Steroid-selective Initiation of Chromatin Remodeling and Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter Is Controlled by the Site of Promoter Integration

Abstract

The mouse mammary tumor virus (MMTV) promoter has target sequences recognized by several steroid receptors. We present evidence for a novel mechanism that confers hormone specificity to this promoter. We show that remodeling of MMTV chromatin and the concomitant activation of the MMTV promoter are induced equally by glucocorticoids and progestins in one chromosomal context but are selective for glucocorticoids in another. Furthermore, increased histone acetylation modulates MMTV promoter regulation disparately at the two chromosomal locations. Together, these data indicate that chromosomal architecture commands a crucial role in gene regulation, imposing locus-specific selectivity between regulators with similar sequence recognition.Many of the concepts that underlie the current understanding of both steroid hormone action and the role of chromatin in the regulation of gene expression have sprung from studies employing the mouse mammary tumor virus (MMTV) 1 long terminal repeat (LTR) as a model promoter. Target sequences related to a TGTTCT motif direct hormone-induced transcription by glucocorticoid receptors (GR), progesterone receptors (PR), and mineralocorticoid and androgen receptors (1-8). In stable chromatin, the MMTV LTR also directs the assembly of an ordered nucleosome array that represses loading of basal transcription factors on the promoter. Activation of GR or PR by hormones promotes the remodeling of the MMTV chromatin and the loading of basal transcription factors. In contrast, nucleosomes are randomly arrayed on transiently transfected MMTV templates. Such templates exhibit elevated constitutive levels of transcription factor loading and promoter activity and, consequently, a reduced induction by hormones (9 -11). Thus, the organization of chromatin and its remodeling by steroid hormones are critical to appropriate steroid-mediated regulation.Although GR and PR recognize the same target elements and each induces certain genes regulated by the other receptor, the two receptors mediate very different actions in tissues such as the mammary epithelium that can express both receptors. GR and PR are expressed at comparable levels in the T47D(A1-2) mammary carcinoma cell line. This cell line also contains 10 copies of a stably transfected MMTV-luciferase (LUC) reporter gene. Surprisingly, luciferase is induced by glucocorticoids but is almost entirely refractory to progestins, whereas a transiently transfected template, MMTV-chloramphenicol acetyltransferase (CAT), is responsive to both hormones. On the MMTV-LUC template, glucocorticoids induce remodeling of chromatin, whereas progestins fail to do so (12).Selectivity between two regulators that recognize the same target element could be a property inherent to a promoter sequence, a property that cannot be reconstituted on transient chromatin. Alternatively, the chromatin at the site of integration of a transgene may impose a selective mechanism on the integrated promoter. Examples of integration site determining whether a gene is expressed or silenced...

Transactivation Specificity of Glucocorticoid VersusProgesterone Receptors

“…Chromatin structure can repress gene expression (11,12), thereby increasing the fold induction by receptor-steroid complexes, especially in cell-free systems (13,14). Chromatin environment can control gene inducibility by PR (15)(16)(17), and chromatin structure has been proposed to be a determinant for PR induction (10,15). However, alterations in chromatin structure do not appear to be a prerequisite for all steroid receptor-induced gene transactivation.…”

The Identification of Genes Differentially Regulated by Progestins and Glucocorticoids in Human Breast Cancer Cells

“…Transient transfections of 4F-derived cell lines were performed using a diethylaminoethyl/dextran method as previously described (Nordeen et al 1998). After 20-24 h, cells were treated with vehicle, dexamethasone (100 nM) or R5020, a synthetic progestin, (10 nM).…”

“…Cells were harvested 44-48 h after treatment. Cell extracts were prepared by first washing the cells, harvesting them in 0-5 ml cell lysis buffer, then pelleting debris (Nordeen et al 1998). For luciferase assays, 25 ul soluble lysate were used and for ß-galactosidase assays 2-5 ul.…”

“…For luciferase assays, 25 ul soluble lysate were used and for ß-galactosidase assays 2-5 ul. Luciferase and ß-galactosidase assays were assessed using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Sparks, MD, USA) as previously described (Nordeen et al 1998). Data are reported as luciferase activity normalized to ß-galactosidase activity in the same transfection.…”

Role of Akt/protein kinase B in the activity of transcriptional coactivator p300

“…While histone deacetylase inhibitors, generally, enhance gene expression from various promoters [4,5,13,14], sodium butyrate inhibits the activation of the tyrosine aminotransferase gene and MMTV promoter [15][16][17][18] through stimulating selective p300 degradation [9]. In addition, sodium butyrate and other histone deacetylase inhibitors affect a variety of cellular processes, such as cell growth, differentiation and apoptosis by mechanisms that are still unknown [19][20][21][22].…”