Cytosine methylation enhances Z-DNA formation in vivo

Abstract

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M -HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M -HhaI than in the absence of M HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.DNA structural microheterogeneity is believed to play a fundamental role in genetic regulatory mechanisms (16,20). Left-handed DNA (Z-DNA) is a structural alternative to right-handed DNA (B-DNA) that has been well characterized in vitro (14,17,21). Recently, it was demonstrated that Z-DNA can exist in living Escherichia coli cells and that a given sequence can coexist in the Z form and the B form in the same cell (7,8,15,22,23). The in vivo B-Z equilibrium is influenced by active biological processes (like transcription, replication, supercoil and toroid formations, and DNAprotein interactions).There is evidence that cytosine methylation may have an important signaling function in eucaryotic gene expression (4, 11). The hypomethylation of gene regulatory regions is often connected to an expressed state of those genes, whereas silent genes are generally overmethylated. In vitro, cytosine methylation also facilitates the B-to-Z transition in synthetic linear DNA polymers (1, 2, 5) and in certain sequences in supercoiled recombinant plasmids (10,25).In previous studies, we employed two independent assays for structural analyses under in vivo conditions. The first assay was based on the observation that a recognition sequence (GAATTC) is not methylated by the corresponding bacterial methyltransferase (M EcoRI) when this site is near or inside a left-handed helix, whereas other EcoRI sites in the same plasmid molecule were completely methylated (8,23). The presence of a Z structure around the unmethylated sites was confirmed by a variety of chemical, enzymatic, and topological approaches (14,16,17,21,24 On this basis, the formation of Z-DNA inside growing bacterial cells could be analyzed by a linking-number assay. The B-to-Z transition inside E. coli cells results in the relaxation of a number of supercoils, in proportion to the length of the Z-region involved. This process causes DNA gyrase to decrease...

Inflammatory responses following direct injection of plasmid DNA into skeletal muscle

“…The insolubility is possibly due to a greater tendency of methylated DNA to form zDNA. 43,44 Additionally, methylation can downregulate promoter activity although there are examples of promoter sequences devoid of CpG motifs, ie mouse mammary tumour virus long terminal repeat (MMTV LTR), which renders such promoters insensitive to methylation. 45 Unfortunately, the expression levels obtained from MMTV␤ following direct injection are lower than that obtained for methylated CMV␤ (Wells KE, unpublished data).…”

Methylation of the rat glial fibrillary acidic protein gene shows tissue‐specific domains

“…Z-DNA is a right-handed helical conformer with 12 bp per turn that differs from the more common left-handed B-DNA helix with 10 bp per turn. Z-DNA is preferentially recognized over B-DNA by DNA methyltransferases (Smith et al, 1991), and the Z-DNA conformation is promoted by methylation at "'C (Zacharias et al, 1990). A possible consequence of Z-DNA is the phasing of nucleosomes, which can, in turn, influence transcription (see Introduction).…”

“…Repression of transcription in vitro by histones can also be enhanced by methylation (Englander et al, 1993). Alternatively, methylation can stabilize Z-DNA structures (Zacharias et al, 1990), which, in turn, can alter nucleosome phasing (Nickol et al, 1982), among other effects on chromatin structure (Tazi and Bird, 1990;Keshet et al, 1986;Selker, 1990). DNA methylation also can influence transcription initiation (Levine et al, 1992) and elongation (Barry et al, 1993).…”

DNA Methylation and Cancer

“…The methyl group in CpG dinucleotides protrudes from the cytosine nucleotide into the major groove of the DNA and has two main effects: it displaces transcription factors that normally bind to the DNA; and it attracts methyl binding proteins, which in turn are associated with gene silencing and chromatin compaction (Fazzari and Greally 2004 ) . Methylation of DNA in the common 'B' form facilitates a conformational change to the 'Z' form, increases the helical pitch of DNA and alters the kinetics of cruciform extrusion (Murchie and Lilley 1989 ;Zacharias et al 1990 ) . Promoters with a few exceptions are generally inactive when the CpG island within them are methylated (an exception, for example, is seen in the case of the H-2K gene promoter, Tanaka et al 1983 ) .…”