Tiling Arrays Research Articles

MeDIP-on-Chip for methylation profiling.

DNA methylation is an important regulatory step in gene expression. Knowledge of the alterations in DNA methylation at a whole genome scale improves our understanding of gene regulation and potential correlations with biological events or disease progression. Methylated DNA immunoprecipitation (MeDIP) uses an antibody that efficiently enriches methylated DNA fragments for downstream locus-specific or genome-wide analyses. MeDIP-on-Chip uses the MeDIP approach in combination with a tiling array for the investigation of genome-wide DNA methylation patterns (or DNA methylomics). The following protocol describes the application of MeDIP to the hybridization of DNA microarrays and data analysis.

DNA-affinity-purified chip (DAP-chip) method to determine gene targets for bacterial two component regulatory systems.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

Long noncoding RNA HOTAIR is associated with human cervical cancer progression

Objectives: The functions of many long noncoding RNAs (lncRNAs) in human cancers remain to be clarified. The lncRNA Hox transcript antisense intergenic RNA (HOTAIR), which was identified from a custom tiling array of the HOXC locus (12q13.13), has been reported to reprogram chromatin organization and promote breast and colorectal cancer metastasis. In this study, we examined the expression and functional role of HOTAIR in cervical carcinoma.

EuGene-PP: a next-generation automated annotation pipeline for prokaryotic genomes.

It is now easy and increasingly usual to produce oriented RNA-Seq data as a prokaryotic genome is being sequenced. However, this information is usually just used for expression quantification. EuGene-PP is a fully automated pipeline for structural annotation of prokaryotic genomes integrating protein similarities, statistical information and any oriented expression information (RNA-Seq or tiling arrays) through a variety of file formats to produce a qualitatively enriched annotation including coding regions but also (possibly antisense) non-coding genes and transcription start sites. EuGene-PP is an open-source software based on EuGene-P integrating a Galaxy configuration. EuGene-PP can be downloaded at eugene.toulouse.inra.fr.

Impacts of TCDD and MeHg on DNA methylation in zebrafish (Danio rerio) across two generations

This study aimed to investigate whether dioxin (TCDD) and methylmercury (MeHg) pose a threat to offspring of fish exposed to elevated concentrations of these chemicals via epigenetic-based mechanisms. Adult female zebrafish were fed diets added either 20μg/kg 2,3,7,8 TCDD or 10mg/kg MeHg for 47days, or 10mg/kg 5-aza-2′-deoxycytidine (5-AZA), a hypomethylating agent, for 32days, and bred with unexposed males in clean water to produce F1 and F2 offspring. Global DNA methylation, promoter CpG island methylation and target gene transcription in liver of adult females and in 3days post fertilization (dpf) F1 and F2 embryos were determined with HPLC, a novel CpG island tiling array containing 54,933 different probes and RT-qPCR, respectively. The results showed that chemical treatment had no significant effect on global DNA methylation levels in F1 (MeHg and TCDD) and F2 (MeHg) embryos and only a limited number of genes were identified with altered methylation levels at their promoter regions. CYP1A1 transcription, an established marker of TCDD exposure, was elevated 27-fold in F1 embryos compared to the controls, matching the high levels of CYP1A1 expression observed in F0 TCDD-treated females. This suggests that maternal transfer of TCDD is a significant route of exposure for the F1 offspring. In conclusion, the selected doses of TCDD and MeHg, two chemicals often found in high concentrations in fish, appear to have only modest effects on DNA methylation in F1 (MeHg and TCDD) and F2 (MeHg) embryos of treated F0 females.

A novel method for identification and quantification of consistently differentially methylated regions.

