Gene Expression In Regions Research Articles

Bioinformatic Identification of Potential RNA Alterations on the Atrial Fibrillation Remodeling from Human Pulmonary Veins.

Atrial fibrillation (AF) is the most frequent persistent arrhythmia. Many genes have been reported as a genetic background for AF. However, most transcriptome analyses of AF are limited to the atrial samples and have not been evaluated by multiple cardiac regions. In this study, we analyzed the expression levels of protein-coding and long noncoding RNAs (lncRNAs) in six cardiac regions by RNA-seq. Samples were donated from six subjects with or without persistent AF for left atria, left atrial appendages, right atria, sinoatrial nodes, left ventricles, right ventricles, and pulmonary veins (PVs), and additional four right atrial appendages samples were collected from patients undergoing mitral valve replacement. In total, 23 AF samples were compared to 23 non-AF samples. Surprisingly, the most influenced heart region in gene expression by AF was the PV, not the atria. The ion channel-related gene set was significantly enriched upon analysis of these significant genes. In addition, some significant genes are cancer-related lncRNAs in PV in AF. A co-expression network analysis could detect the functional gene clusters. In particular, the cancer-related lncRNA, such as SAMMSON and FOXCUT, belong to the gene network with the cancer-related transcription factor FOXC1. Thus, they may also play an aggravating role in the pathogenesis of AF, similar to carcinogenesis. In the least, this study suggests that (1) RNA alteration is most intense in PVs and (2) post-transcriptional gene regulation by lncRNA may contribute to the progression of AF. Through the screening analysis across the six cardiac regions, the possibility that the PV region can play a role other than paroxysmal triggering in the pathogenesis of AF was demonstrated for the first time. Future research with an increase in the number of PV samples will lead to a novel understanding of the pathophysiology of AF.

Genomic Regions Associated With Salinity Stress Tolerance in Tropical Maize (Zea Mays L.).

Being a widely cultivated crop globally under diverse climatic conditions and soil types, maize is often exposed to an array of biotic and abiotic stresses. Soil salinity is one of the challenges for maize cultivation in many parts of lowland tropics that significantly affects crop growth and reduces economic yields. Breeding strategies integrated with molecular approach might accelerate the process of identifying and developing salinity-tolerant maize cultivars. In this study, an association mapping panel consisting of 305 diverse maize inbred lines was phenotyped in a managed salinity stress phenotyping facility at International Center for Biosaline Agriculture (ICBA), Dubai, United Arab Emirates (UAE). Wide genotypic variability was observed in the panel under salinity stress for key phenotypic traits viz., grain yield, days to anthesis, anthesis-silking interval, plant height, cob length, cob girth, and kernel number. The panel was genotyped following the genome-based sequencing approach to generate 955,690 SNPs. Total SNPs were filtered to 213,043 at a call rate of 0.85 and minor allele frequency of 0.05 for association analysis. A total of 259 highly significant (P ≤ 1 × 10–5) marker-trait associations (MTAs) were identified for seven phenotypic traits. The phenotypic variance for MTAs ranged between 5.2 and 9%. A total of 64 associations were found in 19 unique putative gene expression regions. Among them, 12 associations were found in gene models with stress-related biological functions.

