Glucocorticoid hormone-induced receptor localization to the chromatin fibers formed on injected DNA in Xenopus oocytes.
Oocytes from Xenopus laevis have provided a model system for studying the dynamic changes that occur in chromatin during gene activation. We have reconstituted glucocorticoid receptor (GR) induced transcription from the mouse mammary tumor virus (MMTV) promoter by intranuclear injection of an MMTV-driven reporter and cytoplasmic injection of synthetic mRNA(GR) into Xenopus oocytes. Here we investigate the intranuclear distribution of injected DNA, which is assembled into chromatin. We show that this chromatin is organized as an intranuclear fibrous network. Unliganded GR is located in the cytosol and hormone triggers its nuclear translocation and association with the chromatin fibers. Furthermore, we analyze the intranuclear distribution of other factors involved in transcription from the MMTV promoter. Indirect immunofluorescence microscopy on cryostat-sectioned oocytes revealed that BRG1, which is a subunit of the SWI/SNF chromatin remodeling complex, as well as RNA polymerase II and recombinantly expressed Xenopus nuclear factor 1-B, are all associated with the endogenous chromosomes and the chromatin fibers formed on injected DNA. This association does not depend on specific DNA binding sites and appears to be nonspecific.
Gelius
B
B
Laboratory of Molecular Genetics, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Wrange
O
O
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D005938:Glucocorticoids / Q000378:metabolism*; D015151:Immunoblotting; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008845:Microinjections; D008856:Microscopy, Fluorescence; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D016707:Tissue Fixation / Q000379:methods; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D012319:RNA Polymerase II; C513194:Smarca4 protein, mouse; D004265:DNA Helicases