title: The i-motif in the bcl-2 P1 promoter forms an unexpectedly stable structure with a unique 8:5:7 loop folding pattern.
abstract: Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.
authors.affiliation: Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, Arizona 85724, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Kendrick
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Akiyama
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Hurley
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001966:Bromine / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D003857:Deoxyuridine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D020862:GC Rich Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D017384:Sequence Deletion; D013696:Temperature; D013941:Thymine / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D004247:DNA; D013941:Thymine; D001966:Bromine; D003857:Deoxyuridine