Expression and Characterization of a Fusion Protein Between the Catalytic Domain of Poly(ADP-Ribose) polymerase and the DNA Binding Domain of the Glucocorticoid Receptor

Rosenthal, Dean S.; Hong, Tao; Cherney, Barry; Zhang, Shimin; Shima, Thomas B.; Danielsen, Mark; Smulson, Mark E.

doi:10.1006/bbrc.1994.2012

Cited by 6 publications

(6 citation statements)

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“…Studies with a chimeric protein of PARP fused to the glucocorticoid receptor revealed that PARP activity can be targeted to specific DNA sequences and repress gene expression (24) ation might prevent polymerase II-dependent transcription (21). Here, we also demonstrate that a chimeric protein of PARP fused to the Gal4 DNA binding domain represses gene expression of a promoter harboring the Gal4 binding site.…”

Section: Discussionmentioning

confidence: 61%

“…However, these direct interaction models do not explain transcriptional squelching between receptors or the roles of receptor-associated cofactors. Negative transcriptional regulation by TR and RAR is mediated, in part, by their association with a class of silencing mediators termed 21,24,25,27). In addition, at least three distinct classes of receptor cofactors have been identified, including SRC-1, TRIP1, RIP140/160, and TIF1.…”

Section: Mammalian Poly(adp-ribose) Polymerase (Parp) Is a Nuclear Chmentioning

confidence: 99%

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Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

Miyamoto

Kakizawa

Hashizume

1999

Molecular and Cellular Biology

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Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD؉ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed liganddependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXR␣. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADPribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.Nuclear hormone receptors for steroids, retinoids, thyroid hormone, vitamin D 3 , and prostanoids comprise a large family of sequence-specific transcription factors. They play diverse roles in development, differentiation, and homeostasis (18) by modulating gene transcription. Retinoid X receptors (RXR) are members of a superfamily of nuclear hormone receptors and heterodimerize with a variety of other family members, including all-trans-retinoic acid receptor (RAR), thyroid hormone receptor (TR), and vitamin D receptor (VDR), indicating that RXR play a central role in ligand-dependent transcriptional regulation by nuclear receptors (15,35,37). These heterodimers bind to specific DNA sequences and directly regulate transcription of target genes in response to specific ligands. Nuclear receptors are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription machinery (3, 5, 6, 9, 11-14, 16, 30-32).The mechanism of transcriptional regulation by nuclear receptors has been a focus of intense study. The demonstration of direct interactions of receptors with basal transcription factors, such as TFIIB and TBP (19,20,22,23,34), suggests that liganded receptors may directly influence the function of the basal transcription machinery. However, these direct interaction models do not explain transcriptional squelching between receptors or the roles of receptor-associated cofactors. Negative transcriptional regulation by TR and RAR is mediated, in part, by their association with a class of silencing mediators termed 21,24,25,27). In addition, at least three distinct classes of re...

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Section: Discussionmentioning

confidence: 61%

Section: Mammalian Poly(adp-ribose) Polymerase (Parp) Is a Nuclear Chmentioning

confidence: 99%

Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

Miyamoto

Kakizawa

Hashizume

1999

Molecular and Cellular Biology

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“…PARP1 deletion mutants and fusion proteins had been successfully employed before to study different aspects of poly(ADP-ribosyl)ation (31,32). Here, we have generated PARP family chimera to analyze the molecular mechanism of PAR formation.…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Altmeyer

Messner

Hassa

et al. 2009

Nucleic Acids Research

297

290

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Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.

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“…More specifically, a PARP-GR fusion repressed GR-mediated transcription (Rosenthal et al 1994). There have also been two articles implicating a role for PARP during ovulation and oocyte maturation (Aoufouchi & Shall 1997, Murdoch 1998.…”

Section: Parp -Relationship To Transcription and Progesteronementioning

confidence: 99%

Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors

Sartorius¹,

Takimoto²,

Richer³

et al. 2000

Journal of Molecular Endocrinology

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Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD PR ) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD PR fusion protein.Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADPribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK CS . DNA-PK that was 'pulleddown' by DBD PR on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK CS , (2) transphosphorylate DBD PR , and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD CYS ) nor the core DBD of the estrogen receptor (DBD ER ) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/ DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD PR interact, that DBD PR is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.

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Expression and Characterization of a Fusion Protein Between the Catalytic Domain of Poly(ADP-Ribose) polymerase and the DNA Binding Domain of the Glucocorticoid Receptor

Cited by 6 publications

References 0 publications

Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors

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