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.
Highlights
2008 and Italian Ministry of Scientific Research (PRIN 2007). □S The on-line version of this article contains supplemental Table S1 and Figs
This promoter contains a 34-bp G-rich sequence between nucleotides Ϫ322 and Ϫ288 (Ϫ1 is the 3Ј boundary of the exon 0), which is sensitive to nuclease S1 and able to fold into a parallel G-quadruplex conformation
We provide here compelling evidence that myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) are transcription activators and propose that these proteins are recruited to the KRAS promoter by the G-quadruplex structure formed within the GA-element
Summary
Site-specific Mutagenesis; Wild-type and Mutant Plasmid Construction—Plasmid pKRS413 harboring the CAT gene driven by the murine KRAS promoter was subjected to PCR using primers 5Ј-TGCAGCCGCTCCCTCTCTCTCTCCTTCTCTCTCTCCCGCGCG and 5Ј-GAGGGAGCGGCTGCAGCGCTGGGAG (to introduce four G 3 A point mutations) and 5Ј-TGCAGCCGCTCCCTCACTCACTCCTTCCCT and 5Ј-GAGGGAGCGGCTGCAGCGCTGGGAG (to introduce two G 3 T point mutations). The PCR reaction mixture was 1ϫ PCR buffer (from the kit), 0.2 mM dNTPs, 0.4 M primers, 0.04 units of Taq polymerase (EuroTaq Euroclone), and 1⁄10 of the ChIP samples. For cDNA synthesis 5 l of RNA in diethyl pyrocarbonate-treated water (extracted from about 25,000 cells) was heated at 55 °C and placed in ice. The solution was added to 7.5 l of mix containing (final concentrations) 1ϫ buffer, 0.01 M DTT (Invitrogen), 1.6 M primer dT (MWG Biotech, Ebersberg, Germany; d(T)16), 1.6 M Random primers (Promega), 0.4 mM dNTPs solution containing equimolar amounts of dATP, dCTP, dGTP, and dTTP (Euroclone, Pavia, Italy), 0.8 units/l RNase OUT, and 8 units/l of Maloney murine leukemia virus reverse transcriptase (Invitrogen). Polymerase-stop Assay—A linear DNA sequence of 80 nucleotides, named wtR-Mur (Table 1), containing the G-rich element of murine KRAS, was used as a template for Taq polymerase primer-extension reactions. The samples were re-annealed melted again to give the same
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