Journal
BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS
Volume 1860, Issue 2, Pages 175-183Publisher
ELSEVIER
DOI: 10.1016/j.bbagrm.2016.11.002
Keywords
Guanine quadruplex; Oct4; Transcription regulation; Circular dichroism; Luciferase reporter; Human embryonic stem cells
Categories
Funding
- Grant Agency of the Czech Republic [14-33947P, 13-28310S, P205/12/0466, 15-06785S]
- CEITEC project [LQ1601]
- Ministry of Education, Youths and Sports of the Czech Republic within the National Programme for Sustainability II funds
- SoMoPro II Programme - People Programme (Marie Curie Action) of the Seventh Framework Programme of EU [4SGA8580, 291782]
- South-Moravian Region
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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. (C) 2016 Elsevier B.V. All rights reserved.
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