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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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Fixation by ChIP protocols induces PARylation.(A) PAR staining after fixation by three different ChIP protocols or methanol. ChIP fixations induce PAR staining (I–III). Methanol fixation shows no PAR formation (IV). H2O2 and methanol fixation (V) induces granular PAR staining. Focal PAR formation in JLI fixation (I) is reverted to normal distribution if H2O2 is applied in advance (VI). Procedures are indicated below microscopic pictures. Scale bars represent 10 µM. (B) Statistical evaluation of data obtained in (A). Three independent experiments (N = 3) were analyzed with at least 50 cells each data point of one experiment. % PAR positive cells were calculated and analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test; ***P<0.001. Only methanol fixation is significantly different from the others. Error bars represent mean±s.e.m. (C) Mouse 3T3 cells were fixed by JLI protocol. PAR formation was detected in all three lines tested, i.e. wild type (wt), PARP1 knockout (P1ko) and PARP2 knockout (P2ko). PAR-fluorescence intensities of cells from four randomly chosen microscopic fields per cell line were analyzed by ImageJ and normalized to intensity in wt cells (RFU: relative fluorescence units). At least six independent experiments were used for statistical analysis by One-Way-ANOVA with Bonferroni's Multiple Comparisons Test; error bars represent mean±s.e.m. (N≥6), ***P<0.001 wt vs. P1ko, ****P<0.0001 wt vs. P2ko, P1ko vs. P2ko not significant.
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pone-0032914-g002: Fixation by ChIP protocols induces PARylation.(A) PAR staining after fixation by three different ChIP protocols or methanol. ChIP fixations induce PAR staining (I–III). Methanol fixation shows no PAR formation (IV). H2O2 and methanol fixation (V) induces granular PAR staining. Focal PAR formation in JLI fixation (I) is reverted to normal distribution if H2O2 is applied in advance (VI). Procedures are indicated below microscopic pictures. Scale bars represent 10 µM. (B) Statistical evaluation of data obtained in (A). Three independent experiments (N = 3) were analyzed with at least 50 cells each data point of one experiment. % PAR positive cells were calculated and analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test; ***P<0.001. Only methanol fixation is significantly different from the others. Error bars represent mean±s.e.m. (C) Mouse 3T3 cells were fixed by JLI protocol. PAR formation was detected in all three lines tested, i.e. wild type (wt), PARP1 knockout (P1ko) and PARP2 knockout (P2ko). PAR-fluorescence intensities of cells from four randomly chosen microscopic fields per cell line were analyzed by ImageJ and normalized to intensity in wt cells (RFU: relative fluorescence units). At least six independent experiments were used for statistical analysis by One-Way-ANOVA with Bonferroni's Multiple Comparisons Test; error bars represent mean±s.e.m. (N≥6), ***P<0.001 wt vs. P1ko, ****P<0.0001 wt vs. P2ko, P1ko vs. P2ko not significant.

Mentions: Having established the link between PARP activity and low-dose formaldehyde fixation, we focused on the technique of chromatin immunoprecipitation (ChIP). In this method, a 10 minute/1% formaldehyde fixation step is typically used to crosslink proteins with DNA. We analyzed three different published ChIP protocols abbreviated JLI, MLI and UMC, respectively [41]–[43] and tested them for induction of PAR formation (Figure 2A). As summarized in Fig. 2B, nearly 100% of the cells were polymer positive in all three protocols, whereas standard methanol fixation showed no signal. To exclude that PAR formation is confined to HeLa cells and to define, which PARP is the prevalent enzyme performing this reaction, we employed mouse 3T3 fibroblasts and subjected them to JLI fixation procedure (Figure 2C). Wild type (wt) cells also showed massive PAR synthesis with formaldehyde, similar to HeLa cells. Genetic deletion of either PARP1 protein (P1ko) or PARP2 protein (P2ko) reduced PAR levels to a similar extent of about 40%, which suggests that at least these two PARPs are responsible for synthesis of polymer after low-dose formaldehyde treatment. In order to abrogate PAR synthesis, we used the standard treatment to suppress polymer production after DNA damage by applying 2 µM PJ34, a pan-PARP inhibitor, 6 h in advance of fixation (Figure 3). Still, we observed polymer signals in all cells after formaldehyde fixation. In order to determine at which point of the JLI protocol [42] PAR is produced, we fixed the cells with methanol directly after the formaldehyde or the PBS washing step, respectively. Only methanol application directly after formaldehyde fixation reduced PAR formation significantly. Therefore, PARP activity is triggered during formaldehyde incubation and aggravated during PBS washings, as PAR intensity is massively increased. This enzymatic reaction could only be blocked by a combined pre- and post-incubation with the inhibitor (Figure 3A, panel VII). This suggests that the fixation process itself induces PARP activity.


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)

Fixation by ChIP protocols induces PARylation.(A) PAR staining after fixation by three different ChIP protocols or methanol. ChIP fixations induce PAR staining (I–III). Methanol fixation shows no PAR formation (IV). H2O2 and methanol fixation (V) induces granular PAR staining. Focal PAR formation in JLI fixation (I) is reverted to normal distribution if H2O2 is applied in advance (VI). Procedures are indicated below microscopic pictures. Scale bars represent 10 µM. (B) Statistical evaluation of data obtained in (A). Three independent experiments (N = 3) were analyzed with at least 50 cells each data point of one experiment. % PAR positive cells were calculated and analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test; ***P<0.001. Only methanol fixation is significantly different from the others. Error bars represent mean±s.e.m. (C) Mouse 3T3 cells were fixed by JLI protocol. PAR formation was detected in all three lines tested, i.e. wild type (wt), PARP1 knockout (P1ko) and PARP2 knockout (P2ko). PAR-fluorescence intensities of cells from four randomly chosen microscopic fields per cell line were analyzed by ImageJ and normalized to intensity in wt cells (RFU: relative fluorescence units). At least six independent experiments were used for statistical analysis by One-Way-ANOVA with Bonferroni's Multiple Comparisons Test; error bars represent mean±s.e.m. (N≥6), ***P<0.001 wt vs. P1ko, ****P<0.0001 wt vs. P2ko, P1ko vs. P2ko not significant.
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getmorefigures.php?uid=PMC3316553&req=5

pone-0032914-g002: Fixation by ChIP protocols induces PARylation.(A) PAR staining after fixation by three different ChIP protocols or methanol. ChIP fixations induce PAR staining (I–III). Methanol fixation shows no PAR formation (IV). H2O2 and methanol fixation (V) induces granular PAR staining. Focal PAR formation in JLI fixation (I) is reverted to normal distribution if H2O2 is applied in advance (VI). Procedures are indicated below microscopic pictures. Scale bars represent 10 µM. (B) Statistical evaluation of data obtained in (A). Three independent experiments (N = 3) were analyzed with at least 50 cells each data point of one experiment. % PAR positive cells were calculated and analyzed by one-way ANOVA and Bonferroni's Multiple Comparison Test; ***P<0.001. Only methanol fixation is significantly different from the others. Error bars represent mean±s.e.m. (C) Mouse 3T3 cells were fixed by JLI protocol. PAR formation was detected in all three lines tested, i.e. wild type (wt), PARP1 knockout (P1ko) and PARP2 knockout (P2ko). PAR-fluorescence intensities of cells from four randomly chosen microscopic fields per cell line were analyzed by ImageJ and normalized to intensity in wt cells (RFU: relative fluorescence units). At least six independent experiments were used for statistical analysis by One-Way-ANOVA with Bonferroni's Multiple Comparisons Test; error bars represent mean±s.e.m. (N≥6), ***P<0.001 wt vs. P1ko, ****P<0.0001 wt vs. P2ko, P1ko vs. P2ko not significant.
Mentions: Having established the link between PARP activity and low-dose formaldehyde fixation, we focused on the technique of chromatin immunoprecipitation (ChIP). In this method, a 10 minute/1% formaldehyde fixation step is typically used to crosslink proteins with DNA. We analyzed three different published ChIP protocols abbreviated JLI, MLI and UMC, respectively [41]–[43] and tested them for induction of PAR formation (Figure 2A). As summarized in Fig. 2B, nearly 100% of the cells were polymer positive in all three protocols, whereas standard methanol fixation showed no signal. To exclude that PAR formation is confined to HeLa cells and to define, which PARP is the prevalent enzyme performing this reaction, we employed mouse 3T3 fibroblasts and subjected them to JLI fixation procedure (Figure 2C). Wild type (wt) cells also showed massive PAR synthesis with formaldehyde, similar to HeLa cells. Genetic deletion of either PARP1 protein (P1ko) or PARP2 protein (P2ko) reduced PAR levels to a similar extent of about 40%, which suggests that at least these two PARPs are responsible for synthesis of polymer after low-dose formaldehyde treatment. In order to abrogate PAR synthesis, we used the standard treatment to suppress polymer production after DNA damage by applying 2 µM PJ34, a pan-PARP inhibitor, 6 h in advance of fixation (Figure 3). Still, we observed polymer signals in all cells after formaldehyde fixation. In order to determine at which point of the JLI protocol [42] PAR is produced, we fixed the cells with methanol directly after the formaldehyde or the PBS washing step, respectively. Only methanol application directly after formaldehyde fixation reduced PAR formation significantly. Therefore, PARP activity is triggered during formaldehyde incubation and aggravated during PBS washings, as PAR intensity is massively increased. This enzymatic reaction could only be blocked by a combined pre- and post-incubation with the inhibitor (Figure 3A, panel VII). This suggests that the fixation process itself induces PARP activity.

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