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Identification of genes with consistent methylation levels across different human tissues.

ABSTRACT: DNA methylation plays an important role in regulating cell growth and disease development. Methylation profiles are examined by bisulfite conversion; however, the lack of markers for bisulfite conversion efficiency and appropriate internal control genes remains a major challenge. To address these issues, we utilized two bioinformatics approaches, coefficients of variances and resampling tests, to identify probes showing stable methylation levels from several independent microarray datasets. Mass spectrometry validated the consistently high methylation levels of the five probes (N4BP2, EGFL8, CTRB1, TSPAN3, and ZNF690) in 13 human tissue types from 24 cell lines. Linear associations between detected methylation levels and methyl concentrations of DNA samples were further demonstrated in three genes (N4BP2, EGFL8, and CTRB1). To summarize, we identified five genes which may serve as internal controls for methylation studies by analyzing large-scale microarray data, and three of them can be used as markers for evaluating the efficiency of bisulfite conversion.

SUBMITTER: Lu TP

PROVIDER: S-EPMC3950633 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Identification of genes with consistent methylation levels across different human tissues.

Lu Tzu-Pin TP Chen Kevin T KT Tsai Mong-Hsun MH Kuo Kuan-Ting KT Hsiao Chuhsing Kate CK Lai Liang-Chuan LC Chuang Eric Y EY

Scientific reports 20140312

DNA methylation plays an important role in regulating cell growth and disease development. Methylation profiles are examined by bisulfite conversion; however, the lack of markers for bisulfite conversion efficiency and appropriate internal control genes remains a major challenge. To address these issues, we utilized two bioinformatics approaches, coefficients of variances and resampling tests, to identify probes showing stable methylation levels from several independent microarray datasets. Mass ...[more]

PMID: 24619003

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Project description:This study addresses a recurrent biological problem, that is to define a formal clustering structure for a set of tissues on the basis of the relative abundance of multiple alternatively spliced isoforms mRNAs generated by the same gene. To this aim, we have used a model-based clustering approach, based on a finite mixture of multivariate Gaussian densities. However, given we had more technical replicates from the same tissue for each quantitative measurement, we also employed a finite mixture of linear mixed models, with tissue-specific random effects.A panel of human tissues was analysed through quantitative real-time PCR methods, to quantify the relative amount of mRNA encoding different IGF-1 alternative splicing variants. After an appropriate, preliminary, equalization of the quantitative data, we provided an estimate of the distribution of the observed concentrations for the different IGF-1 mRNA splice variants in the cohort of tissues by employing suitable kernel density estimators. We observed that the analysed IGF-1 mRNA splice variants were characterized by multimodal distributions, which could be interpreted as describing the presence of several sub-population, i.e. potential tissue clusters. In this context, a formal clustering approach based on a finite mixture model (FMM) with Gaussian components is proposed. Due to the presence of potential dependence between the technical replicates (originated by repeated quantitative measurements of the same mRNA splice isoform in the same tissue) we have also employed the finite mixture of linear mixed models (FMLMM), which allowed to take into account this kind of within-tissue dependence.The FMM and the FMLMM provided a convenient yet formal setting for a model-based clustering of the human tissues in sub-populations, characterized by homogeneous values of concentrations of the mRNAs for one or multiple IGF-1 alternative splicing isoforms. The proposed approaches can be applied to any cohort of tissues expressing several alternatively spliced mRNAs generated by the same gene, and can overcome the limitations of clustering methods based on simple comparisons between splice isoform expression levels.

Project description:BACKGROUND:DNA methylation is one of the main epigenetic mechanisms for the regulation of gene expression in eukaryotes. In the standard model, methylation in gene promoters has received the most attention since it is generally associated with transcriptional silencing. Nevertheless, recent studies in human tissues reveal that methylation of the region downstream of the transcription start site is highly informative of gene expression. Also, in some cell types and specific genes it has been found that methylation of the first intron, a gene feature typically rich in enhancers, is linked with gene expression. However, a genome-wide, tissue-independent, systematic comparative analysis of the relationship between DNA methylation in the first intron and gene expression across vertebrates has not been explored yet. RESULTS:The most important findings of this study are: (1) using different tissues from a modern fish, we show a clear genome-wide, tissue-independent quasi-linear inverse relationship between DNA methylation of the first intron and gene expression. (2) This relationship is conserved across vertebrates, since it is also present in the genomes of a model pufferfish, a model frog and different human tissues. Among the gene features, tissues and species interrogated, the first intron's negative correlation with the gene expression was most consistent. (3) We identified more tissue-specific differentially methylated regions (tDMRs) in the first intron than in any other gene feature. These tDMRs have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. (4) Lastly, we identified CpGs in transcription factor binding motifs, enriched in the first intron, the methylation of which tended to increase with the distance from the first exon-first intron boundary, with a concomitant decrease in gene expression. CONCLUSIONS:Our integrative analysis clearly reveals the important and conserved role of the methylation level of the first intron and its inverse association with gene expression regardless of tissue and species. These findings not only contribute to our basic understanding of the epigenetic regulation of gene expression but also identify the first intron as an informative gene feature regarding the relationship between DNA methylation and gene expression where future studies should be focused.

Dataset Information

Identification of genes with consistent methylation levels across different human tissues.

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Identification of genes with consistent methylation levels across different human tissues.

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