Two regions of the mouse mammary tumor virus long terminal repeat regulate the activity of its promoter in mammary cell lines.

Abstract

In vivo expression of the mouse mammary tumor virus (MMTV) is restricted to a few organs, with the highest rate of transcription found in the mammary gland. Using a series of mammary and nonmammary murine cell lines, we have identified two regulatory elements, located upstream of the hormone responsive element, that specifically regulate the MMTV promoter. The first element displays an enhancerlike activity and is coincident with the binding of a nuclear factor (designated MP4; position -1078 to -1052 in the long terminal repeat) whose presence is apparently restricted to mammary cell lines. The second regulatory region mediates a repressive activity and is mapped to the long terminal repeat segment from -415 to -483. This repression is specific for a particular subtype of mammary cells (RAC cells) able to grow under two differentiation states (A. Sonnenberg, H. Daams, J. Calafat, and J. Hilgers, Cancer Res. 46:5913-5922, 1986). The MMTV promoter in mammary cell lines thus appears to be modulated by two cis-acting elements that are likely to be involved in tissue-specific expression in vivo.Mouse mammary tumor virus (MMTV) is the etiologic agent of mammary adenocarcinoma in mice. Its association with T-cell leukemia (27, 28), thymomas (1), and kidney adenocarcinomas (17,47) in the same species has also been documented. Although the MMTV genome encodes a large open reading frame of unknown function (15), this gene has not been associated with oncogenic activity, and the virus is currently thought to induce cell transformation by integrating in the vicinity of cellular proto-oncogenes (int genes), causing their deregulated expression (11,37). The integration of a new proviral DNA in the vicinity of the most extensively characterized int genes, wnt-J and int-2, has been observed in numerous mammary tumors and is clearly required for tumorigenesis (35). Integrations at the wnt-J or int-2 locus never alter the coding potential of the cellular gene, suggesting that these genes are activated solely by viral cis-acting sequence(s), while the nonaltered allele remains silent (12,36,46 [40], and transforming growth factor alpha [26]) has been targeted to the mammary gland by driving the transgene with the intact MMTV long terminal repeat (LTR). It is likely that these two properties of MMTV (its tissuespecific expression and its insertional mutagenic activity) are related.Several investigations have indicated the presence of transcriptionally active sequences in the LTR in addition to the well-characterized hormone responsive element (HRE) (-70 to -220). One large stretch of the LTR (-364 to -455) is associated with a repressive activity on basal transcriptional activity in nonmammary transformed cell lines (21,32). A region at the 5' end of the LTR (-1195 to -741) correlated with efficient and tissue-selective expression of the promoter when assayed in LTR-human growth hormone chimeras in transgenic mice. This upstream LTR fragment was by itself sufficient to target expression of the transgene in the same tissues (45).To...

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

“…The dynamic exchange of remodeling proteins on the MMTV array are consistent with our in vitro results obtained from rapid UV laser crosslinking where purified SWI/SNF binds to and is displaced from purified MMTV chromatin (Fletcher et al, 2002;Nagaich et al, 2004). Because the FRAP recovery kinetics of chromatin proteins are directly related to their chromatinbinding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Hager et al, 2002;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005), we conclude that the remodeling proteins with the slowest exchange rate reside longest on the MMTV promoter and associate most strongly with MMTV chromatin. A comparison of the kinetic properties of chromatin-remodeling complexes revealed that BRG1 was more strongly associated with the MMTV array than BRM ( Figure 6D).…”

“…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 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.…”

Nucleoprotein Structure Influences the Response of the Mouse Mammary Tumor Virus Promoter to Activation of the Cyclic AMP Signalling Pathway

“…All cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% charcoal-stripped serum. Transfections were carried out by the calcium phosphate precipitation method in six-well dishes using carrier DNA with 250 to 500 ng of pLTRluc (36). Hormone treatments were carried out for 24 h at a concentration of 100 nM.…”

“…Okadaic acid (Calbiochem) and 8-Br-cAMP (Sigma) were used at concentrations of 50 nM and 1 mM, respectively. Cells were harvested and assayed for luciferase activity as previously described (36).…”