcription factors govern both body plan establishment and the final differentiation of tissues and organs. Three decades later, major advances have been made in our comprehension of how various protein motifs mediate specific DNA recognition, exemplified by recent successes in designing artificial proteins to target a given genomic region. Deciphering the genome of hundreds of species has revealed that transcription factors are an abundant class of molecules (representing 5-10% of the total number of proteins), displaying unexpected levels of evolutionary conservation. It bears reminding that, even in genetically amenable systems, the function of most transcription factors has yet to be discovered. While the general concepts of transcriptional regulation are now well established, a major challenge resides in understanding the logic and physical elements implementing these regulatory interactions within a given cell in a developing organism. A central problem to be addressed is how transcriptional programs are set up and then modified throughout the successive steps of embryonic development. This volume provides a broad range of insights into the molecular determinants of such transcriptional switches during development.

The emergence of new genes throughout evolution requires rewiring and extension of regulatory networks. However, the molecular details of how the transcriptional regulation of new gene copies evolves remain largely unexplored. Here we show how duplication of a transcription factor gene allowed the emergence of two independent regulatory circuits. Interestingly, the ancestral transcription factor was promiscuous and could bind different motifs in its target promoters. After duplication, one paralogue evolved increased binding specificity so that it only binds one type of motif, whereas the other copy evolved a decreased activity so that it only activates promoters that contain multiple binding sites. Interestingly, only a few mutations in both the DNA-binding domains and in the promoter binding sites were required to gradually disentangle the two networks. These results reveal how duplication of a promiscuous transcription factor followed by concerted cis and trans mutations allows e...

The identification of promoters is an essential step in the genome annotation process, providing a framework for gene regulatory networks and their role in transcription regulation. Despite considerable advances in the high-throughput determination of transcription start sites (TSSs) and transcription factor binding sites (TFBSs), experimental methods are still time-consuming and expensive. Instead, several computational approaches have been developed to provide fast and reliable means for predicting the location of TSSs and regulatory motifs on a genome-wide scale. Numerous studies have been carried out on the regulatory elements of mammalian genomes, but plant promoters, especially in gymnosperms, have been left out of the limelight and, therefore, have been poorly investigated. The aim of this study was to enhance and expand the existing genome annotations using computational approaches for genome-wide prediction of TSSs in the four conifer species: loblolly pine, white spruce, N...

Background: Identifying transcriptional enhancers and other cis-regulatory modules (CRMs) is an important goal of post-sequencing genome annotation. Computational approaches provide a useful complement to empirical methods for CRM discovery, but it is critical that we develop effective means to evaluate their performance in terms of estimating their sensitivity and specificity. Results: We introduce here pCRMeval, a pipeline for in silico evaluation of any enhancer prediction tools that are flexible enough to be applied to the Drosophila melanogaster genome. pCRMeval compares the result of predictions with the extensive existing knowledge of experimentally-validated Drosophila CRMs in order to estimate the precision and relative sensitivity of the prediction method. In the case of supervised prediction methods-when training data composed of validated CRMs are used-pCRMeval can also assess the sensitivity of specific training sets. We demonstrate the utility of pCRMeval through evaluation of our SCRMshaw CRM prediction method and training data. By measuring the impact of different parameters on SCRMshaw performance, as assessed by pCRMeval, we develop a more robust version of SCRMshaw, SCRMshaw_HD, that improves the number of predictions while maintaining sensitivity and specificity. Our analysis also demonstrates that SCRMshaw_HD, when applied to increasingly less well-assembled genomes, maintains its strong predictive power with only a minor dropoff in performance. Conclusion: Our pCRMeval pipeline provides a general framework for evaluation that can be applied to any CRM prediction method, particularly a supervised method. While we make use of it here primarily to test and improve a particular method for CRM prediction, SCRMshaw, pCRMeval should provide a valuable platform to the research community not only for evaluating individual methods, but also for comparing between competing methods.

Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis thaliana accessions originating from different geographic regions, when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number, without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over...

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which le...

Although the human genome has been sequenced, progress in understanding gene regulation in humans has been particularly slow. Many computational approaches developed for lower eukaryotes to identify cis-regulatory elements and their associated target genes often do not generalize to mammals, largely due to the degenerate and interactive nature of such elements. Motivated by the switch-like behavior of transcriptional responses, we present a systematic approach that allows adaptive determination of active transcriptional subnetworks (cis-motif combinations, the direct target genes and physiological processes regulated by the corresponding transcription factors) from microarray data in mammals, with accuracy similar to that achieved in lower eukaryotes. Our analysis uncovered several new subnetworks active in human liver and in cell-cycle regulation, with similar functional characteristics as the known ones. We present biochemical evidence for our predictions, and show that the recently discovered G2/M-specific E2F pathway is wider than previously thought; in particular, E2F directly activates certain mitotic genes involved in hepatocellular carcinomas. Additionally, we demonstrate that this method can predict subnetworks in a condition-specific manner, as well as regulatory crosstalk across multiple tissues. Our approach allows systematic understanding of how phenotypic complexity is regulated at the transcription level in mammals and offers marked advantage in systems where little or no prior knowledge of transcriptional regulation is available.