Abstract 465: Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples

Abstract Background RNA sequencing (RNA-Seq) has been widely used for gene expression and fusion detection in fresh frozen samples for research use. In clinical studies, RNA was more commonly derived from FFPE samples, which may degrade or yield low due to preparation methods, storage conditions and time. We evaluate the feasibility, quality, and analytical performance of a targeted region RNA sequencing assay (we called as Master RNA Panel) on clinical FFPE tumor samples for gene fusion detection and expression quantification. Methods Total RNA was extracted from FFPE tumor samples. cDNA synthesis and library preparation were used to prepare next-generation sequencing (NGS) libraries that were captured by a customized panel contains 31 gene fusion/splicing regions and 1813 gene expression regions. NGS libraries were then sequenced on Illumina NexSeq CN 500 or NovaSeq 6000 instrument. Sequencing data were analyzed and annotated with an in-house developed pipeline. A set of experimental and data quality control parameters were set up. Results In both fresh tissue and FFPE samples, all known fusion sites (100%, 7/7) can be detected stably with the RNA input volume as low as 5 ng and 1 Gbp data, the gene expression of 5 ng RNA input volume shows a very high correlation with 100ng even under 1Gb data (R2≥ 0.95). Master RNA Panel can detect as low as 100 copies of the fusion gene with 50 ng RNA and the specificity was 100% based on a test of 10 fusion negative samples. The consistency of fusion gene detection by Master RNA Panel was 96.2% (51/53) compared with RNA-Seq in 44 clinical samples. To validate the accuracy of gene expression, we used both qPCR and RNA-Seq as comparison methods. Gene expression of 12 tumor-related genes (BAG1, BIRC5, CD68, ERBB2, ESR1, GRB7, GSTM1, MKI67, MMP11, MYBL2, PGR, SCUBE2) in 5 samples were profiled with qPCR assay and the correlation for each gene is greater than 0.8. 27 clinical samples were profiled with both Master RNA panel and RNA-Seq, the total correlation of gene expression between the two assay was also greater than 0.8. The precision verification results show that under the inspection of different operation dates, reagent batches and operators, the gene expression correlation between 5 replicates of 2 FFPE samples is greater than 0.99. The consistency of the fusion test for 5 replicates of 2 FFPE samples and 3 standards was 95.3% (81/85, >=100 copies), indicating that the operation date, reagent batch and operator have no significant influence on the test results, and the test method has good precision. Conclusions Master RNA Panel can help to identify both gene fusions and gene quantification in cancer patients with extremely low RNA input and high limit of detection (LoD), which is a good supplement for DNA based NGS assay, especially for gene expression quantification and fusion detection. Citation Format: Jingbo Zhao, Shuang Yang, Hua Dong. Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 465.

Plant 3' Regulatory Regions From mRNA-Encoding Genes and Their Uses to Modulate Expression.

Molecular biotechnology has made it possible to explore the potential of plants for different purposes. The 3’ regulatory regions have a great diversity of cis-regulatory elements directly involved in polyadenylation, stability, transport and mRNA translation, essential to achieve the desired levels of gene expression. A complex interaction between the cleavage and polyadenylation molecular complex and cis-elements determine the polyadenylation site, which may result in the choice of non-canonical sites, resulting in alternative polyadenylation events, involved in the regulation of more than 80% of the genes expressed in plants. In addition, after transcription, a wide array of RNA-binding proteins interacts with cis-acting elements located mainly in the 3’ untranslated region, determining the fate of mRNAs in eukaryotic cells. Although a small number of 3’ regulatory regions have been identified and validated so far, many studies have shown that plant 3’ regulatory regions have a higher potential to regulate gene expression in plants compared to widely used 3’ regulatory regions, such as NOS and OCS from Agrobacterium tumefaciens and 35S from cauliflower mosaic virus. In this review, we discuss the role of 3’ regulatory regions in gene expression, and the superior potential that plant 3’ regulatory regions have compared to NOS, OCS and 35S 3’ regulatory regions.

SegCorr a statistical procedure for the detection of genomic regions of correlated expression

