Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes.

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.     

Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.

Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand.