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PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia.

Martínez-Zamudio RI, Ha HC - Brain Behav (2014)

Bottom Line: Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells.Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines.Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology 337 Basic Science Building, 3900 Reservoir Road, Washington, District of Columbia, 20057.

ABSTRACT

Background: Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells.

Methods: PARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with (32)P-NAD(+). To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA.

Results: Our results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.

Conclusions: Collectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation.

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Related in: MedlinePlus

Histone ADP-ribosylation by PARP1 increases accessibility of nucleosomal DNA. (A) 601 nucleosomes labeled with 32P-NAD+ at the 5′ end of the antisense strand were incubated with PARP1 and the indicated NAD+ concentrations. Reactions were subsequently incubated with DNAse I and analyzed in a denaturing 8% polyacrylamide gel followed by autoradiography. +, DNase I-hypersensitive sites. Lane 1, DNA marker; lane 2, digested DNA control; lane 3, digested nucleosomes. The trace 10 bp repeating pattern observed in lane 2 is due to contamination from lane 1. (B) Schematic representation of the nucleosomes reconstituted with the Il1β proximal promoter indicating the NF-κB binding site and the Sty I restriction site. (C) The Il1β nucleosomes were incubated with PARP1 and the indicated NAD+ concentrations for 20 min at room temperature. Subsequently, 20 units of StyI were added and reactions were incubated at 37°C for 1 h. Reactions were stopped by addition of EDTA and heat inactivation at 65°C for 10 min. Reactions were analyzed by native agarose gel electrophoresis and DNA visualized by SYBR Green staining (B and C). (D) The Il1β nucleosomes were remodeled with the indicated NAD+ concentrations and subsequently incubated with or without p65 for 20 min at room temperature. The blue asterisk indicates the PARP1/nucleosome complex. The yellow star indicates a putative p65/nucleosome complex.
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fig03: Histone ADP-ribosylation by PARP1 increases accessibility of nucleosomal DNA. (A) 601 nucleosomes labeled with 32P-NAD+ at the 5′ end of the antisense strand were incubated with PARP1 and the indicated NAD+ concentrations. Reactions were subsequently incubated with DNAse I and analyzed in a denaturing 8% polyacrylamide gel followed by autoradiography. +, DNase I-hypersensitive sites. Lane 1, DNA marker; lane 2, digested DNA control; lane 3, digested nucleosomes. The trace 10 bp repeating pattern observed in lane 2 is due to contamination from lane 1. (B) Schematic representation of the nucleosomes reconstituted with the Il1β proximal promoter indicating the NF-κB binding site and the Sty I restriction site. (C) The Il1β nucleosomes were incubated with PARP1 and the indicated NAD+ concentrations for 20 min at room temperature. Subsequently, 20 units of StyI were added and reactions were incubated at 37°C for 1 h. Reactions were stopped by addition of EDTA and heat inactivation at 65°C for 10 min. Reactions were analyzed by native agarose gel electrophoresis and DNA visualized by SYBR Green staining (B and C). (D) The Il1β nucleosomes were remodeled with the indicated NAD+ concentrations and subsequently incubated with or without p65 for 20 min at room temperature. The blue asterisk indicates the PARP1/nucleosome complex. The yellow star indicates a putative p65/nucleosome complex.

Mentions: To further understand the role of PARP1 enzymatic in facilitating cytokine expression via the regulation of nucleosome structure, we performed DNAse I hypersensitivity assays with nucleosomes reconstituted with the 601 positioning sequence. The 601 nucleosomes were end-labeled on the antisense strand with 32P and subsequently digested with DNAse I. Under these conditions, the expected 10-11 bp DNA helical digestion pattern (Vitolo et al. 2001) was observed (Fig. 3A, lane 3). Addition to the nucleosomes of either PARP1 (Fig. 3A, lane 4) or NAD+ alone (Fig. S1) did not alter the digestion pattern. However, upon co-addition of NAD+, the digestion pattern significantly changed, resulting in increased digestion particularly in the core particle region (Fig. 3A, lanes 5–8). Of note, the increase in DNAse I sensitivity correlated with NAD+ concentration, and therefore with the extent of histone ADP-ribosylation. These studies suggest that PARP1-catalyzed histone ADP-ribosylation likely alters nucleosome structure by weakening the interaction between histones and DNA, leading to increased accessibility of nucleosomal DNA.


PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia.

Martínez-Zamudio RI, Ha HC - Brain Behav (2014)

Histone ADP-ribosylation by PARP1 increases accessibility of nucleosomal DNA. (A) 601 nucleosomes labeled with 32P-NAD+ at the 5′ end of the antisense strand were incubated with PARP1 and the indicated NAD+ concentrations. Reactions were subsequently incubated with DNAse I and analyzed in a denaturing 8% polyacrylamide gel followed by autoradiography. +, DNase I-hypersensitive sites. Lane 1, DNA marker; lane 2, digested DNA control; lane 3, digested nucleosomes. The trace 10 bp repeating pattern observed in lane 2 is due to contamination from lane 1. (B) Schematic representation of the nucleosomes reconstituted with the Il1β proximal promoter indicating the NF-κB binding site and the Sty I restriction site. (C) The Il1β nucleosomes were incubated with PARP1 and the indicated NAD+ concentrations for 20 min at room temperature. Subsequently, 20 units of StyI were added and reactions were incubated at 37°C for 1 h. Reactions were stopped by addition of EDTA and heat inactivation at 65°C for 10 min. Reactions were analyzed by native agarose gel electrophoresis and DNA visualized by SYBR Green staining (B and C). (D) The Il1β nucleosomes were remodeled with the indicated NAD+ concentrations and subsequently incubated with or without p65 for 20 min at room temperature. The blue asterisk indicates the PARP1/nucleosome complex. The yellow star indicates a putative p65/nucleosome complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Histone ADP-ribosylation by PARP1 increases accessibility of nucleosomal DNA. (A) 601 nucleosomes labeled with 32P-NAD+ at the 5′ end of the antisense strand were incubated with PARP1 and the indicated NAD+ concentrations. Reactions were subsequently incubated with DNAse I and analyzed in a denaturing 8% polyacrylamide gel followed by autoradiography. +, DNase I-hypersensitive sites. Lane 1, DNA marker; lane 2, digested DNA control; lane 3, digested nucleosomes. The trace 10 bp repeating pattern observed in lane 2 is due to contamination from lane 1. (B) Schematic representation of the nucleosomes reconstituted with the Il1β proximal promoter indicating the NF-κB binding site and the Sty I restriction site. (C) The Il1β nucleosomes were incubated with PARP1 and the indicated NAD+ concentrations for 20 min at room temperature. Subsequently, 20 units of StyI were added and reactions were incubated at 37°C for 1 h. Reactions were stopped by addition of EDTA and heat inactivation at 65°C for 10 min. Reactions were analyzed by native agarose gel electrophoresis and DNA visualized by SYBR Green staining (B and C). (D) The Il1β nucleosomes were remodeled with the indicated NAD+ concentrations and subsequently incubated with or without p65 for 20 min at room temperature. The blue asterisk indicates the PARP1/nucleosome complex. The yellow star indicates a putative p65/nucleosome complex.
Mentions: To further understand the role of PARP1 enzymatic in facilitating cytokine expression via the regulation of nucleosome structure, we performed DNAse I hypersensitivity assays with nucleosomes reconstituted with the 601 positioning sequence. The 601 nucleosomes were end-labeled on the antisense strand with 32P and subsequently digested with DNAse I. Under these conditions, the expected 10-11 bp DNA helical digestion pattern (Vitolo et al. 2001) was observed (Fig. 3A, lane 3). Addition to the nucleosomes of either PARP1 (Fig. 3A, lane 4) or NAD+ alone (Fig. S1) did not alter the digestion pattern. However, upon co-addition of NAD+, the digestion pattern significantly changed, resulting in increased digestion particularly in the core particle region (Fig. 3A, lanes 5–8). Of note, the increase in DNAse I sensitivity correlated with NAD+ concentration, and therefore with the extent of histone ADP-ribosylation. These studies suggest that PARP1-catalyzed histone ADP-ribosylation likely alters nucleosome structure by weakening the interaction between histones and DNA, leading to increased accessibility of nucleosomal DNA.

Bottom Line: Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells.Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines.Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular & Cellular Biology 337 Basic Science Building, 3900 Reservoir Road, Washington, District of Columbia, 20057.

ABSTRACT

Background: Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells.

Methods: PARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with (32)P-NAD(+). To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA.

Results: Our results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.

Conclusions: Collectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation.

Show MeSH
Related in: MedlinePlus