BackgroundDetecting local correlations in expression between neighboring genes along the genome has proved to be an effective strategy to identify possible causes of transcriptional deregulation in cancer. It has been successfully used to illustrate the role of mechanisms such as copy number variation (CNV) or epigenetic alterations as factors that may significantly alter expression in large chromosomal regions (gene silencing or gene activation).ResultsThe identification of correlated regions requires segmenting the gene expression correlation matrix into regions of homogeneously correlated genes and assessing whether the observed local correlation is significantly higher than the background chromosomal correlation. A unified statistical framework is proposed to achieve these two tasks, where optimal segmentation is efficiently performed using dynamic programming algorithm, and detection of highly correlated regions is then achieved using an exact test procedure. We also propose a simple and efficient procedure to correct the expression signal for mechanisms already known to impact expression correlation. The performance and robustness of the proposed procedure, called SegCorr, are evaluated on simulated data. The procedure is illustrated on cancer data, where the signal is corrected for correlations caused by copy number variation. It permitted the detection of regions with high correlations linked to epigenetic marks like DNA methylation.ConclusionsSegCorr is a novel method that performs correlation matrix segmentation and applies a test procedure in order to detect highly correlated regions in gene expression.

Abstract 112: Concurrent analysis between copy number variation and gene expression of non-smoking lung cancer females in Taiwan

Abstract Although smoking has been proved to be an important factor for lung cancer, the etiology and molecular mechanisms of non-smoking female lung cancer remain unclear. We collected paired normal and tumor lung tissues from 44 female non-smoking lung cancer patients. Copy number variation and Gene expression profiles were analyzed by Affymetrix U133plus 2.0 expression arrays and Affymetrix SNP 6.0 single nucleotide polymorphism (SNP) arrays. The data from two platforms were integrated for concurrent analyses in order to elucidate the putative molecular mechanisms. We first identified the probes located at the same region of gene expression and copy number variation (CNV). Next, we calculated the Pearson correlation coefficient to assess the association between log intensity values of mRNA expression and the corresponding averaged intensity values of SNP probes in both tumor and normal tissues. Genes were then selected based on the following two criteria: (1) the difference in expression-SNP correlations between normal and tumor tissues > 0.7, and (2) the absolute expression-SNP correlation of either normal or tumor >0.3. The selected genes were further analyzed by Ingenuity Pathway Analysis (IPA) to explore the related pathways and networks. Results of IPA showed that mitochondrial dysfunction, IGF-1 signaling, and cell cycle: G1/S checkpoint regulation pathways were significantly dysregulated in tumor tissues. The associations between lung cancer and genomic alternations in these enriched pathways have been reported previously, such as overexpression of IGF1R in IGF-1 signaling pathway might resist EGFR inhibitors used in lung cancer treatment. In summary, with the integrated data of copy number variation and expression, the concurrent analyses help to dissect lung carcinogenesis of non-smoking lung cancer females in Taiwan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 112.

Differential Regional Expression of Genes in the Developing Brain of Ciona intestinalis Embryos

Our previous transcriptome analysis identified 565 genes that are preferentially expressed in the developing brain of Ciona intestinalis larvae. Here, we show by in-situ hybridization that the spatial expression patterns of these brain-specific genes fall into different categories depending on the regions where the gene is expressed. For example, Ci-opsin3 and Ci-Dkk3 are expressed in the entire brain, Ci-tyrosinase and Ci-TYRP1 in the dorsal region, and Ci-synaptotagmin3, Ci-ZF399, and Ci-PTFb in the ventral region. Other genes are specific to the posterior, anterior, central, posterior and ventral, or anterior-ventral region of the brain. This regional expression of genes in the Ciona brain is not always associated with cell lineage, suggesting that complex mechanisms control the regionalized expression of brain-specific genes.

Genomic structure and promoter analysis of pathogen‐induced repat genes from Spodoptera exigua

The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5'-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5'-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5'-untranslated region in the enhancement of gene expression.

Genome-wide Transcriptional Dependence on TAF1 Functional Domains

Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise approximately 90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes.

Region-specific constitutive gene expression in the adult porcine meniscus

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.

