Cellular Factors Binding to a Novel cis-Acting Element Mediate Steroid Hormone Responsiveness of Mouse Mammary Tumor Virus Promoter

Abstract

Steroid hormone receptors regulate mouse mammary tumor virus (MMTV) gene expression by binding to hormone response DNA elements present in the long terminal repeat. Tissue-specific expression of MMTV is unlikely to be regulated by steroid hormone-receptor complex alone, and mammary cell-specific factors might play a role in the hormone-induced transcriptional activation. In this report we have investigated the function of a novel cis-acting element designated Kil (؊204 to ؊188) which is located adjacent to the distal glucocorticoid response element, in steroid hormone-induced transcription of MMTV. Electrophoretic mobility shift assays indicate that cellular factors bind to the Kil element, and dexamethasone stimulation results in alterations in the binding pattern of proteins in this region. By transient transfection assays using wild type and deletion mutants of the Kil element, we show that this novel cis-acting element is necessary for hormone-induced transcription of MMTV and functions in mammary tumor cells but not in NIH/3T3 cells. Mutagenesis of the Kil sequence suggests that the entire Kil element functioning as one unit is necessary for hormone-induced transcription of MMTV. When placed in the context of heterologous promoters, neither Kil element nor glucocorticoid response element is able to induce significant hormone-induced transcription of MMTV. The presence of both the DNA elements in tandem results in optimal induction of transcription in the presence of steroid hormones. Our results also indicate that the Kil element functions in human breast carcinoma cell lines such as T47D and MCF-7. These results suggest that Kil element in combination with distal glucocorticoid response element functions as a mammary cell-specific enhancer to regulate MMTV transcription.Steroid hormone receptors are cytoplasmic proteins, which, upon binding to their ligands, translocate into the nucleus and bind to DNA elements in a sequence-specific manner and modulate the transcription of cellular genes (1). Transcription from the long terminal repeat (LTR) 1 of mouse mammary tumor virus (MMTV) has been used extensively to understand the regulation of gene expression by steroid hormones. Glucocorticoids, progesterones, and androgens induce the transcription of MMTV (2-4) upon binding of their respective receptors to the hormone response elements. These elements are located between Ϫ202 and Ϫ59 bases upstream of the transcription initiation site in the MMTV promoter, which can be divided into the proximal and distal glucocorticoid response elements (GREs). The GRE consists of four repeats of the hexanucleotide 5Ј-TGTTCT-3Ј to which the steroid hormone receptors were shown to bind and exert their effects on transcription. The proximal glucocorticoid responsive element (GRE) contains three repeats of the hexanucleotide, while the distal GRE (Ϫ188 to Ϫ170) contains only one of the hexanucleotide. Apart from steroid hormone-receptor complex, which is a major determinant of MMTV transcription, cellular factors have been implic...

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

“…7B]). Various groups have reported detection of regulatory elements upstream of the minimal HRE (17,27,44,45,49), including binding sites for the glucocorticoid receptor (57), and an effect of this region on the hormonal response of MMTV has been documented (17,27,33,44,45,49). Cooperative loading of transcription factors to an entire stretch of chromatin could partly explain the linkage between sites in the HSR.…”

“…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, OTF-1, and TFIID (5,19,43,59,76).…”

Gene Expression Assay for Hazard Assessment of Chemicals.

“…For example, the mouse mammary tumor virus (MMTV) promoter is convenient for this purpose, because it includes both the minimal promoter and multiple binding sites for GR/MR/ PR 35,36) . Such a regulatory fragment is usually several hundred nucleotides in length, and includes various elements other than the receptor binding site of interest, so that the possible effects of other signaling pathways should be taken into consideration.…”