RRC ID 6867
Author Cogoi S, Paramasivam M, Membrino A, Yokoyama KK, Xodo LE.
Title The KRAS promoter responds to Myc-associated zinc finger and poly(ADP-ribose) polymerase 1 proteins, which recognize a critical quadruplex-forming GA-element.
Journal J Biol Chem
Abstract 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.
Volume 285(29)
Pages 22003-16
Published 2010-7-16
DOI 10.1074/jbc.M110.101923
PII M110.101923
PMID 20457603
PMC PMC2903372
MeSH Animals Base Sequence Chromatin Immunoprecipitation DNA-Binding Proteins / metabolism* Electrophoretic Mobility Shift Assay Fluorescence Resonance Energy Transfer G-Quadruplexes* Guanine / metabolism* Indoles / chemistry Ligands Mice Molecular Sequence Data Mutant Proteins / metabolism NIH 3T3 Cells Nucleic Acid Conformation Poly(ADP-ribose) Polymerases / metabolism* Promoter Regions, Genetic* Protein Binding Protein Stability Proto-Oncogene Proteins p21(ras) / genetics* Transcription Factors / metabolism* Transcription, Genetic
IF 4.238
Times Cited 72
DNA material pGEX-hMAZ (RDB03219) pCMV-MAZ (RDB05701).