Acacia mangium is one of the most important species in forest plantation industry in Southeast Asia. Lignin is a complex polymer found in plant which has to be removed using harsh and toxic chemicals for the production of high quality paper. Understanding the complex pathway that underlies the regulation of lignin biosynthetic genes requires in depth knowledge of the genes involved and it’s regulatory elements. Using Thermal Asymmetric Interlaced PCR, a 770bp promoter of pseudoCOMTAm and 619bp of AmCCR1 promoter were isolated. Bioinformatics analysis revealed the presence of cis acting elements commonly found in lignin biosynthetic genes such as TATA box, CAAT box, W box, AC-I and AC-11 elements on both promoters.

Trehalose-6-phosphate phosphatase (TPP) genes take part in trehalose metabolism and also in stress tolerance, which has been well documented in many species but poorly understood in wheat. The present research has identified a family of 31 TPP genes in Triticum aestivum L. through homology searches and classified them into five clades by phylogenetic tree analysis, providing evidence of an evolutionary status with Hordeum vulgare, Brachypodium distachyon and Oryza sativa. The exon-intron distribution revealed a discrete evolutionary history and projected possible gene duplication occurrences. Furthermore, different computational approaches were used to analyze the physical and chemical properties, conserved domains and motifs, subcellular and chromosomal localization, and three-dimensional (3-D) protein structures. Cis-regulatory elements (CREs) analysis predicted that TaTPP promoters consist of CREs related to plant growth and development, hormones, and stress. Transcriptional anal...

Acacia mangium is one of the most important species in forest plantation industry in Southeast Asia. Lignin is a complex polymer found in plant which has to be removed using harsh and toxic chemicals for the production of high quality paper. Understanding the complex pathway that underlies the regulation of lignin biosynthetic genes requires in depth knowledge of the genes involved and it’s regulatory elements. Using Thermal Asymmetric Interlaced PCR, a 770bp promoter of pseudoCOMTAm and 619bp of AmCCR1 promoter were isolated. Bioinformatics analysis revealed the presence of cis acting elements commonly found in lignin biosynthetic genes such as TATA box, CAAT box, W box, AC-I and AC-11 elements on both promoters.

Trehalose-6-phosphate phosphatase (TPP) genes take part in trehalose metabolism and also in stress tolerance, which has been well documented in many species but poorly understood in wheat. The present research has identified a family of 31 TPP genes in Triticum aestivum L. through homology searches and classified them into five clades by phylogenetic tree analysis, providing evidence of an evolutionary status with Hordeum vulgare, Brachypodium distachyon and Oryza sativa. The exon-intron distribution revealed a discrete evolutionary history and projected possible gene duplication occurrences. Furthermore, different computational approaches were used to analyze the physical and chemical properties, conserved domains and motifs, subcellular and chromosomal localization, and three-dimensional (3-D) protein structures. Cis-regulatory elements (CREs) analysis predicted that TaTPP promoters consist of CREs related to plant growth and development, hormones, and stress. Transcriptional anal...

Trichomonas vaginalis is the causative agent of trichomonosis, a sexually transmitted disease (STD) that affects over 180 million people worldwide. This parasite is capable to infect the urogenital tract of women and men, both microenvironments might affect the expression of key genes that may be involved in the parasite pathogenesis. The processing of 3 0 end of mRNA promotes mRNA stability in many eukaryotes, however in T. vaginalis this molecular machinery is under research. By means of an in silico analysis we identified putative proteins of the 3 0 end mRNA processing machinery of T. vaginalis, and by RT-PCR assays we evaluated the expression of eight of these genes in a female and male T. vaginalis isolates. According to the in silico analysis, the T. vaginalis 3 0 end mRNA processing machinery, comprises a similar complex and protein factors that those described in Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae and Entamoeba histolytica. The complex contains several subcomplexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CFIm) and cleavage factor II (CFIIm). We demonstrated that genes tvpsf2p, tvcfi25, tvcpsf160, tvcpsf73, tvfip1, tvpap1, tvpc4 and tvpabp are expressed in male or female T. vaginalis isolates. Besides we identify two different isoforms of TvPC4. T. vaginalis genome contains most of genes encoding for 3 0 end mRNA processing, which may be transcriptionally active and could be involved in the capping, splicing, cleavage and polyadenylation of mRNAs in this parasite. Further studies are necessary to elucidate the biological meaning of our findings.

Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen Mycoplasma hyopneumoniae. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from M. hyopneumoniae strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5' upstream regions of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays rev...

The emergence of new genes throughout evolution requires rewiring and extension of regulatory networks. However, the molecular details of how the transcriptional regulation of new gene copies evolves remain largely unexplored. Here we show how duplication of a transcription factor gene allowed the emergence of two independent regulatory circuits. Interestingly, the ancestral transcription factor was promiscuous and could bind different motifs in its target promoters. After duplication, one paralogue evolved increased binding specificity so that it only binds one type of motif, whereas the other copy evolved a decreased activity so that it only activates promoters that contain multiple binding sites. Interestingly, only a few mutations in both the DNA-binding domains and in the promoter binding sites were required to gradually disentangle the two networks. These results reveal how duplication of a promiscuous transcription factor followed by concerted cis and trans mutations allows e...

Hereditary transthyretin amyloidosis is the most common form of hereditary amyloidosis, with an autosomal dominant inheritance and a variable penetrance. ATTRv amyloidosis can present as a progressive, axonal sensory autonomic and motor neuropathy or as an infiltrative cardiomyopathy. The definition of biomarkers for the early diagnosis of ATTRv is particularly important in the current era of emerging treatments. In this sense, metabolomics could be an instrument able to provide metabolic profiles with their related metabolic pathways, and we would propose them as possible fluid biomarkers. The aim of this study is to identify altered metabolites (free fatty acids and amino acids) in subjects with a confirmed pathogenic TTR variant. Out of the studied total free fatty acids and amino acids, the serum values of palmitic acid are significantly lower in the ATTRv patients compared to the recruited healthy subjects. The metabolic remodeling identified in this neurogenetic disorder could...

Methionine adenosyltransferase is a ubiquitous enzyme that catalyzes the only known route of biosynthesis of S-adenosylmethionine, the major methyl group donor in cell metabolism. In mammals, two different methionine adenosyltransferases exist: one is confined to the liver, and the other one is distributed in extrahepatic tissues. In the present study, we report the cloning of the 5-flanking region of liver-specific methionine adenosyltransferase gene from rat. Two closely spaced sites for transcriptional initiation were identified by primer extension analysis. The major transcription start site was determined to be 29 nucleotides downstream from the putative TATA box. Transient transfection assays of constructs containing sequentially deleted 5flanking sequences fused to the luciferase gene showed that rat hepatic methionine adenosyltransferase promoter was able to efficiently drive reporter expression not only in liver-type cells (rat hepatoma H35 cells and human hepatoblastoma HepG2 cells) but also in Chinese hamster ovary cells. Two regions spanning nucleotides ؊1251 to ؊958 and ؊197 to ؉65 were found to be crucial for the promoter efficiency. The distal upstream region contains elements that positively regulate promoter activity in H35 and HepG2 cells but are ineffective in Chinese hamster ovary cells. Eight protein binding sites were characterized in both regions by DNase I footprinting analysis. Two of these elements, sites A and B, located in the distal region, were found to be essential for the regulation of promoter activity. Electrophoretic mobility shift assays and competition experiments showed that site A is recognized by an NF1 protein. Site B was able to interact with a member of HNF-3 family when nuclear extracts from rat liver and H35 cells were used in the in vitro assay, but an additional binding activity to an NHF1-like protein was obtained with the hepatoma cell extracts. It is suggested that this differential binding can contribute to the cell specificity of promoter function.

Transcriptional regulation, a primary mechanism for controlling the development of multicellular organisms, is carried out by transcription factors (TFs) that recognize and bind to their cognate binding sites. In Caenorhabditis elegans, our knowledge of which genes are regulated by which TFs, through binding to specific sites, is still very limited. To expand our knowledge about the C. elegans regulatory network, we performed a comprehensive analysis of the C. elegans, Caenorhabditis briggsae, and Caenorhabditis remanei genomes to identify regulatory elements that are conserved in all genomes. Our analysis identified 4959 elements that are significantly conserved across the genomes and that each occur multiple times within each genome, both hallmarks of functional regulatory sites. Our motifs show significant matches to known core promoter elements, TF binding sites, splice sites, and poly-A signals as well as many putative regulatory sites. Many of the motifs are significantly corr...