The Position and Length of the Steroid-Dependent Hypersensitive Region in the Mouse Mammary Tumor Virus Long Terminal Repeat Are Invariant despite Multiple Nucleosome B Frames

Abstract

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5-most receptor binding site within the HRE is downstream of ؊190, hypersensitive sites were found further upstream to at least ؊295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.Regulation of transcription of the mouse mammary tumor virus (MMTV) by steroid hormones is mediated through a hormone response element (HRE) located between positions Ϫ70 and Ϫ190 (16,28,33,39). Four binding sites for steroid receptors have been mapped within the HRE (57,61,68,82). Binding sites for other factors have also been detected within (30,41,73,74) and immediately upstream of (17,27,44,45,49) this region, and they participate in the regulation of MMTV by steroids (17,27,44,45,74). Loading of transcription factors downstream of the HRE occurs upon activation of the promoter by the glucocorticoid or the progesterone receptors, including NF-1, and TFIID (5,19,43,59,76). Concomitant with this activation, hypersensitivity to DNase I, methidiumpropyl-EDTA ⅐ iron(II) [MPE-Fe(II)], and restriction enzymes is detected in the general location of the HRE (3, 9, 62, 67, 76, 88).To understand the chromatin transition leading to hypersensitivity and thus the regulation of MMTV by steroids, it is necessary to understand the chromatin organization of the promoter in both the uninduced and induced states. The long terminal repeat (LTR) of MMTV is organized in an array of six nucleosomes termed Nuc-A through Nuc-F; the HRE and the transcription initiation site are in the Nuc-B and Nuc-A regions, respectively (67). Nucleosomes in these two regions of the LTR occupy multiple frames; that is, different copies of the LTR possess Nuc-A and Nuc-B in different positions (26). Although treatment with dexamethasone, a synthetic glucocort...

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

“…In 3134 (murine mammary adenocarcinoma) cells, 200 copies of the MMTV-LTR with 800 -1200 GR-binding sites are stably integrated in a head-to-tail orientation near the centromere of chromosome 4. Previous studies have indicated that the hormone response of the MMTV-LTR array as a unit is indistinguishable from that of a single integrated copy of the viral LTR (Fragoso et al, 1998;Kramer et al, 1999;McNally et al, 2000). The array, therefore, provides a good model system to examine the in vivo recruitment of chromatin-remodeling complexes to their target promoter.…”

“…GFP-BRM and GFP BRG1 have been previously described (Reyes et al, 1997;de la Serna et al, 2001) and was provided by Christian Muchardt, (Institute Pasteur, Paris, France). The full-length MMTV-LTR driving transcription of the luciferase gene, pRSV-GR (glucocorticoid receptor) and pCMV ␤-Galactosidase (internal control) has been described previously (Lefebvre et al, 1991;Fragoso et al, 1998). All cloned vectors were confirmed by sequencing.…”

“…In the presence of hormone, this region becomes more accessible to a variety of chemical and enzymatic nucleases, the hallmark of a chromatin-remodeling event. We and others have used the restriction endonuclease, SacI, which cuts within the nucleosome B-C region, as a measure of this chromatin transition (Fragoso et al, 1998;Fryer and Archer, 1998;Fletcher et al, 2002). We compared the extent of SacI cleavage in 3617 mouse mammary adenocarcinoma cells (which stably expresses GFP-GR in a tetracycline-repressible system; Walker et al, 1999) with SacI cleavage in the 5555 cell line (generated by stable transfection of 3617 cells with a Flag-tagged BRG1-K-R under the control of the same tetra- cycline regulator).…”

“…Upon hormone binding, nuclear hormone receptors such as the glucocorticoid receptor (GR) bind hormone response elements and regulate transcription at their target genes through the recruitment of a variety of coactivators, corepressors, chromatin remodeling activities, and components of the basal transcription machinery (Fragoso et al, 1998;Giangrande et al, 2000;Dilworth and Chambon, 2001;McKenna and O'Malley, 2002;Schaaf and Cidlowski, 2003;Belandia and Parker, 2003;Metivier et al, 2003;Hager et al, 2006;Carroll and Brown, 2006;Lee et al, 2006). To further understand the process by which chromatin-remodeling complexes are recruited and regulate target genes to modulate transcription, we have directly visualized the sequence of gene expression events involving the SWI/SNF complex in mouse mammary adenocarcinoma cells that contain a tandem repeat of stably integrated copies of the MMTV-LTR (mouse mammary tumor virus-long terminal repeat).…”

“…In fact, this modulation is one of the key mechanisms essential for the functional role of nuclear hormone receptors (Stenoien et al, 2001;Schaaf and Cidlowski, 2003;Stavreva et al, 2004;Elbi et al, 2004b;Farla et al, 2005;Rayasam et al, 2005). In vivo FRAP has been used to study the dynamic properties of chromatin proteins and that the FRAP recovery kinetics of chromatin proteins are directly related to their chromatin-binding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005). Because BRG1 and BRM were selectively recruited to the MMTV array in response to hormone (Figures 2 and 3), we used FRAP to study the binding kinetics of chromatin-remodeling complexes at the amplified MMTV target in vivo.…”