The KRAS promoter responds to Myc-associated zinc finger and poly(ADP-ribose) polymerase 1 proteins, which recognize a critical quadruplex-forming GA-element.
The murine KRAS promoter contains a G-rich nuclease hypersensitive element (GA-element) upstream of the transcription start site that is essential for transcription. Pulldown and chromatin immunoprecipitation assays demonstrate that this GA-element is bound by the Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) proteins. These proteins are crucial for transcription, because when they are knocked down by short hairpin RNA, transcription is down-regulated. This is also the case when the poly(ADP-ribosyl)ation activity of PARP-1 is inhibited by 3,4-dihydro-5-[4-(1-piperidinyl) butoxyl]-1(2H) isoquinolinone. We found that MAZ specifically binds to the duplex and quadruplex conformations of the GA-element, whereas PARP-1 shows specificity only for the G-quadruplex. On the basis of fluorescence resonance energy transfer melting and polymerase stop assays we saw that MAZ stabilizes the KRAS quadruplex. When the capacity of folding in the GA-element is abrogated by specific G --> T or G --> A point mutations, KRAS transcription is down-regulated. Conversely, guanidine-modified phthalocyanines, which specifically interact with and stabilize the KRAS G-quadruplex, push the promoter activity up to more than double. Collectively, our data support a transcription mechanism for murine KRAS that involves MAZ, PARP-1 and duplex-quadruplex conformational changes in the promoter GA-element.
Cogoi
Susanna
S
Department of Biomedical Science and Technology, University of Udine, School of Medicine, Piazzale Kolbe 4, 33100 Udine, Italy.
Paramasivam
Manikandan
M
Membrino
Alexandro
A
Yokoyama
Kazunari K
KK
Xodo
Luigi E
LE
D000818:Animals; D001483:Base Sequence; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D054856:G-Quadruplexes; D006147:Guanine / Q000378:metabolism*; D007211:Indoles / Q000737:chemistry; D054833:Isoindoles; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D009690:Nucleic Acid Conformation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055550:Protein Stability; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D007211:Indoles; D054833:Isoindoles; D008024:Ligands; D050505:Mutant Proteins; D014157:Transcription Factors; C076035:c-MYC-associated zinc finger protein; D006147:Guanine; D011065:Poly(ADP-ribose) Polymerases; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras); C013647:phthalocyanine
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