ERK modulates DNA bending and enhancesome structure by phosphorylating HMG1-boxes 1 and 2 of the RNA polymerase I transcription factor UBF.
Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding of the enhancesome.
Cancer Research Centre and Department of Medical Biology of Laval University, Centre de Recherche de l'Hôtel-Dieu de Québec, 9 rue McMahon G1R 2J6 Québec, QC, Canada.
Victor Y
VY
Stefanovsky
Frédèric
F
Langlois
David
D
Bazett-Jones
Guillaume
G
Pelletier
Tom
T
Moss
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D024243:HMGB1 Protein / Q000378:metabolism*; D024261:HMGB2 Protein / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000378:metabolism
D045566:DNA, Cruciform; D024243:HMGB1 Protein; D024261:HMGB2 Protein; D035463:Pol1 Transcription Initiation Complex Proteins; D011994:Recombinant Proteins; C063844:transcription factor UBF; D004247:DNA; D020929:Mitogen-Activated Protein Kinase Kinases; D012318:RNA Polymerase I