ABSTRACT
Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation.
Subject(s)
DNA-Binding Proteins/metabolism , Epidermal Growth Factor/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Pol1 Transcription Initiation Complex Proteins , Ribosomes/genetics , Transcription Factors/metabolism , Transcription, Genetic/physiology , Animals , DNA/metabolism , DNA-Binding Proteins/genetics , Enzyme Activation , Genes, Reporter , Humans , Mice , Mitogen-Activated Protein Kinase 3 , Mutation , Phosphorylation , Protein Structure, Secondary , RNA Polymerase I/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effectsABSTRACT
The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.
Subject(s)
DNA, Ribosomal/chemistry , DNA, Ribosomal/metabolism , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Nucleic Acid Conformation , Pol1 Transcription Initiation Complex Proteins , RNA Polymerase I/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , DNA, Ribosomal/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dimerization , High Mobility Group Proteins/metabolism , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Quaternary , Ribosomes/metabolism , TATA-Box Binding Protein , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, Genetic/genetics , Xenopus laevis/geneticsABSTRACT
RNA polymerase I (PolI) transcription is activated by the HMG box architectural factor UBF, which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. Here we show that the acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation "flip-flop." Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb.
Subject(s)
DNA-Binding Proteins/metabolism , Histone Deacetylases/metabolism , Nuclear Proteins/metabolism , Pol1 Transcription Initiation Complex Proteins , Retinoblastoma Protein/metabolism , Ribosomes/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, Genetic , 3T3 Cells , Acetylation , Animals , Binding, Competitive , CREB-Binding Protein , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , DNA Footprinting , DNA-Binding Proteins/chemistry , Enzyme Activation , Histone Deacetylases/chemistry , Mice , Models, Genetic , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary , RNA Polymerase I/metabolism , Rats , Retinoblastoma Protein/antagonists & inhibitors , Substrate Specificity , Trans-Activators/antagonists & inhibitors , Trans-Activators/chemistry , Transcription Factors/chemistry , Xenopus laevis/geneticsABSTRACT
The formation of a near complete loop of DNA is a striking property of the architectural HMG-box factor xUBF. Here we show that DNA looping only requires a dimer of Nbox13, a C-terminal truncation mutant of xUBF containing just HMG-boxes 1-3. This segment of xUBF corresponds to that minimally required for activation of polymerase I transcription and is sufficient to generate the major characteristics of the footprint given by intact xUBF. Stepwise reduction in the number of HMG-boxes to less than three significantly diminishes DNA bending and provides an estimate of bend angle for each HMG-box. Together the data indicate that a 350 +/- 16 degree loop in 142 +/- 30 bp of DNA can be induced by binding of the six HMG-boxes in an Nbox13 dimer and that DNA looping is probably achieved by six in-phase bends. The positioning of each HMG-box on the DNA does not predominantly involve DNA sequence recognition and is thus an intrinsic property of xUBF.
