In vivo monitoring of a cAMP-stimulated DNA-binding activity

Abstract

The transcriptional activity of the tyrosine aminotransferase (TAT) gene is influenced by two major signal transduction pathways, by glucocorticoids and by glucagon acting via cAMP. We analyzed the effect of cAMP on protein-DNA interactions in vivo and on the transcription rate of the TAT gene. We demonstrate that a cAMP-responsive element (CRE) is located in a tissue-specific DNase I-hypersensitive region, 3.6 kb upstream of the start site of transcription. By using the genomic footprinting technique, we show that this sequence is occupied by protein in uninduced cells and that the in vivo footprint is transiently increased upon cAMP induction. Protein binding at the TAT-CRE correlates with the rate of transcription of the TAT gene. Cycloheximide treatment reveals that the genomic footprint is subject to rapid turnover; however, subsequent cAMP induction in the continued presence of cycloheximide restores the footprint partially. We conclude that as a part of the signal transduction pathway, a cAMP-dependent, post-translational modification increases the DNA-binding activity of a protein to the TAT-CRE and thereby stimulates the transcription rate of the TAT gene.