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Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark.

Lodhi N, Kossenkov AV, Tulin AV - Nucleic Acids Res. (2014)

Bottom Line: Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence.After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent.Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, PA, 19111 USA.

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PARP-1 is associated with chromatin during mitosis. (A) Equal amounts of total chromatin proteins extracted from asynchronous cells and cells arrested in mitosis were analyzed after PAGE on western blot using anti-PARP-1, anti-RNAP (RNA polymerase II), anti-H3 and anti-phospho-Ser10-H3 (marker of mitotic chromatin) antibodies (B–D). Panel (B) shows typical single mitotic chromosome used for Immuno-EM analysis. Immuno-EM analysis of PARP-1 (C) and H1 histone (D) localization in mitotic chromatin. Scale bars correspond to 1 mm (B) and 100 nm (C and D). (E) Confocal microscopy analysis of PARP-1 (red) and H1 histone (green) localization in mitotic chromatin. Lack of overlap between red and green areas indicates the extent to which PARP-1 and H1 occupy different genomic domains. Scale bars correspond to 5 nm. (F–H) The identification of PARP-1 protein binding in mitotic (M) and interphase (asynchronous) (A) chromatin using ChIP-Seq assays. UCSC genome browser view is presented based on ChIP-Seq data. Red box represents the area of promoter sequences. Significant peaks are shown as green. (F) PARP-1 binding in the previously reported PARP-1-dependent gene PVALB. (G) Absence of PARP-1 binding along PARP-1-independent GAPDH locus. (H) PARP-1 binds along KDM2A gene sequences.
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Figure 1: PARP-1 is associated with chromatin during mitosis. (A) Equal amounts of total chromatin proteins extracted from asynchronous cells and cells arrested in mitosis were analyzed after PAGE on western blot using anti-PARP-1, anti-RNAP (RNA polymerase II), anti-H3 and anti-phospho-Ser10-H3 (marker of mitotic chromatin) antibodies (B–D). Panel (B) shows typical single mitotic chromosome used for Immuno-EM analysis. Immuno-EM analysis of PARP-1 (C) and H1 histone (D) localization in mitotic chromatin. Scale bars correspond to 1 mm (B) and 100 nm (C and D). (E) Confocal microscopy analysis of PARP-1 (red) and H1 histone (green) localization in mitotic chromatin. Lack of overlap between red and green areas indicates the extent to which PARP-1 and H1 occupy different genomic domains. Scale bars correspond to 5 nm. (F–H) The identification of PARP-1 protein binding in mitotic (M) and interphase (asynchronous) (A) chromatin using ChIP-Seq assays. UCSC genome browser view is presented based on ChIP-Seq data. Red box represents the area of promoter sequences. Significant peaks are shown as green. (F) PARP-1 binding in the previously reported PARP-1-dependent gene PVALB. (G) Absence of PARP-1 binding along PARP-1-independent GAPDH locus. (H) PARP-1 binds along KDM2A gene sequences.

Mentions: We first compared the PARP-1 protein distribution in interphase chromatin and metaphase-arrested chromosomes (Figure 1A), confirming the metaphase status of DNA by co-staining with phosphoH3/Ser10, a well-known marker of mitotic chromatin (43) (Supplementary Figure S1). PARP-1 remained bound to condensed chromatin during mitosis (Figure 1A). To assess the distribution PARP-1 in metaphase chromatin, we performed ultrastructural analysis of metaphase chromosomes using immuno-electron microscopy (EM) (37) and immunofluorescence. EM immunocytochemistry deploying anti-PARP-1 and the antibody to linker histone H1 (Figures 1B–D) revealed a number of well-defined domains in the condensed chromatin occupied by PARP-1. Confocal microscopy (Figure 1E) confirmed that PARP-1 and H1 localized to distinct non-overlapping blocks in mitotic chromatin (Figure 1E). Similar anti-correlation in PARP-1 and H1 distributions has been previously observed in interphase chromatin (28,35).


Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark.

