Protein kinase A-dependent recruitment of RNA polymerase II, C/EBP beta and NF-Y to the rat GTP cyclohydrolase I proximal promoter occurs without alterations in histone acetylation.
Cyclic-AMP stimulation of GTP cyclohydrolase I (GCH1) gene transcription was investigated in PC12 cells, the protein kinase A-deficient PC12 cell line 126-1B2 and C6 cells using transient transfection assays of proximal promoter reporter constructs and wild type or dominant negative proteins, chromatin immunoprecipitation and real-time quantitative PCR. These studies show that protein kinase A is necessary and sufficient for cAMP-dependent transcription conferred by both the cAMP regulatory element and the adjacent CCAAT-box. In intact cells these cis-elements were shown to bind cAMP response element binding protein, CCAAT-enhancer binding protein beta and nuclear factor-Y, with each protein controlling a different aspect of the cAMP response. Cyclic-AMP acting through protein kinase A stimulated promoter recruitment of CCAAT-enhancer binding protein beta, nuclear factor-Y and RNA polymerase II while depleting the promoter of cyclic-AMP response element binding protein. Stimulation of transcription by cAMP was not associated with increased acetylation of histones H3 and H4 at proximal promoter nucleosomes, indicating that histone acetyltransferases are not involved in this response. Nonetheless, pharmacological inhibition of histone deacetylase activity did increase histone H4 acetylation and the recruitment of RNA polymerase II, indicating that histone acetyltransferases are normally associated with the proximal promoter. Only in C6 cells, however, did inhibition of histone deacetylases stimulate transcription and synergize with cAMP. These experiments provide the first glimpse of the GCH1 gene promoter functioning within intact cells and supply evidence for the involvement of histone acetyltransferase-containing complexes in GCH1 gene transcription.
Kapatos
Gregory
G
Department of Pharmacology and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA. gkapato@med.wayne.edu
Vunnava
Prashanthi
P
Wu
Yanning
Y
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods; D000242:Cyclic AMP / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000172:deficiency / Q000502:physiology*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006136:GTP Cyclohydrolase / Q000235:genetics*; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D023081:CCAAT-Binding Factor; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D006657:Histones; D012333:RNA, Messenger; D014157:Transcription Factors; C402244:nuclear factor Y; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D012319:RNA Polymerase II; D006655:Histone Deacetylases; D006136:GTP Cyclohydrolase