A Gain-of-Function Mutant of patched Dissects Different Responses to the Hedgehog Gradient

The Hedgehog (Hh) signal has an inductive role during Drosophila development. Patched is part of the Hedgehog–receptor complex and shows a repressive function on the signaling cascade, which is alleviated in the presence of Hh. Herein, we identify the first dominant gain-of-function allele of patched, Confused (patchedCon). Analysis of the patchedCon allele led us to uncover novel features of the reception and function of the Hh signal. At least three different regions of gene expression were identified and a gradient of cell affinities was established in response to Hh. A new state of Cubitus interruptus activity responsible for the activation of araucan and caupolican genes of the iroquois complex, independent of Fused kinase function, was shown. In the disc, patchedCon behaved like fused mutants and was rescued by Suppressor of fused mutations. However, fused mutants are embryonic lethal while patchedCon is not, suggesting that Patched could interpret Hedgehog signaling differently in the embryo and in the adult.

Spatially distinct head and heart inducers within the Xenopus organizer region

Background: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus, can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues.Results: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus-expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction.Conclusions: In Xenopus, the cerberus-expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.

Isolation and characterization of three mRNAs enriched in embryos of the direct-developing sea urchin Heliocidaris erythrogramma: evolution of larval ectoderm.

The Australian sea urchin Heliocidaris erythro-gramma utilizes a derived direct developmental mode that evolved 8-12 million years ago. From a differential screen we have isolated a small set of cDNAs corresponding to genes more greatly expressed in embryos of H. erythrogramma than in those of its indirect-developing nearest relative, H. tuberculata. The method was biased towards abundant transcripts and did not allow detection of modifications of usage of highly conserved gene family members. Three differentially expressed abundant transcripts were found that potentially encode secreted proteins. Two of these, the arylsulfatase HeARS and the putative lectin HeEL-1, were identifiable as homologues of known proteins. Another gene, HeET-1, may be exclusively expressed in the H. erythrogramma embryo. In situ hybridization experiments demonstrate that all three transcripts are localized to the ectoderm. Two of them, HeET-1 and HeEL-1, are transcribed in an identical domain comprising the larval ectoderm. This region of gene expression has acquired a novel columnar cytology during the evolution of the H. erythrogramma embryo. The third sequence, HeARS, encodes an arylsulfatase homologue. Its expression is uniform in the gastrula, but as the rudiment develops it accumulates to the greatest extent in the invaginating vestibular ectoderm. Through comparisons with indirect-developing species, we show that this concentration of arylsulfatase mRNA in the rudiment is a novel feature of H. erythrogramma development. These data suggest that H. erythrogramma has a unique arrangement of ectodermal gene expression territories. We propose that these reflect larval adaptations that have occurred in the lineage leading to H. erythrogramma, and enabled the evolution of direct development.

Functional analysis of sigma-70 consensus promoters in Pseudomonas aeruginosa and Escherichia coli.

A series of synthetic promoters, based upon the Escherichia coli sigma 70 consensus promoter sequence, was constructed upstream of the lacZ reporter gene in the modified broad-host-range vector pQF52. The role of the intervening spacer region in gene expression in Pseudomonas aeruginosa and E. coli was studied by insertions and deletions within this region. In P. aeruginosa and E. coli the patterns of gene expression were identical with maximum beta-galactosidase activity being measured from promoters possessing 19 bp in their intervening regions, presumably as a result of impeded promoter clearance with the consensus 17-bp promoter. In P. aeruginosa a second occurrence of enhanced activity, which could not be attributed to the involvement of the alternative sigma factor RpoN (sigma 54), was evident with the promoter having a 16-bp spacer.

Interaction of E. coli RNA polymerase with promotors of coliphage T5

The genome of virulent coliphage T5 contains about 30 sites which form stable complexes with E. coli RNA polymerase. Some of these sites bind RNA polymerase with high rates, others form extremely stable complexes as compared with promotors of other E. coli systems. The transcriptional activity of these promotors in vivo and in vitro reflects the rate of complex formation with RNA polymerase rather than the stability of the enzyme/promotor complex. The fastest, i.e. the most active promotors are found in the "early" region of gene expression followed by promotors of the "preearly" class. The few binding sites for the E. coli holoenzyme within the "late" region react more slowly with the enzyme.