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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

The expression of h PARP-1 in vivo increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-beta-galactosidase expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-parp-1 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).
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pone-0042690-g003: The expression of h PARP-1 in vivo increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-beta-galactosidase expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-parp-1 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).

Mentions: Because ChIP experiments suggested that PARP-1 binds to both forms of the h c-myc promoter, we experimentally tested how PARP-1 is influencing the promoter activity of the h c-myc gene. Therefore reporter assays were set up as described in the Materials and Methods section. Three days after transfections, cell lysates were prepared and their luciferase and beta-galactosidase activities were assayed. Fig. 3 shows that as a consequence of h PARP-1 expression the beta-galactosidase normalized luciferase activities were increased from 47.4±11.1 units to 187.5±45.2 units representing a strong and significant increase in the h c-myc promoter activity of the reporter plasmid.


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)

The expression of h PARP-1 in vivo increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-beta-galactosidase expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-parp-1 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412819&req=5

pone-0042690-g003: The expression of h PARP-1 in vivo increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-beta-galactosidase expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-parp-1 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).
Mentions: Because ChIP experiments suggested that PARP-1 binds to both forms of the h c-myc promoter, we experimentally tested how PARP-1 is influencing the promoter activity of the h c-myc gene. Therefore reporter assays were set up as described in the Materials and Methods section. Three days after transfections, cell lysates were prepared and their luciferase and beta-galactosidase activities were assayed. Fig. 3 shows that as a consequence of h PARP-1 expression the beta-galactosidase normalized luciferase activities were increased from 47.4±11.1 units to 187.5±45.2 units representing a strong and significant increase in the h c-myc promoter activity of the reporter plasmid.

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