Limits...
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: 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.Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed.

View Article: PubMed Central - PubMed

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

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066802&req=5

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066802&req=5

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: 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.Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus