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PubMed2005

As antisense RNA gets intronic.

Recent work describing the transcriptional output of the human genome points to the existence of a significant number of non-coding RNA transcripts coming from intronic regions, with a fraction of these being oriented antisense relative to the protein-coding mRNA of the known gene. In this article, we survey the main findings of the large-scale expression analysis projects that led to the identification of antisense intronic messages and which demonstrate their ubiquitous expression in the human genome. We review the current knowledge on long, unspliced, intronic antisense transcripts, a new class of non-coding RNAs, recently described by our group to be correlated with the degree of tumor differentiation in prostate cancer, which we postulate is involved in the fine tuning of gene expression in eukaryotes. Possible mechanisms of antisense intronic transcript biogenesis and function in gene expression regulation are discussed, as is their involvement in human diseases. While there is still no conclusive evidence demonstrating a functional role for these long, intronic antisense messages, the far-reaching implications of their existence for the mechanisms regulating gene expression certainly warrant further experimentation.

Alternative Splicing↗
PubMed2006

Global analysis of the transcriptional network controlling Xenopus endoderm formation.

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.

Animals↗
PubMed2004

P2Y receptors activate neuroprotective mechanisms in astrocytic cells.

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.

Animals↗
PubMed2005

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains.

Small RNA (sRNA) genes are expressed in all organisms, primarily as regulators of translation and message stability. We have developed comparative genomic approaches to identify sRNAs that are expressed by Staphylococcus aureus, the most common cause of hospital-acquired infections. This study represents an in-depth analysis of the RNome of a Gram-positive bacterium. A set of sRNAs candidates were identified in silico within intergenic regions, and their expression levels were monitored by using microarrays and confirmed by Northern blot hybridizations. Two sRNAs were also detected directly from purification and RNA sequence determination. In total, at least 12 sRNAs are expressed from the S. aureus genome, five from the core genome and seven from pathogenicity islands that confer virulence and antibiotic resistance. Three sRNAs are present in multiple (two to five) copies. For the sRNAs that are conserved throughout the bacterial phylogeny, their secondary structures were inferred by phylogenetic comparative methods. In vitro binding assays indicate that one sRNA encoded within a pathogenicity island is a trans-encoded antisense RNA regulating the expression of target genes at the posttranscriptional level. Some of these RNAs show large variations of expression among pathogenic strains, suggesting that they are involved in the regulation of staphylococcal virulence.

Base Pairing↗
PubMed2004

Identification of the K efflux channel coupled to the gastric H-K-ATPase during acid secretion.

Genomic microarray analysis of genes specifically expressed in a pure cell isolate from a heterocellular organ identified the likely K efflux channel associated with the gastric H-K-ATPase. The function of this channel is to supply K to the luminal surface of the pump to allow H for K exchange. KCNQ1-KCNE2 was the most highly expressed and significantly enriched member of the large variety of K channels expressed in the gastric epithelium. The function of this K channel in acid secretion was then shown by inhibition of secretion in isolated gastric glands with specific KCNQ inhibitors and by colocalization of the channel with the H-K-ATPase in the secretory canaliculus of the parietal cell. KCNQ1-KCNE2 appears to be the K efflux channel that is essential for gastric acid secretion.

Acridine Orange↗
PubMed2006

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes.

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T(d-w), temperature of maximum specific dissociation rate constant), obtained from probe-target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

Base Pair Mismatch↗
PubMed2003

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications.

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.

CpG Islands↗
PubMed2004

Rituximab induces different but overlapping sets of genes in human B-lymphoma cell lines.

The therapeutic unconjugated anti-CD20 Mab rituximab is used for the treatment of B-non-Hodgkin's lymphomas. We have studied the direct biological effects, signalling and gene expression profiles induced by rituximab in two human B-lymphoma cell lines, DHL4 and BJAB, using microarray, quantitative PCR and gel shift analysis. Rituximab alone inhibited thymidine uptake and induced homotypic adhesion in DHL4 only, but not BJAB. Analysis of Affymetrix microchips carrying probes for about 10,000 human cDNAs, allowed us to identify 16 genes in DHL4 and 12 in BJAB induced by rituximab at 4 h. Eleven and seven of these genes were specific for DHL4 and BJAB, respectively; whereas the remaining five were up-regulated in both cell lines. Mean induction ranged from 2- to 16-fold. Real time PCR analysis allowed us to confirm up-regulation of all genes identified, except one in BJAB. Time course of induction of eight genes was studied, showing peak induction in most cases at 4 h. The up-regulation of 5/5 genes was also observed with the F(ab')(2) fragment of rituximab. Analysis of three further B-cell lymphoma lines showed that gene induction is not restricted to BJAB and DHL4. Finally, we show that rituximab alone can induce AP1 activation in both cell lines and provide evidence that the ERK1/2 pathway is involved in the rituximab-mediated up-regulation of gene expression. These data demonstrate that rituximab alone has direct signalling capacity in different B-lymphoma lines, inducing distinct but overlapping sets of genes which may play a role in the biological and/or therapeutic effect of the antibody.

