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Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

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PARP protein is localized to Cajal body (CB) and histone locus body (HLB) and interacts with HLB/CB components.N indicates nucleolus. A. The dissected salivary glands expressing PARPe-EGFP (green) transgenic construct were sectioned to ultra thin sections and stained with rabbit anti-Coilin antibody (red). Position of HLB/CB is indicated with arrow. Inset. Magnification of the chromatin block containing CB is shown. B. The dissected salivary glands co-expressing PARP1-DsRed (red) and EYFP-LSM11 (green) transgenic constructs were stained with the DNA binding dye Draq5 (blue). Position of HLB/CB is indicated with arrow. C. The dissected salivary glands from wild-type larvae were fixed and sectioned, followed by immunostaining with rabbit anti-pADPr (Red); Guinea Pig anti-Coilin antibody (green) and mouse anti-histone H1 (blue). Position of CB/HLB is indicated with arrow. Accumulation of pADPr in CB is clearly shown. Inset. Magnification of the chromatin block containing CB is shown. D. Immunoprecipitation assays using Guinea Pig anti-Coilin antibody. Drosophila stocks expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: rabbit anti-Coilin; rabbit anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); rabbit anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin. E. Immunoprecipitation assays using rabbit anti-GFP antibody. Drosophila stock expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin; mouse anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); mouse anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin.
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pgen-1000387-g001: PARP protein is localized to Cajal body (CB) and histone locus body (HLB) and interacts with HLB/CB components.N indicates nucleolus. A. The dissected salivary glands expressing PARPe-EGFP (green) transgenic construct were sectioned to ultra thin sections and stained with rabbit anti-Coilin antibody (red). Position of HLB/CB is indicated with arrow. Inset. Magnification of the chromatin block containing CB is shown. B. The dissected salivary glands co-expressing PARP1-DsRed (red) and EYFP-LSM11 (green) transgenic constructs were stained with the DNA binding dye Draq5 (blue). Position of HLB/CB is indicated with arrow. C. The dissected salivary glands from wild-type larvae were fixed and sectioned, followed by immunostaining with rabbit anti-pADPr (Red); Guinea Pig anti-Coilin antibody (green) and mouse anti-histone H1 (blue). Position of CB/HLB is indicated with arrow. Accumulation of pADPr in CB is clearly shown. Inset. Magnification of the chromatin block containing CB is shown. D. Immunoprecipitation assays using Guinea Pig anti-Coilin antibody. Drosophila stocks expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: rabbit anti-Coilin; rabbit anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); rabbit anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin. E. Immunoprecipitation assays using rabbit anti-GFP antibody. Drosophila stock expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin; mouse anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); mouse anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin.

Mentions: To study PARP1 protein nuclear localization during Drosophila development, we used Drosophila stock ubiquitously expressing PARP1-DsRed recombinant protein. Previously, we biologically validated this construct by testing its ability to rescue a ParpCH1 mutation phenotype and by using immunofluorescence to assess recombinant protein localization to chromatin [6],[8]. We also demonstrated that the expression level of PARP1-DsRed transgene does not exceed the level of endogenous PARP1 expression [5]. In wild-type nuclei, the automodified PARP1 protein form is represented by a very minor and transient fraction of the total PARP1 protein. Therefore, at all stages of development and in all tissues, most of the PARP1 protein pool is shown to be associated with chromatin. However, using high resolution laser confocal microscopy, we often observed small PARP1-positive (Figure S1A) and pADPr-positive extrachromosomal particles (Figure S1B), the structural properties, size and localization of which all suggested certain similarities to Cajal bodies (CBs) [20] and the related organelle, histone locus body (HLB) [21],[22]. In order to test this hypothesis, we compared localization of the Coilin protein, which is a classical marker of CBs and HLBs [19], and snRNP-specific protein LSM11, which has been reported as a histone locus body (HLB) marker in Drosophila [23], with localization of recombinant PARP protein. Wild-type nuclei typically showed single CB and single HLB [22], while PARP protein demonstrated colocalization with LSM11- and Coilin-positive particles in nuclei of et least 20 salivary glands taken for analysis (Figure 1A, B). Moreover, co-immunostaining of wild-type Drosophila tissues demonstrated strong colocalization of pADPr and Coilin protein (Figure 1C) Taken together, these observations suggest that PARP1 protein, as well as poly(ADP-ribosyl)ated proteins, are residents of CB and HLB in Drosophila.


Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

PARP protein is localized to Cajal body (CB) and histone locus body (HLB) and interacts with HLB/CB components.N indicates nucleolus. A. The dissected salivary glands expressing PARPe-EGFP (green) transgenic construct were sectioned to ultra thin sections and stained with rabbit anti-Coilin antibody (red). Position of HLB/CB is indicated with arrow. Inset. Magnification of the chromatin block containing CB is shown. B. The dissected salivary glands co-expressing PARP1-DsRed (red) and EYFP-LSM11 (green) transgenic constructs were stained with the DNA binding dye Draq5 (blue). Position of HLB/CB is indicated with arrow. C. The dissected salivary glands from wild-type larvae were fixed and sectioned, followed by immunostaining with rabbit anti-pADPr (Red); Guinea Pig anti-Coilin antibody (green) and mouse anti-histone H1 (blue). Position of CB/HLB is indicated with arrow. Accumulation of pADPr in CB is clearly shown. Inset. Magnification of the chromatin block containing CB is shown. D. Immunoprecipitation assays using Guinea Pig anti-Coilin antibody. Drosophila stocks expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: rabbit anti-Coilin; rabbit anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); rabbit anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin. E. Immunoprecipitation assays using rabbit anti-GFP antibody. Drosophila stock expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin; mouse anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); mouse anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin.
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pgen-1000387-g001: PARP protein is localized to Cajal body (CB) and histone locus body (HLB) and interacts with HLB/CB components.N indicates nucleolus. A. The dissected salivary glands expressing PARPe-EGFP (green) transgenic construct were sectioned to ultra thin sections and stained with rabbit anti-Coilin antibody (red). Position of HLB/CB is indicated with arrow. Inset. Magnification of the chromatin block containing CB is shown. B. The dissected salivary glands co-expressing PARP1-DsRed (red) and EYFP-LSM11 (green) transgenic constructs were stained with the DNA binding dye Draq5 (blue). Position of HLB/CB is indicated with arrow. C. The dissected salivary glands from wild-type larvae were fixed and sectioned, followed by immunostaining with rabbit anti-pADPr (Red); Guinea Pig anti-Coilin antibody (green) and mouse anti-histone H1 (blue). Position of CB/HLB is indicated with arrow. Accumulation of pADPr in CB is clearly shown. Inset. Magnification of the chromatin block containing CB is shown. D. Immunoprecipitation assays using Guinea Pig anti-Coilin antibody. Drosophila stocks expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: rabbit anti-Coilin; rabbit anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); rabbit anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin. E. Immunoprecipitation assays using rabbit anti-GFP antibody. Drosophila stock expressing PARP1-ECFP and PARPe-EGFP were used. Wild type Drosophila stock was used as a control. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin; mouse anti-GFP (to detect PARP1-ECFP and PARPe-EGFP); mouse anti-Fibrillarin; mouse anti-Dlg; and mouse anti-Actin.
Mentions: To study PARP1 protein nuclear localization during Drosophila development, we used Drosophila stock ubiquitously expressing PARP1-DsRed recombinant protein. Previously, we biologically validated this construct by testing its ability to rescue a ParpCH1 mutation phenotype and by using immunofluorescence to assess recombinant protein localization to chromatin [6],[8]. We also demonstrated that the expression level of PARP1-DsRed transgene does not exceed the level of endogenous PARP1 expression [5]. In wild-type nuclei, the automodified PARP1 protein form is represented by a very minor and transient fraction of the total PARP1 protein. Therefore, at all stages of development and in all tissues, most of the PARP1 protein pool is shown to be associated with chromatin. However, using high resolution laser confocal microscopy, we often observed small PARP1-positive (Figure S1A) and pADPr-positive extrachromosomal particles (Figure S1B), the structural properties, size and localization of which all suggested certain similarities to Cajal bodies (CBs) [20] and the related organelle, histone locus body (HLB) [21],[22]. In order to test this hypothesis, we compared localization of the Coilin protein, which is a classical marker of CBs and HLBs [19], and snRNP-specific protein LSM11, which has been reported as a histone locus body (HLB) marker in Drosophila [23], with localization of recombinant PARP protein. Wild-type nuclei typically showed single CB and single HLB [22], while PARP protein demonstrated colocalization with LSM11- and Coilin-positive particles in nuclei of et least 20 salivary glands taken for analysis (Figure 1A, B). Moreover, co-immunostaining of wild-type Drosophila tissues demonstrated strong colocalization of pADPr and Coilin protein (Figure 1C) Taken together, these observations suggest that PARP1 protein, as well as poly(ADP-ribosyl)ated proteins, are residents of CB and HLB in Drosophila.

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

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Related in: MedlinePlus