Abstract
Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR. Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.
Highlights
The chromatin organization of a gene is an important determinant of transcriptional regulation
Nucleosome Ϫ2 of the PHO5 promoter was replaced with the mammary tumor virus (MMTV) B nucleosome, and in another, the B nucleosome was inserted into the full-length PHO5 promoter
Activation of PHO5 UAS elements and glucocorticoid response elements (GRE) contained within the B nucleosome could be achieved by growth in medium lacking phosphate and by addition of hormone, respectively
Summary
The chromatin organization of a gene is an important determinant of transcriptional regulation. Activation of the PHO5 promoter is accompanied by the disruption of four positioned nucleosomes, and many requirements for this remodeling have been defined [3] Both the glucocorticoid receptor (GR) and nuclear factor I (NFI) are essential for activation of the MMTV LTR in response to glucocorticoids. This simple model, the facilitation of NFI binding by GR-mediated nucleosome disruption, was subsequently modified, when Truss et al [13] showed that the B nucleosome persists following receptor binding in vivo This observation was consistent with similar results obtained in earlier in vitro studies, showing that GR can bind to a nucleosome without disrupting it [14], but the precise composition of the B nucleosome in the activated MMTV promoter remains undefined [15]. Further refinement of the yeast experiments has made it possible to demonstrate a synergy between GR and NFI activation, which was dependent on the presence of a modified nucleosome in the MMTV promoter [16, 17]
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