Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

CpG island density and its correlations with genomic features in mammalian genomes

Background  

CpG islands, which are clusters of CpG dinucleotides in GC-rich regions, are considered gene markers and represent an important feature of mammalian genomes. Previous studies of CpG islands have largely been on specific loci or within one genome. To date, there seems to be no comparative analysis of CpG islands and their density at the DNA sequence level among mammalian genomes and of their correlations with other genome features.     

Species-specific non-expression of ribosomal RNA genes in a mammalian hybrid,the mule

The expression of nucleolus organizer activity in diploid cells was investigated in a model system for mammalian hybrids, the horse-ass cross (mule), by means of sequential Ag-NOR and chromomycin A3/distamycin ADAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes (PBL). As a rule we found species-specific nonexpression of the horse-derived NOR chromosomes in the mule, whereas the ass-derived NOR chromosomes were active. The results of PBL interphase studies were compatible with the data gained from mitotic metaphase analyses.     

Nonrandom frequency patterns of synonymous substitutions in homologous mammalian genes

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous positions of quartet and duet (fourfold and twofold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable) according to whether they show no change, one change, or more than one change, respectively. We observed (1) that the frequencies of conserved, intermediate, and variable positions of quartet and duet codons are different in different genes; (2) that the frequencies of the three classes are significantly different from expectations based on a random substitution process in the majority of genes (especially for GC-rich genes) for quartet codons and in a minority of genes for doublet codons; and (3) that the frequencies of the three classes of positions of quartet codons are correlated with those of duet codons, the conserved positions of quartet and duet codons being, in addition, correlated with the degree of amino acid conservation. Our main conclusions are that synonymous substitution frequencies: (1) are gene-specific; (2) are not simply the result of a stochastic process in which nucleotide substitutions accumulate at random, over time; and (3) are correlated in quartet and duet codons.     

Specific compositional patterns of synonymous positions in homologous mammalian genes

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous (ie., third) positions of quartet (fourfold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable), according to whether they show no change, one change, and more than one change, respectively. The three classes were analyzed in their compositional patterns. In the majority of GC-rich genes, the three classes of positions (but especially conserved positions) exhibited significantly different base compositions compared to expectations based on a random substitution process from the ancestral (consensus) sequence to the present-day (actual) sequences. Significant differences were rare in GC-poor genes.An analysis of the present results indicates that natural selection plays a role in the synonymous nucleotide substitution process, especially in GC-rich genes which represent the vast majority of mammalian genes.This paper is dedicated to the memory of Dr. Motoo Kimura (1924–1994)Correspondence to: Giorgio Bernardi     

Evaluation of single CpG sites as proxies of CpG island methylation states at the genome scale

Methylation of a CpG island is a faithful marker of silencing of its associated gene. Different approaches report the methylation status of a CpG island based on the determination of one or a few CpG sites by assuming the homogeneity of methylation along the element. This strategy is frequently applied in both locus-specific and genome-wide studies, but often without a validation of the representativeness of the interrogated CpG site compared with the whole element. We have evaluated the predictive informativeness of the HpaII sites located in CpG islands using data from high-resolution methylome maps, which offer the possibility to assess the methylation homogeneity of each CpG island and to determine the reporter accuracy of single sites as surrogate markers. An excellent correlation was observed between the HpaII and CpG island methylation levels (r > 0.93). At the qualitative level, the predictive sensitivity of HpaII was >95% with >92% specificity for methylated CpG islands and >90% sensitivity with >95% specificity for unmethylated CpG islands. This analysis provides a global validation framework for strategies based on the use of the methylation-sensitive HpaII restriction enzyme.     

CpG island libraries from human Chromosomes 18 and 22: landmarks for novel genes

CpG islands are found at the 5′ end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5′ end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5′ end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5′ gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5′ gene sequences from specific human chromosomes. Received: 15 November 1999 / Accepted: 31 January 2000     

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin–biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.     

MicroRNA regulation constrains the organization of target genes on mammalian chromosomes

The regulation of microRNAs (miRNAs) is a complicated process requiring a large number of molecular events to be coordinated in both space and time. It is not known whether this complicated regulation process constrains the organization of target genes on mammalian chromosomes. We performed a genome-wide analysis to provide a better picture of chromosomal organization of miRNA target genes. Our results showed clustering of the target genes (TGs) of miRNAs on mammalian chromosomes, and further revealed that the particular gene organization is constrained by miRNA regulation. The clustering pattern of TGs provides an insight into the complexity of miRNA regulation.