Transcriptional consequences of targeting the i-motif structure of the c-Myc promoter with TMPyP4

3169 The nuclease hypersensitivity element III1 (NHE III1) upstream of both the P1 and P2 promoters in the c-Myc gene controls the majority of transcriptional activity (80â\#8364;“90%) of the gene and has been shown to form secondary DNA structures. It has also been shown as the target for the cationic porphyrin TMPyP4 molecule, which has been previously shown to downregulate c-Myc gene expression (Siddiqui-Jain et al., Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 11593). Through a series of experiments, an additional target to the G-quadruplex has been identified for TMPyP4 binding, which is the i-motif structure that forms from the pyrimidine-rich strand of the NHE. Using a cleavage assay, it has been shown that TMPyP4 binds specifically to the top of the i-motif structure. This binding of TMPyP4 increases the melting temperature of the i-motif, making it more stable, as seen by CD spectrometry. Using the ChIP assay it was shown that binding of TMPyP4 to the i-motif structure prevents the association of hnRNP K, a transcription factor known to regulate the expression of c-Myc. In addition, NMR confirms the binding of TMPyP4 to the i-motif structure. Finally, gene transcription was detected using real-time PCR, illustrating the effects of TMPyP4 in cells. These experiments suggest that an important target for TMPyP4, in addition to the G-quadruplex, is the i-motif and that stabilization of this structure prevents binding of hnRNP K, resulting in transcriptional silencing.