Antibodies, Monoclonal↗
PubMed2004

A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.

Adenosine Triphosphatases↗
PubMed2002

Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.

UV radiation is the most important environmental skin aggressor, causing cancer and other problems. This paper reports the use of oligonucleotide microarray technology to determine changes in gene expression in human keratinocytes after UVB treatment. Examination of the effects of different doses at different times after irradiation gave a global picture of the keratinocyte response to this type of insult. Five hundred thirty-nine regulated transcripts were found and organized into nine different clusters depending on behavior patterns. Classification of these genes into 23 functional categories revealed that several biological processes are globally affected by UVB. In addition to confirming a majority up-regulation of the transcripts related to the UV-specific inflammatory and stress responses, significant increases were seen in the expression of genes involved in basal transcription, splicing, and translation as well as in the proteasome-mediated degradation category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly downregulated. These results demonstrate the complexity of the transcriptional profile of the UVB response, describe several cellular processes previously not known to be affected by UV irradiation, and serve as a basis for the global characterization of UV-regulated genes and pathways.

3T3 Cells↗
PubMed2004

Development and evaluation of an Arabidopsis whole genome Affymetrix probe array.

We describe the development of a high-density Arabidopsis'whole genome' oligonucleotide probe array for expression analysis (the Affymetrix ATH1 GeneChip probe array) that contains approximately 22 750 probe sets. Precedence on the array was given to genes for which either expression evidence or a credible database match existed. The remaining space was filled with 'hypothetical' genes. The new ATH1 array represents approximately 23 750 genes of which 60% were detected in RNA from cultured seedlings. Sensitivity of the array, determined using spiking controls, was approximately one transcript per cell. The array demonstrated high technical reproducibility and concordance with real-time PCR results. Indole-3 acetic acid (IAA)-induced changes in gene expression were used for biological validation of the array. A total of 222 genes were significantly upregulated and 103 significantly downregulated by exposure to IAA. Of the genes whose products could be functionally classified, the largest specific classes of upregulated genes were transcriptional regulators and protein kinases, many fewer of which were represented among the downregulated genes. Over one-third of the auxin-regulated genes have no known function, although many belong to gene families with members that have previously been shown to be auxin regulated. For the 6714 genes represented both on this and the earlier Arabidopsis Genome (AG) array, both signal intensities and gene expression ratios were very similar. Mapping of the oligonucleotides on the ATH1 array to the latest (version 4.0) annotation showed that over 95% of the probe sets (based on version 2.0 annotation) still fully represented their original target genes.

Arabidopsis↗
PubMed2004

Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA.

Microarray-based gene expression analysis plays a pivotal role in modern biology and is poised to enter the field of molecular diagnostics. Current microarray-based gene expression systems typically require enzymatic conversion of mRNA into labeled cDNA or cRNA. Conversion to cRNA involves a target amplification step that overcomes the low sensitivity associated with commonly used fluorescent detection methods. Herein, we present a novel enzyme-free, microarray-based gene expression system that uses unamplified total human RNA sample as the target nucleic acid. The detection of microarray-bound RNA molecules is accomplished by targeting the poly-A tail with an oligo-dT20 modified gold nanoparticle probe, signal amplification by autometallography, and subsequent measurement of nanoparticle-mediated light scattering. The high sensitivity afforded by the nanoparticle probes allows differential gene expression from as little as 0.5 microg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.

Biosensing Techniques↗
PubMed2006

ARID3B induces malignant transformation of mouse embryonic fibroblasts and is strongly associated with malignant neuroblastoma.

ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, plays an essential role in the survival of neural crest during embryogenesis. Here, we report evidence that ARID3B is involved in the development of malignant neuroblastoma, a childhood tumor derived from neural crest. (a) ARID3B is expressed by all five cell lines derived from neuroblastoma tested by us. (b) Analysis of published DNA microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only in 9% of stage I-III+IVs tumors. (c) In vitro growth of several neuroblastoma cell lines is suppressed significantly by antisense as well as siRNA treatment. (d) An increase of the ARID3B expression level by transfection in the SY5Y neuroblastoma cell line enhances the malignancy in tumor growth assays in nu/nu mice. (e) ARID3B by itself can immortalize mouse embryonic fibroblasts (MEFs) in vitro and confers malignancy to MEF when transfected together with MYCN, the best characterized oncogene for neuroblastoma. Thus, ARID3B seems to play a key role in the malignant transformation of neuroblastoma and may serve not only as a marker of malignancy but also as a potential target for cancer therapy of stage IV neuroblastoma for which there is currently no effective treatment available.

