Many detailed studies on the roles of chromatin structure in transcriptional regulation have compared active and inactive promoters in simple eukaryotic organisms, such as yeast (16
). However, the mechanisms by which endogenous human CpG island promoters in mammalian cells are regulated is less well understood (32
). This paucity of information led us to study the composition of the CpG island promoters of p16/CDKN2A
Conventional approaches are not sensitive enough to discern the organization of individual promoter molecules. While high resolution techniques involving primer extension showed multiple nucleosome translational frames (4
) as was found in the present work, these still rely on nuclease digestion. By definition, this means that a promoter could not be studied as a single intact entity until now. The method presented here to footprint DNA–protein interactions at single DNA molecule resolution relies on the known resistance of nucleosomal DNA to M.SssI modification (18
) coupled with the exquisite sensitivity afforded by the cloning and sequencing of individual progeny molecules.
Several lines of evidence suggest that the 120–180 bp inaccessible patches we observed in the p16/CDKN2A
promoter correspond to the presence of nucleosomes. First, their mean size reflects the expected size of a core nucleosome. These patches were also present on nucleosomes reconstituted using purified histones. The differences in patch sizes may be attributed to varying degrees of accessibility of M.SssI to the DNA as it approaches the nucleosomal pseudodyad (18
). Second, the average patch positions correspond to the patterns detected by traditional MNase digestion. Finally, pre-washing with 0.4 M salt, which is known to remove most non-histone binding proteins, did not markedly alter the patch distribution. However, regions upstream of the TISs became slightly more accessible to M.SssI, especially in high p16 expressing J82 cells (), indicating that DNA binding proteins may also restrict M.SssI accessibility (34
). In some of the promoters in which we find patches which are significantly larger than 120–170 bp, we believe these to reflect compaction of nucleosomes in a way which did not enable M.SssI access to the linker DNA. Thus, we conclude that the patches correlate with the approximate positions of nucleosomes on single molecules. This new approach substantially increases the resolution of analysis of protein–DNA interactions. It is limited by the fact that it relies on the density of CpG sites found in the area analyzed and that only sites unmethylated before M.SssI treatment can be analyzed. However, while the mammalian genome is generally CpG repressed (36
), ~40% of mammalian genes have promoters and exonic regions containing CpG islands (37
), which are normally unmethylated (38
), meaning that they can be analyzed by this approach. Since organisms such as Saccharomyces cerevisiae
and Drosophila melanogaster
are not CpG depleted (36
), this method should allow for a high resolution analysis of nucleosomal positioning in these species. Another limitation is that while the outer limits of patch size can be determined, the exact sizes cannot. This problem may be overcome in the future by combining CpG-specific (M.SssI) and GpC-specific methyltransferases (M.CviPI) (39
), to increase the density of methylated sites.
Application of this method, which reflects the state of a single promoter molecule ‘frozen’ in time, revealed different dynamics of the promoters of two cell types with markedly different levels of p16
gene expression. Many of the promoters in the low expressing LD419 cells had nucleosomes organized in a quite conserved pattern which correlated to nucleosomes I, II and III mapped by the MNase assay. However, in the highly expressing J82 cells only one or two nucleosomes were detected, which were more dynamically organized. Since nucleosomes are often moved or disassembled (13
) to allow the transcriptional machinery access (3
) this could reflect nucleosomal remodeling which may enable the increased level of p16
Apart from analyzing nucleosome positioning this method may be useful to detect footprints of regulatory proteins acting on various promoters. Furthermore, the combination of cross linking prior to treatment with M.SssI might provide an even higher detailed profile of the promoters analyzed (E. Nili Gal-Yam, S. Jeong, P.A. Jones, unpublished data).
In summary, we have modified a technique by Kladde and Simpson (18
) to analyze promoters at single molecule resolution. The method does not rely on cutting with nucleases, so that the relevant nucleosomes and transcription factor footprints are maintained on the replica DNA molecules allowing us to see how the promoter functions as a unit. This approach provides a powerful tool to investigate dynamic changes involved in nucleosome remodeling and transcriptional activation.