The orphan nuclear receptor TR2 interacts directly with both class I and class II histone deacetylases.
A combination of in vivo and in vitro assays was employed to describe the ligand-independent interaction of the orphan nuclear receptor TR2 and histone deacetylase proteins. The repressive effect of TR2 on transcription of a luciferase reporter driven by a promoter containing a direct repeat-5 (DR5) derived from the human RARbeta gene was suppressed by the addition of the histone deacetylase inhibitor trichostatin A. Immunoprecipitation with FLAG-epitope (MDYKDDDDK)-tagged histone deacetylase proteins was used to demonstrate that TR2 and histone deacetylases 3 or 4 are present in the same immunoprecipitated complex. Deacetylase activity was demonstrated for these coimmunoprecipitates, further confirming the in vivo interaction of TR2 and histone deacetylases. Immunoprecipitation with anti-TR2 antibody was used to demonstrate interaction of TR2 with endogenously expressed histone deacetylases 3 and 4 in COS-1 cells. Dissection of TR2 domains showed that the DNA binding domain of the receptor was responsible for interaction with both histone deacetylases 3 and 4 in glutathione-S-transferase pull-down assays, while the ligand binding domain did not interact. The pull-down data were confirmed with far Western blots that also showed a direct interaction between labeled histone deacetylase proteins and TR2. It is suggested that repression mediated by unliganded TR2 is mediated, in part, by a direct interaction of this receptor with histone deacetylase proteins.
Franco
P J
PJ
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Farooqui
M
M
Seto
E
E
Wei
L N
LN
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007163:Immunosorbent Techniques; D008156:Luciferases / Q000235:genetics; D051379:Mice; D057107:Nuclear Receptor Subfamily 2, Group C, Member 1; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C539822:NR2C1 protein, human; C539823:Nr2c1 protein, mouse; D057107:Nuclear Receptor Subfamily 2, Group C, Member 1; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C012589:trichostatin A; D004247:DNA; D004492:Edetic Acid; D008156:Luciferases; D005982:Glutathione Transferase; D006655:Histone Deacetylases; D015032:Zinc