Animals↗
PubMed2005

Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococci, GAS) is a human pathogen which probably varies its multiplication rate and thus, growth phases in association with the type of infection caused in its host. To create a basis for future determinations of such associations, the genome-wide growth phase-related GAS transcriptome was assessed in the present study. Therefore, the published serotype M1 S. pyogenes genome sequence as well as the partially sequenced serotype M18 and M49 GAS genomes were used to produce DNA microarrays that carried 2256 oligonucleotide probes matching 3662 open reading frames (ORFs). With these microarrays, the transcriptome of the serotype M49 GAS strain 591 grown to the exponential, transition, and early stationary growth phases was assessed in seven independent experiments. The gained data were compared to real-time RT-PCR assays. Data analysis was refined by a novel approach, i.e. grouping of expressed genes to four classes according to relative transcript abundance and gene functions. At the different growth phases, 86.7%, 79.5% and 55.7% of the at least 1883 ORFs contained in the serotype M49 genome were expressed above the defined detection level. Contrary to the general trend, transcript amounts of genes in the functional groups of transport and membrane proteins as well as stress response factors peaked at the transition phase. The most prominent changes in the transcript abundances were predominantly observed for sugar compound transport and turnover-related ORFs. The majority of known virulence genes had their maximum expression during the transition phase, consistent with the proposed associated change in virulence behavior of the bacteria. With these results, it will now be feasible to assess the in situ growth phase of a given GAS strain during any type of infection by measuring the expression of selected marker genes.

DNA, Bacterial↗
PubMed2005

A robust method for the amplification of RNA in the sense orientation.

BACKGROUND: Small quantities of RNA (1-4 microg total RNA) available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity. RESULTS: To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility. CONCLUSION: The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip arrays.

Algorithms↗
PubMed2004

Microarray analysis of gene expression in human donor sclera.

PURPOSE: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes in this tissue-type, and to identify candidate genes for scleral disorders. METHODS: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7-(dT) 24 primer. The resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16 h hybridization step with oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were scaled to the same target intensity using all probe sets. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate the microarray results. RESULTS: There were 3,751 genes with "present" calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed scleral expression in 7 extracellular matrix genes examined. CONCLUSIONS: This study demonstrated the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides an extended list of genes expressed in human sclera. Identification of genes expressed in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.

Adult↗
PubMed2005

Transcriptional analysis of testis maturation using trout cDNA macroarrays.

The project seeks to identify genes involved in key stages of trout spermatogenesis and their regulation. Within the framework of the French project of farm animal genomics (AGENAE) we produced an original normalised trout testis cDNA library and obtained 1152 trout ESTs corresponding to 967 potential genes. To study the expression of those genes throughout first stages of spermatogenesis, we used nylon macroarray. Gonads in stage of immaturity (stage I), or at initiation of spermatogonial proliferation (stage II), meiosis (stage III) or spermiogenesis were selected by histological analysis. Total RNA was extracted and then used to produce complex targets labelled with [33P]dCTP and hybridised with cDNA arrays. After filtering and normalisation of hybridisation signals, genes presenting differential expression as revealed by ANOVA analysis were submitted to k-means clustering and hierarchical classification. Genes were separated into five clusters which presented distinct profiles. One cluster overexpressed in stage I could be involved in the initial events of spermatogenesis as seminiferous tubule organisation. The second cluster displays a transient increase at the beginning of testicular recrudescence (stage II). Three other clusters group several genes involved in cell proliferation and protein synthesis and modification. One is particularly down-expressed during stage I, the two others show increased expression during stages III and IV and appear to be involved in spermatogonial and meiotic proliferation and in protein metabolism linked to cellular growth. This allows us to plan further experiments to better understand the functional implication of some of the genes that are found to be significantly regulated like CDC2, hematological and neurological expressed gene 1-like protein, HCDI protein, Mago Nashi, a BMP-like, and a steroid receptor binding protein. These data demonstrate the applicability of the array based technology using our trout cDNA arrays and highlight genes that are potential targets for the control of puberty and fertility in farmed fish.

Amino Acid Sequence↗
PubMed2004

Microarray-based AMASE as a novel approach for mutation detection.

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Alleles↗