The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5′-CG-3′ dinucleotides

In eukaryotic cells, RNA polymerase II- and Illtranscribed promoters can be inactivated by sequencespecific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motifprotein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in nondenaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the Hpall (5′-CCGG-3′), the FnuDII (5′-CGCG-3′), or the CpG DNA methyltransferase from Spiroplasma species (M-Sssl; 5′-CG-3'′. Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E.coli dcm (5′-CCA/TGG-3′) sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNAbound transcription factors.