Koichi Matsuo

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF‐κB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by α‐amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replication‐dependent, possibly passive mechanism.

Evidence for erosion of mouse CpG islands during mammalian evolution

In housekeeping and many tissue-specific genes, the promoter is embedded in a so-called CpG island. We have compared the available human and mouse DNA sequences with respect to their CpG island properties. While mouse sequences showed a simple gradient distribution of G+C content and CpG densities, man had a distinct peak of sequences with typical CpG island characteristics. Pairwise comparison of 23 orthologous genes revealed that mouse almost always had a less pronounced CpG island than man, or none at all. In both species the requirements for a functional CpG island may be similar in that most DNA regions with a density of six or more CpG per 100 bp remain unmethylated. However, the mouse has apparently experienced more accidental CpG island methylation, suggested by local TpG and CpA excess. We propose that: (1) in mouse the CpG islands do not represent the ancestral state but have been eroded during evolution, and (2) this erosion may be related to the mouses small body mass and short life-span, allowing for a more relaxed control of gene activity.

Transcriptional repression by methylation: Cooperativity between a CpG cluster in the promoter and remote CpG-rich regions

Cytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA‐VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation‐free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer.

Complex demethylation patterns at Sp1 binding sites in F9 embryonal carcinoma cells

The ubiquitous transcription factor Sp1 has been implicated in the mechanism which maintains CpG islands methylation‐free. Plasmids containing GC boxes (Sp1 sites) were in vitro methylated at every CpG dinucleotide. After stable introduction into F9 embryonal carcinoma cells, we analysed the methylation of the sequence around the GC boxes with bisulphite sequencing. In agreement with restriction site analysis by other labs, we found preferential demethylation at GC box DNA versus control DNA. However, the bisulphite sequencing which permits the analysis of every CpG site on a given DNA molecule, revealed a complex pattern of methylated and unmethylated sites. Upon prolonged culture the pattern became simpler, with most sites demethylated but certain sites being consistently methylated.

Periodicity of eight nucleotides in purine distribution around human genomic CpG dinucleotides

Mammalian genomes, unlike the genomes of Drosophila and yeast, are characterized by CpG methylation and concomitant CpG depletion, which is caused by the enhanced mutation rate of 5-methylcytosine. To find out whether local nucleotide sequences around existing methylated CpG dinucleotides have common patterns, we analyzed a large population of CpG-poor regions in human DNA, which are typically methylated. We detected a novel periodic variation in the numbers of purine bases around CpGs in the noncoding parts of these sequences. This periodicity of eight nucleotides gradually diminished over 64 nucleotides on each side of the central CpG. Furthermore, the frequencies of the 5′ and 3′ nearest neighbors of CpGs in CpG-poor regions were biased towards cytosine and guanine, respectively. Such biased sequence contexts may have helped to stabilize CpGs against depletion during mammalian evolution.