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Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation.

Beneke S, Meyer K, Holtz A, Hüttner K, Bürkle A - PLoS ONE (2012)

Bottom Line: Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites.Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose).By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.

View Article: PubMed Central - PubMed

Affiliation: Molecular Toxicology, University of Konstanz, Konstanz, Germany. sascha.beneke@vetpharm.uzh.ch

ABSTRACT
Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.

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Low-dose formaldehyde induces γH2AX formation.Cells were fixed with 1% or 4% paraformaldehyde and analyzed by confocal microscopy. (A) γH2AX foci were counted using one confocal slice after reducing background staining by ImageJ software. Reduction parameters were identical for respective pictures. Cells were split into three groups with (i) less than 5, (ii) between 5 and 20, (iii) more than 20 foci, and percent of total cells was calculated. 10 min 1% paraformaldehyde (1% FA) induces more than sevenfold increase in cells with more than 20 foci, and a decrease in cells with less than 5 foci compared to 4% paraformaldehyde (4% FA). Error bars represent mean±s.e.m. (N = 3), ***P<0.001; data were analyzed with one-way ANOVA and Dunnett's Multiple Comparison Test. (B) All pictures from a z-stack were analyzed for γH2AX foci intensity and normalized to cells fixed for 20 min with 4% paraformaldehyde. Intensity increases eightfold with 1% FA compared to 4% FA. Error bars represent mean±s.e.m. (N = 3), *P = 0.016; data were analyzed by unpaired t-test.
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pone-0032914-g004: Low-dose formaldehyde induces γH2AX formation.Cells were fixed with 1% or 4% paraformaldehyde and analyzed by confocal microscopy. (A) γH2AX foci were counted using one confocal slice after reducing background staining by ImageJ software. Reduction parameters were identical for respective pictures. Cells were split into three groups with (i) less than 5, (ii) between 5 and 20, (iii) more than 20 foci, and percent of total cells was calculated. 10 min 1% paraformaldehyde (1% FA) induces more than sevenfold increase in cells with more than 20 foci, and a decrease in cells with less than 5 foci compared to 4% paraformaldehyde (4% FA). Error bars represent mean±s.e.m. (N = 3), ***P<0.001; data were analyzed with one-way ANOVA and Dunnett's Multiple Comparison Test. (B) All pictures from a z-stack were analyzed for γH2AX foci intensity and normalized to cells fixed for 20 min with 4% paraformaldehyde. Intensity increases eightfold with 1% FA compared to 4% FA. Error bars represent mean±s.e.m. (N = 3), *P = 0.016; data were analyzed by unpaired t-test.

Mentions: In the next step, we investigated if formaldehyde fixation induces DNA damage and related signaling by analyzing γH2AX formation with confocal microscopy. We detected a more than sevenfold increase in cells with high amounts of γH2AX foci if treated with 1% formaldehyde compared to 4% formaldehyde, reflecting massively induced DNA strand-break signaling (Figure 4A). The overall γH2AX intensity in the whole nucleus increased eightfold (Figure 4B). Co-localization analysis in HeLa S3 cells as well as VH7 normal human fibroblasts revealed that both DNA damage markers work independently (Figure S1A–D, see Materials and Methods S1 for experimental setup), with very low if any overlap. Additionally, short term PJ34 application before fixation suppressed synthesis of PAR, but not of γH2AX foci, whereas 3.7% formaldehyde abrogated appearance of both DNA damage markers (Figure S1A–D). Thus, PARP activity seems to be induced by the formation of different DNA lesions than γH2AX, but both signaling processes are abrogated at high concentrations of formaldehyde.


Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation.

Beneke S, Meyer K, Holtz A, Hüttner K, Bürkle A - PLoS ONE (2012)

Low-dose formaldehyde induces γH2AX formation.Cells were fixed with 1% or 4% paraformaldehyde and analyzed by confocal microscopy. (A) γH2AX foci were counted using one confocal slice after reducing background staining by ImageJ software. Reduction parameters were identical for respective pictures. Cells were split into three groups with (i) less than 5, (ii) between 5 and 20, (iii) more than 20 foci, and percent of total cells was calculated. 10 min 1% paraformaldehyde (1% FA) induces more than sevenfold increase in cells with more than 20 foci, and a decrease in cells with less than 5 foci compared to 4% paraformaldehyde (4% FA). Error bars represent mean±s.e.m. (N = 3), ***P<0.001; data were analyzed with one-way ANOVA and Dunnett's Multiple Comparison Test. (B) All pictures from a z-stack were analyzed for γH2AX foci intensity and normalized to cells fixed for 20 min with 4% paraformaldehyde. Intensity increases eightfold with 1% FA compared to 4% FA. Error bars represent mean±s.e.m. (N = 3), *P = 0.016; data were analyzed by unpaired t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316553&req=5

pone-0032914-g004: Low-dose formaldehyde induces γH2AX formation.Cells were fixed with 1% or 4% paraformaldehyde and analyzed by confocal microscopy. (A) γH2AX foci were counted using one confocal slice after reducing background staining by ImageJ software. Reduction parameters were identical for respective pictures. Cells were split into three groups with (i) less than 5, (ii) between 5 and 20, (iii) more than 20 foci, and percent of total cells was calculated. 10 min 1% paraformaldehyde (1% FA) induces more than sevenfold increase in cells with more than 20 foci, and a decrease in cells with less than 5 foci compared to 4% paraformaldehyde (4% FA). Error bars represent mean±s.e.m. (N = 3), ***P<0.001; data were analyzed with one-way ANOVA and Dunnett's Multiple Comparison Test. (B) All pictures from a z-stack were analyzed for γH2AX foci intensity and normalized to cells fixed for 20 min with 4% paraformaldehyde. Intensity increases eightfold with 1% FA compared to 4% FA. Error bars represent mean±s.e.m. (N = 3), *P = 0.016; data were analyzed by unpaired t-test.
Mentions: In the next step, we investigated if formaldehyde fixation induces DNA damage and related signaling by analyzing γH2AX formation with confocal microscopy. We detected a more than sevenfold increase in cells with high amounts of γH2AX foci if treated with 1% formaldehyde compared to 4% formaldehyde, reflecting massively induced DNA strand-break signaling (Figure 4A). The overall γH2AX intensity in the whole nucleus increased eightfold (Figure 4B). Co-localization analysis in HeLa S3 cells as well as VH7 normal human fibroblasts revealed that both DNA damage markers work independently (Figure S1A–D, see Materials and Methods S1 for experimental setup), with very low if any overlap. Additionally, short term PJ34 application before fixation suppressed synthesis of PAR, but not of γH2AX foci, whereas 3.7% formaldehyde abrogated appearance of both DNA damage markers (Figure S1A–D). Thus, PARP activity seems to be induced by the formation of different DNA lesions than γH2AX, but both signaling processes are abrogated at high concentrations of formaldehyde.

Bottom Line: Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites.Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose).By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.

View Article: PubMed Central - PubMed

Affiliation: Molecular Toxicology, University of Konstanz, Konstanz, Germany. sascha.beneke@vetpharm.uzh.ch

ABSTRACT
Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.

Show MeSH
Related in: MedlinePlus