Advances in biotechnology have resulted in large-scale studies of DNA methylation. A differentially methylated region (DMR) is a genomic region with multiple adjacent CpG sites that exhibit different methylation statuses among multiple samples. Many so-called “supervised” methods have been established to identify DMRs between two or more comparison groups. Methods for the identification of DMRs without reference to phenotypic information are, however, less well studied. An alternative “unsupervised” approach was proposed, in which DMRs in studied samples were identified with consideration of nature dependence structure of methylation measurements between neighboring probes from tiling arrays. Through simulation study, we investigated effects of dependencies between neighboring probes on determining DMRs where a lot of spurious signals would be produced if the methylation data were analyzed independently of the probe. In contrast, our newly proposed method could successfully correct for this effect with a well-controlled false positive rate and a comparable sensitivity. By applying to two real datasets, we demonstrated that our method could provide a global picture of methylation variation in studied samples. R source codes to implement the proposed method were freely available at http://www.csjfann.ibms.sinica.edu.tw/eag/programlist/ICDMR/ICDMR.html.

A ChIP-on-chip tiling array approach detects functional histone-free regions associated with boundaries at vertebrate HOX genes.

Hox genes impart segment identity to body structures along the anterior–posterior axis and are crucial for proper development. A unique feature of the Hox loci is the collinearity between the gene position within the cluster and its spatial expression pattern along the body axis. However, the mechanisms that regulate collinear patterns of Hox gene expression remain unclear, especially in higher vertebrates. We recently identified novel histone-free regions (HFRs) that can act as chromatin boundary elements demarcating successive murine Hox genes and help regulate their precise expression domains (Srivastava et al., 2013). In this report, we describe in detail the ChIP-chip analysis strategy associated with the identification of these HFRs. We also provide the Perl scripts for HFR extraction and quality control analysis for this custom designed tiling array dataset.

Large sex differences in chicken behavior and brain gene expression coincide with few differences in promoter DNA-methylation.

While behavioral sex differences have repeatedly been reported across taxa, the underlying epigenetic mechanisms in the brain are mostly lacking. Birds have previously shown to have only limited dosage compensation, leading to high sex bias of Z-chromosome gene expression. In chickens, a male hyper-methylated region (MHM) on the Z-chromosome has been associated with a local type of dosage compensation, but a more detailed characterization of the avian methylome is limiting our interpretations. Here we report an analysis of genome wide sex differences in promoter DNA-methylation and gene expression in the brain of three weeks old chickens, and associated sex differences in behavior of Red Junglefowl (ancestor of domestic chickens). Combining DNA-methylation tiling arrays with gene expression microarrays we show that a specific locus of the MHM region, together with the promoter for the zinc finger RNA binding protein (ZFR) gene on chromosome 1, is strongly associated with sex dimorphism in gene expression. Except for this, we found few differences in promoter DNA-methylation, even though hundreds of genes were robustly differentially expressed across distantly related breeds. Several of the differentially expressed genes are known to affect behavior, and as suggested from their functional annotation, we found that female Red Junglefowl are more explorative and fearful in a range of tests performed throughout their lives. This paper identifies new sites and, with increased resolution, confirms known sites where DNA-methylation seems to affect sexually dimorphic gene expression, but the general lack of this association is noticeable and strengthens the view that birds do not have dosage compensation.

Genome-wide mapping of Painting of fourth on Drosophila melanogaster salivary gland polytene chromosomes.

The protein Painting of fourth (POF) in Drosophila melanogaster specifically targets and stimulates expression output from the heterochromatic 4th chromosome, thereby representing an autosome specific protein [[1], [2]]. Despite the high specificity for chromosome 4 genes, POF is occasionally observed binding to the cytological region 2L:31 in males and females [3] and two loci on the X-chromosome, PoX1 and PoX2 only in females [4]. Here we provide a detailed description of the experimental design and analysis of the tiling array data presented by Lundberg and colleagues in G3: Genes, Genomes, Genetics 2013 [4], where the female specific POF binding to PoX1 and PoX2 loci on the X chromosome was reported. We show the genome-wide high resolution binding profile of the POF protein where these different POF binding sites are detected. The complete data set is available at http://www.ncbi.nlm.nih.gov/geo/ (accession: GSE45402)

Microarray-based optimization to detect genomic deletion mutations.

We performed array comparative genome hybridization (aCGH) analyses of five Arabidopsis thaliana mutants with genomic deletions ranging in size from 4 bp to > 5 kb. We used the Roche NimbleGen Arabidopsis CGH 3 × 720 K whole genome custom tiling array to optimize deletion detection. Details of the microarray design and hybridization data have been deposited at the NCBI GEO repository with accession number GSE55327.

Evaluating the Role of Epigenetic Histone Modifications in the Metabolic Memory of Type 1 Diabetes

We assessed whether epigenetic histone posttranslational modifications are associated with the prolonged beneficial effects (metabolic memory) of intensive versus conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term Epidemiology of Diabetes Interventions and Complications (EDIC) study. We performed chromatin immunoprecipitation linked to promoter tiling arrays to profile H3 lysine-9 acetylation (H3K9Ac), H3 lysine-4 trimethylation (H3K4Me3), and H3K9Me2 in blood monocytes and lymphocytes obtained from 30 DCCT conventional treatment group subjects (case subjects: mean DCCT HbA1c level >9.1% [76 mmol/mol] and progression of retinopathy or nephropathy by EDIC year 10 of follow-up) versus 30 DCCT intensive treatment subjects (control subjects: mean DCCT HbA1c level <7.3% [56 mmol/mol] and without progression of retinopathy or nephropathy). Monocytes from case subjects had statistically greater numbers of promoter regions with enrichment in H3K9Ac (active chromatin mark) compared with control subjects (P = 0.0096). Among the patients in the two groups combined, monocyte H3K9Ac was significantly associated with the mean HbA1c level during the DCCT and EDIC (each P < 2.2E-16). Of note, the top 38 case hyperacetylated promoters (P < 0.05) included >15 genes related to the nuclear factor-κB inflammatory pathway and were enriched in genes related to diabetes complications. These results suggest an association between HbA1c level and H3K9Ac, and a possible epigenetic explanation for metabolic memory in humans.

The differential response of the Pben promoter of Pseudomonas putida mt‐2 to BenR and XylS prevents metabolic conflicts in m‐xylene biodegradation

Pseudomonas putida mt-2 encompasses two alternative and potentially conflicting routes for benzoate metabolism, one meta pathway encoded by xyl genes of the pWW0 plasmid and mastered by the Pm promoter and XylS, and one chromosomally encoded ortho pathway initiated by Pben and the BenR protein. Any cross-activation of Pben promoter by XylS ought to cause a metabolic conflict during the degradation of m-xylene because 3-methylbenzoate (3MBz) generated as an intermediate can be channelled through the ortho pathway and produce toxic dead-end metabolites. The activation of Pben by XylS was revisited using both reporter technology and tiling arrays targeted to the sequences of interest around messenger RNA initiation of both Pben and Pm promoters. Analysis of supersensitive luxCDABE fusions, inspection of xylX versus benA transcripts and growth tests of benR mutants indicated that the natural expression ranges of XylS under various conditions are insufficient to cause a significant cross-regulation of Pben whether cells face endogenous or exogenous 3MBz. This seems to stem from the nature of the operators for binding either transcriptional factor, which in the case of the Pben promoter of P. putida mt-2 appear to have evolved for avoiding a strong interaction with XylS.

Role for Tissue-Dependent Methylation Differences in the Expression of FOXE1 in Nontumoral Thyroid Glands

Discordance of monozygotic twins for thyroid dysgenesis suggests that epigenetic mechanisms may underlie defects in thyroid gland development. This prompted us to evaluate whether differentially methylated regions (DMRs) can be found between human thyroids (either eutopic or ectopic) and matched leukocytes. To compare the genome-wide methylation profile of thyroids and leukocytes, immunoprecipitated methylated DNA was interrogated on human promoter plus CpG island tiling arrays. In addition, the methylation profile of the human FOXE1, PAX8, and NKX2.1 promoter was examined using bisulfite sequencing. Finally, the functional impact of CpG methylation of the promoter on FOXE1 expression was assessed with luciferase assays. Genome-wide methylation profiling and bisulfite sequencing of CpG islands of PAX8 and NKX2.1 promoters revealed no DMR between thyroid and leukocytes. However, bisulfite sequencing revealed that the methylation level of two consecutive CpG dinucleotides (CpG14 and CpG15, which were not covered by the genome-wide array) in one CpG island of the FOXE1 promoter (-1600 to -1140 from the transcription start site) is significantly higher in leukocytes than in eutopic or ectopic thyroid tissues, suggesting that methylation of this region may decrease FOXE1 gene expression. Indeed, luciferase activities were decreased when FOXE1 promoter constructs were methylated in vitro. Moreover, derepression of luciferase activity was observed when the methylation of CpG14 and CpG15 was prevented by mutations. We report a tissue-dependent DMR in the FOXE1 promoter. This DMR contains two consecutive CpG dinucleotides, which are epigenetic modifiers of FOXE1 expression in nontumoral tissues.

