Formation of higher-order secondary and tertiary chromatin structures by genomic mouse mammary tumor virus promoters.
Agarose multigel electrophoresis has been used to characterize the structural features of isolated genomic mouse mammary tumor virus (MMTV) promoters. The mouse 3134 cells used for these studies contain approximately 200 stably integrated tandem repeats of a 2.4-kb MMTV promoter fragment. Inactive, basally active, and hormonally activated genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in low-salt nuclear extracts, and electrophoresed in multigels consisting of nine individual agarose running gels. Specific bands were detected and characterized by Southern and Western blotting. We find that transcriptionally inactive promoters contain TBP and high levels of histone H1, and are present to varying extents in both untreated and dexamethasone (DEX)-treated 3134 cells. In contrast, the basally active promoter, present in untreated cells, is bound to RNA Pol II, TBP, and Oct1, contains acetylated H3 tail domains, and is depleted of histone H1. The DEX-activated promoter possessed similar composition as the basal promoter, but also contains stably bound Brg1. Strikingly, all forms of the MMTV promoter condense into higher-order secondary and/or tertiary chromatin structures in vitro in the presence of Mg2+. Thus, genomic MMTV promoter chromatin retains the ability to form classical higher-order structures under physiological salt conditions, even after dissociation of H1 and binding of several transcription factors and multiprotein complexes. These results suggest that transcriptionally active eukaryotic promoters may function in a locally folded chromatin environment in vivo.
Georgel
Philippe T
PT
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
Fletcher
Terace M
TM
Hager
Gordon L
GL
Hansen
Jeffrey C
JC
D000107:Acetylation; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D016678:Genome; D006657:Histones / Q000032:analysis; D051863:Host Cell Factor C1; D008274:Magnesium / Q000494:pharmacology; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000032:analysis; D009707:Nucleosomes / Q000737:chemistry; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000032:analysis; D035181:TATA-Box Binding Protein / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002843:Chromatin; D004268:DNA-Binding Proteins; C497693:Hcfc1 protein, mouse; D006657:Histones; D051863:Host Cell Factor C1; D009687:Nuclear Proteins; D009707:Nucleosomes; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D003907:Dexamethasone; D012319:RNA Polymerase II; C513194:Smarca4 protein, mouse; D004265:DNA Helicases; D008274:Magnesium