Effect of differential gene expression on the chromatin structure of the DHFR gene domain in vivo.
Photoactivated psoralen was used to probe region-specific chromatin structure in Chinese hamster ovary (CHO) cells. Specifically, the chromatin structure of six regions within the dihydrofolate reductase (DHFR) gene was probed with photoactivated psoralen in cells cultured in such ways as to differentially express the DHFR gene. Cells were irradiated with X-rays prior to the psoralen photocross-linking reaction in order to eliminate the influence of any DNA torsional tension on the psoralen binding and the sequence-specificity of psoralen binding was adjusted for. It was found that a region encompassing the promoter of the serum-regulated DHFR gene was about 50% more accessible to psoralen photocross-linking in serum-stimulated cells and about 90% more accessible in serum-starved cells than the other five regions of the DHFR gene analyzed and the genome overall. Treating serum-stimulated cells with the RNA polymerase II transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) or the topoisomerase I inhibitor camptothecin reversed the elevated accessibility of the DHFR promoter region. These results suggest that the accessible chromatin structure of the DHFR promoter is not dependent on serum-stimulated poising of the gene for transcription, but may reflect the ability of the RNA polymerase to clear the promoter.
Ljungman
M
M
Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor 48109-0582, USA. ljungman@umich.edu
D000818:Animals; D016466:CHO Cells; D002166:Camptothecin / Q000494:pharmacology; D002843:Chromatin / Q000737:chemistry*; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives / Q000494:pharmacology; D011564:Furocoumarins / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013873:Thionucleotides / Q000494:pharmacology
D002843:Chromatin; D003432:Cross-Linking Reagents; D011564:Furocoumarins; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D004004:Dichlororibofuranosylbenzimidazole; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D002166:Camptothecin