Large-Scale Immunopurification of Ribonucleoprotein Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays

It is of interest to be able to define sets of cellular RNAs associated with specific RNA-binding proteins. This "guilt by association" can lead to new insights into how RNA-binding proteins modulate posttranscriptional gene expression of specific target RNAs. To identify these RNAs, antibodies against RNA-binding proteins can be used to immunopurify endogenous RNA-protein complexes from cells, and then the associated RNAs can be characterized. The method described here was developed to identify binding regions on nuclear transcripts for Drosophila heterogeneous nuclear ribonucleoproteins (hnRNPs). An antibody is added to an RNP extract and incubated to allow antigen-antibody complexes to form. The antibody-antigen complexes are then retrieved by binding of the antibody constant region to staphylococcal Protein A immobilized on Sepharose beads. The bead-immobilized complexes are then washed and RNA is prepared. The RNA is used to generate random-primed cDNA, cRNA, and biotinlyated cDNA probes for use on Affymetrix whole-genome Drosophila tiling arrays.

Glial Wingless/Wnt Regulates Glutamate Receptor Clustering and Synaptic Physiology at theDrosophilaNeuromuscular Junction

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene. We demonstrate that Repo regulates wg expression in vivo and that local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ.

Overlapping transcription and bacterial RNA removal

The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5). However, because transcriptome studies have been performed using microgram amounts of RNA purified from millions of bacterial cells instead of RNA purified from a single bacterium, the presence of overlapping sense/antisense RNAs from a genomic region does not necessarily mean that both sense and antisense transcripts are simultaneously present in the same bacteria. Hence, it might be possible that a subgroup in the bacterial population synthesized the sense transcript, another subgroup synthesized the antisense transcript, and consequently overlapping transcripts would never be together in the same cell. A report in PNAS by Lybecker et al. (6) provides clear evidences that both sense and antisense transcripts can be present simultaneously within the same bacterial cell. Using a monoclonal antibody that recognizes double-stranded RNA molecules (dsRNA) irrespectively of the nucleotide sequence, the authors perform immunoprecipitation assays to pull down dsRNA molecules (IP-dsRNA) from a total RNA sample extracted from Escherichia coli, and identified the purified dsRNA by RNA-seq.

Control of Cdc28 CDK1 by a Stress-Induced lncRNA

Genomic analysis has revealed the existence of a large number of long noncoding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Cells display dramatic changes of gene expression upon environmental changes. Upon osmostress, hundreds of stress-responsive genes are induced by the stress-activated protein kinase (SAPK) p38/Hog1. Using whole-genome tiling arrays, we found that Hog1 induces a set of lncRNAs upon stress. One of the genes expressing a Hog1-dependent lncRNA in antisense orientation is CDC28, the cyclin-dependent kinase 1 (CDK1) that controls the cell cycle in yeast. Cdc28 lncRNA mediates the establishment of gene looping and the relocalization of Hog1 and RSC from the 3' UTR to the+1 nucleosome to induce CDC28 expression. The increase in the levels of Cdc28 results in cells able to reenter the cell cycle more efficiently after stress. This may represent a general mechanism to prime expression of genes needed after stresses are alleviated.

Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.

Broad shifts in gene expression during early postnatal life are associated with shifts in histone methylation patterns.

During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration.