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 ϳ50-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.

Nuclear Factor 1 and Octamer Transcription Factor 1 Binding Preset the Chromatin Structure of the Mouse Mammary Tumor Virus Promoter for Hormone Induction

“…We previously showed that NF1 binds to the MMTV promoter within a wide range of intranuclear concentrations but did not have any effect on MMTV transcription. However, NF1 did stimulate MMTV basal and hormone-induced transcription when coexpressed with Oct1 (18). This posed the question of to what extent different Oct1 concentrations affected the hormone-activated MMTV transcription.…”

“…In vitro nucleosome reconstitution experiments with MMTV LTR DNA from the B-nucleosome region demonstrated a strongly preferred frame of rotational positioning (16,17) that was similar to the rotational position seen in vivo (13,18). The in vitro nucleosome reconstitution experiments also indicated a preferred translational nucleosome positioning similar to the organization seen in vivo, but they were conducted on rather small DNA segments (16,17,19,20).…”

“…Assuming an intranuclear volume of 40 nl, this will result in an intranuclear concentration of 2.5, 0.25, and 1.5 M for GR, NF1, and Oct1, respectively. Quantification of MMTV transcription by S1-nuclease has been described previously (18). All analyses were done in duplicate or triplicate.…”

“…Relative amounts GR and Oct1 were estimated by quantification on a Fuji Bio-Imaging analyzer BAS-2500 using the Image Gauge version 3.3 software with correction of the methionine content in the respective proteins as previously described for calculation of intranuclear concentration of NF1 (18). An aliquot was also analyzed by immunoblotting with GR antiserum together with known amounts of GR purified from rat liver (16) to serve as a standard curve for calculation of absolute amounts of Oct1 that followed a linear correlation between injected RNA and translated protein.…”

“…The lack of prepositioned nucleosomes in oocytes suggested that the constitutive translational nucleosome positioning seen in other cells is driven by factor(s) that are absent in Xenopus oocytes. We recently demonstrated that NF1 is able to bind to its DNA site in the inactive promoter (18). However, NF1 binding was weak in the absence of GR activation, and a 50-fold higher NF1 binding efficiency was seen in the hormoneinduced state.…”