immunodetection of 5'-methylcytosine on Giemsa-stained chromosomes

摘要

In higher vertebrates, the major, if not the only, covalent modification of DNA is a methyl-group present at the C5 position of cytosine yielding 5'-methylcytosine (5'-MC). This modification occurs only in the context of CpG dinucleotides, short sequences that are significantly underrepresented in the genome but are found frequently concentrated within so called "CpG island" regions as well as in repetitive sequences (1). Several reasons might account for the statistical underrepre-sentation of CpG dinucleotides in the mammalian genome, one being that 5'-MC was shown to be a hotspot for mutation through deamination (2), Another aspect that has recently emerged addresses the issue of immu-nostimulation by unmethylated CpG sequences present in bacterial DNA, allowing the immune system to discriminate between sterile tissue damage and infectious danger (3). Previous studies on DNA methylation have already revealed a heterogeneous distribution of hypo- and hypermethylated areas on human metaphase chromosomes (4,5), CpG islands are predominantly found at the 5' ends of genes as well as in intron-ic regions, serving two major functions: (z) regulating the transcription levels of genes and (ii) protecting the host organisms against expression of undesired sequences (e.g., noncoding, repetitive, or parasitic ones) (6). Expressed genes are generally unmethylated, and aberrant methylation has been shown to be associated with many serious pathological consequences (7). For example, hypermethylation of tumorsuppressor gene promoters is often associated with transcriptional down-regulation by interfering with the binding of, for example, transcription factors (8,9) Several methods for obtaining meth-ylation-patterns on a genomic scale are currently established, yet most of them are restricted to the analysis of purified, complex DNA preparations (6), On the other hand, immunodetection offers the possibility to obtain spatially resolved information on the distribution of 5'-MC on metaphase chromosomes (10,11). We describe now an advanced experimental technique that renders the immunodetection of 5'-MC on human metaphase chromosome spreads previously stained by the Giemsa method possible. Due to the harsh conditions generally used during karyotype analysis, several modifications had to be introduced to maintain the chromosomal morphology during Giemsa staining, the destaining and DNA-denaturation procedures, and to facilitate the subsequent immunodetection of 5'-MC as outlined below.