The guanine-quadruplex structure in the human c-myc gene's promoter is converted into B-DNA form by the human poly(ADP-ribose)polymerase-1.

The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes

Influence of nuclear proteins on the kinetics of unfolding of the h <i>c-myc</i> GQ structure, assayed by measuring the decrease in FRET activities in the presence of the complementary DNA strand.

*<p>decrease in FRET activity during one minute (% of decrease in the maximal height of the FRET peak measured at 0 time).</p>‡<p>number of experiments.</p><p>F-<i>myc</i> GQ-R double labeled FRET oligonucleotide (25 pmol) was incubated either alone or with the tested proteins for one minute in a volume of 50 µl, than its FRET spectra was taken (0 min value). The complementary, antiparallel oligonucleotide to the GQ structure (in a tenfold molar excess) was admixed and FRET spectra was recorded at 1, 3 and 5 minutes after annealing has been initiated. The kinetics of the decrease of the FRET peak heights were calculated and shown as ΔFRET peak value during one minute of annealing as percentage of the zero minute FRET peak height values. Excitation was at 485 nm and emissions were recorded between 500–650 nms.</p

ChIP-qPCR analysis of <i>in vivo</i> binding of h PARP-1 to the NHE III₁ region present in the promoter of the h <i>c-myc</i> gene, and which is presumably either in the GQ form or in the double stranded B-DNA form.

<p>ChIP-qPCR analysis was carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a> section using a polyclonal antibody raised against PARP-1 (Santa Cruz, H250; 2 µg/extract of one million cells) as precipitation agent. The treatment modalities and the ratio of PARP-1 bound promoter DNAs, isolated from differently treated cells (HeLa and HL60), and amplified by qPCR and calculated from the fluorescence of the Eva-Green complexes formed with the double-stranded PCR products, are shown. The c1/c2 values represent the ratio of the concentrations of PARP-1 bound <i>c-myc</i> promoter DNAs present in the ChIP products obtained from the treated (1) and from the non-treated (2) cell populations and calculated using the c1/c2 = 2^{n2 – n1} formula, where n1 and n2 are the number of PCR cycles needed to reach the same fluorescence value in the logarithmic phase of the PCR curves.</p

<i>In vitro</i> binding of h PARP-1 to the h <i>c-myc</i> GQ structure and the effect of TMPyP4 on that binding.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#pone-0042690-g001" target="_blank">Figure 1A</a> Effect of TMPyP4 on the binding of PARP-1 to the <i>c-myc</i> GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h <i>c-myc</i> GQ oligonucleotide (wild type GQ, 5′-biotin-TGG GGA GGG TGG GGA GGG TGG GGA AGG) in the absence and in the presence of different concentrations of the cationic porphyrin compound TMPyP4 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a>. Binding is expressed as % of binding measured in the absence of competing TMPyP4 and is shown on the ordinate. TMPyP4 concentrations (0, 0.58, 2.9, 14.4, and 72 µM) are shown on the abscissa. Asterisks represent samples where the extent of binding are significantly different (p<0.05, Student t-test). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#pone-0042690-g001" target="_blank">Figure 1B </a><i>In vitro</i> binding of h PARP-1 to various GQ structures and the competing effect of TMPyP4 on that binding. 0.45 pmol aliquots of h PARP-1 were incubated either with 5 pmols h telomeric GQ structure (h-telomeric GQ, 5′-biotin-TTA GGG TTA GGG TTA GGG TTA GGG) or with wild type <i>c-myc</i> GQ structure (wild type GQ, 5′-biotin-TGG GGA GGG TGG GGA GGG TGG GGA AGG) or its mutants (mutant-1 GQ, 5′-biotin-TGG GGA GGG TG<b>A</b> GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TG<b>A</b> GGA GGG TGG GGA G<b>A</b>G TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a>. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, <i>c-myc</i> GQ, G-131A and G-140, -126A) expressed in percentage. The amount of PARP-1 bound to telomeric GQ is taken as 100%. Insert shows the applied concentrations of TMPyP4. Asterisks represent samples where the difference in binding is significantly different (p<0.05).</p

The expression of h PARP-1 <i>in vivo</i> increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.

<p>Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-<i>beta-galactosidase</i> expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-<i>parp-1</i> plasmid). After two days of incubation cells were splitted into six-well plates and grown for a day further. Than cells were harvested, lysed and their luciferase and beta-galactosidase enzyme activities were determined. The luciferase enzyme activities were normalized for beta-galactosidase activities and are shown in the figure. Asterisks show significant difference in the reporter enzyme activities between the two pools of sample (p<0.05).</p