title: G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression.
abstract: The Oct4 gene codes for a transcription factor that plays a critical role in the maintenance of pluripotency in embryonic and cancer stem cells. Its expression thus has to be tightly regulated. We performed biophysical characterization of the promoter region using a combination of UV absorption, CD, and NMR spectroscopies, native PAGE and chemical probing, which was followed by functional studies involving luciferase reporter assays performed in osteosarcoma and human embryonic stem cell lines. We have shown that the evolutionarily conserved G-rich region close to the Oct4 transcription start site in the non-template strand forms a parallel G-quadruplex structure. We characterized its structure and stability upon point mutations in its primary structure. Functional studies then revealed that whereas the wild type quadruplex sequence ensures high reporter gene expression, the expression of mutated variants is significantly decreased proportionally to the destabilizing effect of the mutations on the quadruplex. A ligand, N-methyl mesoporphyrin IX that increases the stability of formed quadruplex rescued the reporter expression of single-mutated variants to the level of wild-type, but it has no effect on a mutated variant that cannot form quadruplex. These data indicate that the quadruplex acts as a strong, positive regulator of Oct4 expression and as such it might serve as a potential target for therapeutic intervention.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Kralovopolska 135, 612 65 Brno, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Renčiuk
authors.affiliation: Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Ryneš
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Kralovopolska 135, 612 65 Brno, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Kejnovská
authors.affiliation: Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic.
authors.forename: Silvie
authors.initials: S
authors.lastname: Foldynová-Trantírková
authors.affiliation: Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; Department of Physiology and Pharmacology, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Michael
authors.initials: M
authors.lastname: Andäng
authors.affiliation: Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic. Electronic address: lukas.trantirek@ceitec.muni.cz.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Trantírek
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i, Kralovopolska 135, 612 65 Brno, Czech Republic. Electronic address: mifi@ibp.cz.
authors.forename: Michaela
authors.initials: M
authors.lastname: Vorlíčková
mesh_terms: D045744:Cell Line, Tumor; D002942:Circular Dichroism / Q000379:methods; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism; D054856:G-Quadruplexes / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008279:Magnetic Resonance Imaging / Q000379:methods; D008652:Mesoporphyrins / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D012516:Osteosarcoma / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D024363:Transcription Initiation Site / Q000187:drug effects / Q000502:physiology
chemical_list: D008652:Mesoporphyrins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C031468:mesoporphyrin IX
keywords: Circular dichroism; Guanine quadruplex; Human embryonic stem cells; Luciferase reporter; Oct4; Transcription regulation