Recruitment of the NCoA/SRC-1/p160 Family of Transcriptional Coactivators by the Aryl Hydrocarbon Receptor/Aryl Hydrocarbon Receptor Nuclear Translocator Complex

ABSTRACT The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.

[1]  C. Elferink,et al.  Maximal aryl hydrocarbon receptor activity depends on an interaction with the retinoblastoma protein. , 2001, Molecular pharmacology.

[2]  G. Perdew,et al.  Differential Recruitment of Coactivator RIP140 byAh and Estrogen Receptors , 1999, The Journal of Biological Chemistry.

[3]  C. Glass,et al.  Transcriptional Activation by NF-κB Requires Multiple Coactivators , 1999, Molecular and Cellular Biology.

[4]  S. Safe,et al.  Interactions of nuclear receptor coactivator/corepressor proteins with the aryl hydrocarbon receptor complex. , 1999, Archives of biochemistry and biophysics.

[5]  M. Garabedian,et al.  GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors , 1997, Molecular and cellular biology.

[6]  Christopher K. Glass,et al.  The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function , 1997, Nature.

[7]  S. Weitzman,et al.  p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter* , 1999, The Journal of Biological Chemistry.

[8]  J. Pelletier,et al.  The murine Sim-2 gene product inhibits transcription by active repression and functional interference , 1997, Molecular and cellular biology.

[9]  G. Semenza,et al.  Purification and Characterization of Hypoxia-inducible Factor 1 (*) , 1995, The Journal of Biological Chemistry.

[10]  Alan P. Wolffe,et al.  Targeting Chromatin Disruption: Transcription Regulators that Acetylate Histones , 1996, Cell.

[11]  D. Livingston,et al.  Binding and modulation of p53 by p300/CBP coactivators , 1997, Nature.

[12]  Thorsten Heinzel,et al.  A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors , 1996, Cell.

[13]  J. Hogenesch,et al.  The PAS superfamily: sensors of environmental and developmental signals. , 2000, Annual review of pharmacology and toxicology.

[14]  F. Sauvé,et al.  CIA, a Novel Estrogen Receptor Coactivator with a Bifunctional Nuclear Receptor Interacting Determinant , 2001, Molecular and Cellular Biology.

[15]  O. Hankinson,et al.  Two Murine Homologs of the Drosophila Single-minded Protein That Interact with the Mouse Aryl Hydrocarbon Receptor Nuclear Translocator Protein* , 1997, The Journal of Biological Chemistry.

[16]  A. Lamond,et al.  Structure and function in the nucleus. , 1998, Science.

[17]  O. Hankinson,et al.  Regulatory gene product of the Ah locus. Characterization of receptor mutants among mouse hepatoma clones. , 1982, The Journal of biological chemistry.

[18]  J. Huff,et al.  Carcinogenicity of TCDD: experimental, mechanistic, and epidemiologic evidence. , 1994, Annual review of pharmacology and toxicology.

[19]  L. Wartman,et al.  Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha. , 1998, Gene expression.

[20]  O. Gotoh,et al.  Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis , 1996, Molecular and cellular biology.

[21]  L. Szekely,et al.  The Promyelocytic Leukemia Gene Product (PML) Forms Stable Complexes with the Retinoblastoma Protein , 1998, Molecular and Cellular Biology.

[22]  A. Matera,et al.  Nuclear bodies: multifaceted subdomains of the interchromatin space. , 1999, Trends in cell biology.

[23]  R. Evans,et al.  Nuclear Receptor Coactivator ACTR Is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300 , 1997, Cell.

[24]  O. Hankinson,et al.  Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT) , 1994, Molecular and cellular biology.

[25]  H. Li,et al.  Transcriptional activation function of the mouse Ah receptor nuclear translocator. , 1994, The Journal of biological chemistry.

[26]  Y Fujii-Kuriyama,et al.  CBP/p300 functions as a possible transcriptional coactivator of Ah receptor nuclear translocator (Arnt). , 1997, Journal of biochemistry.

[27]  C. Allis,et al.  Steroid receptor coactivator-1 is a histone acetyltransferase , 1997, Nature.

[28]  C. Bradfield,et al.  Ligand-dependent Interaction of the Aryl Hydrocarbon Receptor with a Novel Immunophilin Homolog in Vivo* , 1997, The Journal of Biological Chemistry.

[29]  G. Perdew,et al.  Differential recruitment of coactivator RIP140 by Ah and estrogen receptors. Absence of a role for LXXLL motifs. , 1999, The Journal of biological chemistry.

[30]  C. Bradfield,et al.  Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[32]  Y Fujii-Kuriyama,et al.  A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[33]  B. O’Malley,et al.  Sequence and Characterization of a Coactivator for the Steroid Hormone Receptor Superfamily , 1995, Science.

[34]  R J Fletterick,et al.  Structure and specificity of nuclear receptor-coactivator interactions. , 1998, Genes & development.

[35]  L. Poellinger,et al.  Evidence That the Co-chaperone p23 Regulates Ligand Responsiveness of the Dioxin (Aryl Hydrocarbon) Receptor* , 1999, The Journal of Biological Chemistry.

[36]  P. Meltzer,et al.  AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. , 1997, Science.

[37]  M. Rivera,et al.  A Role for CREB Binding Protein and p300 Transcriptional Coactivators in Ets-1 Transactivation Functions , 1998, Molecular and Cellular Biology.

[38]  D. Livingston,et al.  Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. , 1996, Genes & development.

[39]  R. Evans,et al.  Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Hogenesch,et al.  Characterization of a Subset of the Basic-Helix-Loop-Helix-PAS Superfamily That Interacts with Components of the Dioxin Signaling Pathway* , 1997, The Journal of Biological Chemistry.

[41]  R. Pollenz,et al.  The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. , 1994, Molecular pharmacology.

[42]  R. Evans,et al.  Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[43]  P. Chambon,et al.  TIF2, a 160 kDa transcriptional mediator for the ligand‐dependent activation function AF‐2 of nuclear receptors. , 1996, The EMBO journal.

[44]  J. P. Whitlock,et al.  A Novel Cytoplasmic Protein That Interacts with the Ah Receptor, Contains Tetratricopeptide Repeat Motifs, and Augments the Transcriptional Response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin* , 1997, The Journal of Biological Chemistry.

[45]  C. Bradfield,et al.  Ah receptor signaling pathways. , 1996, Annual review of cell and developmental biology.

[46]  G. Perdew,et al.  Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex. , 1994, The Journal of biological chemistry.

[47]  S. Bhattacharya,et al.  An essential role for p300/CBP in the cellular response to hypoxia. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Masatoshi Hagiwara,et al.  Phosphorylated CREB binds specifically to the nuclear protein CBP , 1993, Nature.

[49]  O. Hankinson.  The aryl hydrocarbon receptor complex. , 1995, Annual review of pharmacology and toxicology.

[50]  G. Perdew,et al.  Nuclear receptor coactivator SRC-1 interacts with the Q-rich subdomain of the AhR and modulates its transactivation potential. , 1999, Gene expression.

[51]  F. Gonzalez,et al.  The aryl hydrocarbon receptor: studies using the AHR-null mice. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[52]  L. Poellinger,et al.  Redox-Regulated Recruitment of the Transcriptional Coactivators CREB-Binding Protein and SRC-1 to Hypoxia-Inducible Factor 1α , 2000, Molecular and Cellular Biology.

[53]  C. Glass,et al.  Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. , 1998, Genes & development.

[54]  O. Hankinson,et al.  Intracellular location of the Ah receptor , 1986, Journal of cellular physiology.