Lodhi N, Kossenkov AV, Tulin AV - Nucleic Acids Res. (2014)

PARP-1 is associated with chromatin during mitosis. (A) Equal amounts of total chromatin proteins extracted from asynchronous cells and cells arrested in mitosis were analyzed after PAGE on western blot using anti-PARP-1, anti-RNAP (RNA polymerase II), anti-H3 and anti-phospho-Ser10-H3 (marker of mitotic chromatin) antibodies (B–D). Panel (B) shows typical single mitotic chromosome used for Immuno-EM analysis. Immuno-EM analysis of PARP-1 (C) and H1 histone (D) localization in mitotic chromatin. Scale bars correspond to 1 mm (B) and 100 nm (C and D). (E) Confocal microscopy analysis of PARP-1 (red) and H1 histone (green) localization in mitotic chromatin. Lack of overlap between red and green areas indicates the extent to which PARP-1 and H1 occupy different genomic domains. Scale bars correspond to 5 nm. (F–H) The identification of PARP-1 protein binding in mitotic (M) and interphase (asynchronous) (A) chromatin using ChIP-Seq assays. UCSC genome browser view is presented based on ChIP-Seq data. Red box represents the area of promoter sequences. Significant peaks are shown as green. (F) PARP-1 binding in the previously reported PARP-1-dependent gene PVALB. (G) Absence of PARP-1 binding along PARP-1-independent GAPDH locus. (H) PARP-1 binds along KDM2A gene sequences.
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Figure 1: PARP-1 is associated with chromatin during mitosis. (A) Equal amounts of total chromatin proteins extracted from asynchronous cells and cells arrested in mitosis were analyzed after PAGE on western blot using anti-PARP-1, anti-RNAP (RNA polymerase II), anti-H3 and anti-phospho-Ser10-H3 (marker of mitotic chromatin) antibodies (B–D). Panel (B) shows typical single mitotic chromosome used for Immuno-EM analysis. Immuno-EM analysis of PARP-1 (C) and H1 histone (D) localization in mitotic chromatin. Scale bars correspond to 1 mm (B) and 100 nm (C and D). (E) Confocal microscopy analysis of PARP-1 (red) and H1 histone (green) localization in mitotic chromatin. Lack of overlap between red and green areas indicates the extent to which PARP-1 and H1 occupy different genomic domains. Scale bars correspond to 5 nm. (F–H) The identification of PARP-1 protein binding in mitotic (M) and interphase (asynchronous) (A) chromatin using ChIP-Seq assays. UCSC genome browser view is presented based on ChIP-Seq data. Red box represents the area of promoter sequences. Significant peaks are shown as green. (F) PARP-1 binding in the previously reported PARP-1-dependent gene PVALB. (G) Absence of PARP-1 binding along PARP-1-independent GAPDH locus. (H) PARP-1 binds along KDM2A gene sequences.
Mentions: We first compared the PARP-1 protein distribution in interphase chromatin and metaphase-arrested chromosomes (Figure 1A), confirming the metaphase status of DNA by co-staining with phosphoH3/Ser10, a well-known marker of mitotic chromatin (43) (Supplementary Figure S1). PARP-1 remained bound to condensed chromatin during mitosis (Figure 1A). To assess the distribution PARP-1 in metaphase chromatin, we performed ultrastructural analysis of metaphase chromosomes using immuno-electron microscopy (EM) (37) and immunofluorescence. EM immunocytochemistry deploying anti-PARP-1 and the antibody to linker histone H1 (Figures 1B–D) revealed a number of well-defined domains in the condensed chromatin occupied by PARP-1. Confocal microscopy (Figure 1E) confirmed that PARP-1 and H1 localized to distinct non-overlapping blocks in mitotic chromatin (Figure 1E). Similar anti-correlation in PARP-1 and H1 distributions has been previously observed in interphase chromatin (28,35).

Bottom Line: Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence.After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent.Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, PA, 19111 USA.

Show MeSH
Related in: MedlinePlus