The possibility that histone H1 binds preferentially to DNA containing 5-methylcytosine in the dinucleotide CpG is appealing, as it could help to explain the repressive effects of methylation on gene activity. In this study, the affinity of purified H1 for methylated and non-methylated DNA sequences has been tested using both naked DNA and chromatin. Based on a variety of assays (bandshifts, filter-binding assays, Southwestern blots, and nuclease sensitivity assays), we conclude that H1 has no significant preference for binding to naked methylated DNA. Similarly, H1 showed the same affinities for methylated and non-methylated DNA when assembled into chromatin in a Xenopus oocyte extract. Thus potential cooperative interaction of H1 with polynucleosomal complexes is not enhanced by the presence of DNA methylation.The major DNA modification in vertebrates is methylation at the 5 position of cytosine in the dinucleotide CpG. About 60 -90% of genomic CpGs contain 5-methyl cytosine (m 5 C). Transfection assays with methylated genes have shown that CpG methylation often causes repression of transcription (1-4). The severity of repression depends on a number of parameters, particularly the location of methylation relative to the promoter (1, 5; but see Ref. 6), the local density of methyl-CpG pairs, and the strength of the promoter under test (7). In general, high density methylation strongly inhibits transcription, whereas low density methylation can only inhibit weak promoters.Several lines of evidence suggest that proteins that bind preferentially to methylated DNA are important components of the repression mechanism. Indeed, binding of the methyl-CpGbinding protein (MeCP) 1 MeCP1 (8) shows the same dependence on local density of methylation as does transcriptional repression (4, 7). Methylation-associated transcriptional repression may also arise by other routes. CpG methylation is known to prevent binding of some transcription factors, and this is likely to contribute to repression in some cases (9, 10). Another potential cause of methylation-mediated repression is direct alteration of chromatin structure due to the presence of m 5 C. Involvement of chromatin in mediating the effects of DNA methylation is suggested by the experiments of Buschhausen et al. (3,11). In addition, Keshet et al. found that methylated DNA is preferentially assembled into nuclease-resistant chromatin after transfection (12). While these results could be explained by interaction between known methyl-CpG-binding proteins and chromatin, it is also possible that an ubiquitous component of chromatin, for example histones, interacts differentially with methylated DNA leading to an altered higher order structure that is incompatible with gene expression.Attempts to detect an effect of DNA methylation on nucleosomal cores have not been successful (13,14). The linker histone H1, however, is an attractive candidate for mediator of the effects of methylation on chromatin. H1 plays a role in chromatin condensation (15, 16). It is depleted in CpG i...