Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors.
Using in vivo dimethylsulfate footprinting, we have analyzed protein-DNA interactions within two regions upstream of the tyrosine aminotransferase (TAT) gene that are characterized by an altered chromatin structure in TAT-expressing as compared to nonexpressing cells. All the identified protein contacts to DNA are found exclusively in the TAT-expressing hepatoma cells. In vitro analyses of specific DNA-binding factors in crude nuclear extracts yield DNAase I footprints that correlate well with the binding sites in vivo. Surprisingly, all DNA-binding activities are present in nuclei of TAT-expressing and nonexpressing cells, indicating that the mere presence of factors is not sufficient for their interaction with a binding site in vivo. Genomic sequencing reveals methylation of CpG dinucleotides in the regions analyzed in nonexpressing cells, whereas no methylation is found in TAT-expressing cells. In vitro methylation at a cytosine residue within a footprint region prevents the interaction of a factor with its binding site.
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000648:ultrastructure; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005796:Genes; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D014158:Transcription, Genetic; D014444:Tyrosine Transaminase / Q000235:genetics*
D002843:Chromatin; D004268:DNA-Binding Proteins; D004247:DNA; D014444:Tyrosine Transaminase