Chromatin remodelling and gene regulation

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Cell and Molecular Biology

Sammanfattning: Chromatin plays an important role in gene regulation, as it forms an obstacle for a number of proteins that need access to the DNA. Several protein complexes that open up the chromatin structure have been identified and some of these are important for gene regulation. The mouse mammary tumour virus (MMTV) promoter is used as a model for studies of transcription activation-dependent chromatin remodelling. Transcription from the MMTV promoter is activated by the hormone-bound glucocorticoid receptor (GR). Xenopus oocytes have provided an in vivo model system for studying gene regulation coupled to changes in chromatin structure. The MMTV promoter is microinjected into the nucleus of Xenopus oocytes as singlestranded DNA, and the second DNA strand is synthesised in a process forming chromatin that is repressive to basal transcription. In the absence of hormone the nucleosomes in the MMTV promoter are randomly positioned. Hormone treatment activates GR-mediated transcriptio from the MMTV promoter. This results in a major change in the nucleosome organisation from a random structure to an active translational positioning of the nucleosomes. The nucleosome in the promoter harbouring the GR binding sites is remodelled. The hormoneinduced nucleosome positioning and chromatin remodelling in the MMTV promoter are uncoupled from transcription and from the binding of basal transcription factors. In an attempt to block the glucocorticoid hormone-induced transcription, we used the glucocorticoid antagonists RU43044 and RU486. Hormone-induced transcription from the MMTV promoter was inhibited by both antagonists. This resulted in almost complete loss of translational nucleosome positioning and reversal of chromatin structure to the structure seen in the absence of hormone. In the presence of the antagonist RU43044 GR did not bind DNA in the MMTV promoter, whereas the antagonist RU486 resulted in partial binding of GR. We have characterised a chromatin remodelling activity, the BRG1 -containing SWI/SNF complex, in Xenopus oocytes. The SWI/SNF complex was purified and enriched by sequential chromatography. ATP-dependent nucleosome remodelling activity co-eluted with the BRG l immunogenic activity as well as with a related protein, termed imitation of SW12 (ISWI), which is present in other complexes with nucleosome remodelling activity. Xenopus BRG1 was predominantly nuclear and was present during all stages of oogenesis and embryogenesis. DNA injected into Xenopus oocytes was organised as an intranuclear fibrous network throughout the nucleoplasm. Immunofluorescence microscopy on cryostat-sectioned oocytes revealed that BRG I was co-localised with the injected DNA, forming a network throughout the nucleoplasm and at the nuclear margin. The GR was present in the cytoplasm and translocated into the nucleus after hormone binding, where it associated with the endogenous chromosomes and the injected DNA. BRG1 and GR were also localised at the margin of the nucleus, which might be a storage compartment in the oocyte.

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