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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.

Fekete A, Kenesi E, Hunyadi-Gulyas E, Durgo H, Berko B, Dunai ZA, Bauer PI - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
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.

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Related in: MedlinePlus

In vitro binding of h PARP-1 to the h c-myc GQ structure and the effect of TMPyP4 on that binding.Figure 1A Effect of TMPyP4 on the binding of PARP-1 to the c-myc GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h c-myc 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 Materials and Methods. 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). Figure 1BIn vitro 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 c-myc 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 TGA GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TGA GGA GGG TGG GGA GAG TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in Materials and Methods. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, c-myc 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).
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pone-0042690-g001: In vitro binding of h PARP-1 to the h c-myc GQ structure and the effect of TMPyP4 on that binding.Figure 1A Effect of TMPyP4 on the binding of PARP-1 to the c-myc GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h c-myc 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 Materials and Methods. 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). Figure 1BIn vitro 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 c-myc 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 TGA GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TGA GGA GGG TGG GGA GAG TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in Materials and Methods. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, c-myc 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).

Mentions: PARP-1 is known to recognize the murine K-ras and the human c-kit GQ structures [11], [24]. To characterize the possible interaction between the h PARP-1 protein and the h c-myc GQ structure a binding assay was developed. As it is shown in Fig. 1A, PARP-1 binds to the wild type h c- myc GQ structure, while it displays only a very limited binding activity towards to the mutated h myc GQ sequences, the standard deviation of the binding assay is roughly equal to the measured values of binding (Fig. 1B). The wild type h c-myc GQ oligonucleotide has a sequence identical with the DNA sequence of the h c-myc gene's promoter region, spanning between positions −142 to −116. In the mutant-1 h c-myc GQ sequence a guanine base is replaced with an adenine base at position -131. The mutant-2 h c-myc GQ is a double mutant of the wild type of sequence bearing two guanine to adenine replacements, at positions −140 and at −126. The human telomeric GQ-DNA also displayed PARP-1 binding. The cationic porphyrine TMPyP4 binds to the highest and the lowest guanine tetrad layers and stabilizes the GQ structure. Because PARP-1 was shown to bind to the base part of the cruciform structure [25], we carried experiments to show, whether h PARP-1 binding to the wild type h c-myc GQ structure is influenced by TMPyP4. As it is shown in Fig. 1A, TMPyP4 competes out the binding of PARP-1 to the GQ structure.


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.

Fekete A, Kenesi E, Hunyadi-Gulyas E, Durgo H, Berko B, Dunai ZA, Bauer PI - PLoS ONE (2012)

In vitro binding of h PARP-1 to the h c-myc GQ structure and the effect of TMPyP4 on that binding.Figure 1A Effect of TMPyP4 on the binding of PARP-1 to the c-myc GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h c-myc 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 Materials and Methods. 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). Figure 1BIn vitro 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 c-myc 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 TGA GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TGA GGA GGG TGG GGA GAG TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in Materials and Methods. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, c-myc 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412819&req=5

pone-0042690-g001: In vitro binding of h PARP-1 to the h c-myc GQ structure and the effect of TMPyP4 on that binding.Figure 1A Effect of TMPyP4 on the binding of PARP-1 to the c-myc GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h c-myc 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 Materials and Methods. 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). Figure 1BIn vitro 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 c-myc 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 TGA GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TGA GGA GGG TGG GGA GAG TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in Materials and Methods. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, c-myc 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).
Mentions: PARP-1 is known to recognize the murine K-ras and the human c-kit GQ structures [11], [24]. To characterize the possible interaction between the h PARP-1 protein and the h c-myc GQ structure a binding assay was developed. As it is shown in Fig. 1A, PARP-1 binds to the wild type h c- myc GQ structure, while it displays only a very limited binding activity towards to the mutated h myc GQ sequences, the standard deviation of the binding assay is roughly equal to the measured values of binding (Fig. 1B). The wild type h c-myc GQ oligonucleotide has a sequence identical with the DNA sequence of the h c-myc gene's promoter region, spanning between positions −142 to −116. In the mutant-1 h c-myc GQ sequence a guanine base is replaced with an adenine base at position -131. The mutant-2 h c-myc GQ is a double mutant of the wild type of sequence bearing two guanine to adenine replacements, at positions −140 and at −126. The human telomeric GQ-DNA also displayed PARP-1 binding. The cationic porphyrine TMPyP4 binds to the highest and the lowest guanine tetrad layers and stabilizes the GQ structure. Because PARP-1 was shown to bind to the base part of the cruciform structure [25], we carried experiments to show, whether h PARP-1 binding to the wild type h c-myc GQ structure is influenced by TMPyP4. As it is shown in Fig. 1A, TMPyP4 competes out the binding of PARP-1 to the GQ structure.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus