Chromatin-Mediated Restriction of Nuclear Factor 1/CTF Binding in a Repressed and Hormone-Activated Promoter In Vivo

Abstract

Mouse mammary tumor virus (MMTV) promoter-driven transcription is induced by glucocorticoid hormone via binding of the glucocorticoid receptor (GR). The MMTV promoter also harbors a binding site for nuclear factor 1 (NF1). NF1 and GR were expressed in Xenopus oocytes; this revealed GR-NF1 cooperativity both in terms of DNA binding and chromatin remodeling but not transcription. A fraction of NF1 sites were occupied in a hormone-dependent fashion, but a significant and NF1 concentration-dependent fraction were constitutively bound. Activation of the MMTV promoter resulted in an ∼50-fold increase in the NF1 accessibility for its DNA site. The hormone-dependent component of NF1 binding was dissociated by addition of a GR antagonist; however, the antagonist RU486, which supports partial GR-DNA binding, also maintained partial NF1 binding. Hence GR-NF1 cooperativity is independent of agonist-driven chromatin remodeling. NF1 induced the formation of a micrococcal-nuclease-resistant protein-DNA complex containing the DNA segment from −185 to −55, the MMTV enhanceosome. Coexpression of NF1 and Oct1 resulted in a significant stimulation of hormone-induced MMTV transcription and also in increased basal transcription. We propose that hormone-independent NF1 binding may be involved in maintaining transcriptional competence and establishment of tissue-specific gene networks.

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Chromatin-Mediated Restriction of Nuclear Factor 1/CTF Binding in a Repressed and Hormone-Activated Promoter In Vivo

This work was supported by the Swedish Cancer Foundation (project 2222-B03-19XCC) and Knut and Alice Wallenberg's foundation. P.-H. Holmqvist was supported by the Cell Signaling program of the Swedish Foundation for Strategic Research.

We thank U. Björk for skillful technical assistance and R. Morgenstern for helpful discussions concerning NF1 binding kinetics. We are grateful to L. Poellinger, M. Beato, and R. M. Gronostajski for providing cDNA coding for the human Oct1, the porcine NF1-C1, and the murine NF1-B2, respectively. We are grateful to K. Sousa for construction of the pigNF1-C1/RN3P plasmid. We thank B. Gelius at KaroBio for the generous gift of RU43044.