The i-motif in the bcl-2 P1 promoter forms an unexpectedly stable structure with a unique 8:5:7 loop folding pattern

Samantha Kendrick, Yoshitsugu Akiyama, Sidney Hecht, Laurence H. Hurley

Research output: Contribution to journalArticlepeer-review

93 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)17667-17676
Number of pages10
JournalJournal of the American Chemical Society
Volume131
Issue number48
DOIs
StatePublished - Dec 9 2009

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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