Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus

Abstract

Transcription from the promoter of mouse mammary tumor virus is subject to induction by several classes of steroid hormones as well as to repression by a negative regulatory element present in the long terminal repeats of proviral DNA. In order to characterize the functional elements of the promoter that in some way must respond to these regulatory signals, a number of promoter mutations were constructed, including a set of linker-scanning mutations across the entire promoter region. Analysis of these mutated promoters with a transient-transfection assay defined at least three mutation-sensitive promoter elements that are required for both basal and hormone-induced transcription. One mutation-sensitive region contains a TATA element located at approximately position-30 with respect to the start of transcription. A second mutation-sensitive region contains two 10-base-pair direct repeats located between positions-60 and-38, within which are embedded three copies of octamer-related sequences; complete disruption of this region of the promoter leads to a more severe decrease in transcription than do any of the linker-scanning mutations, suggesting that the repeated sequences may be at least partially functionally redundant. Gel electrophoresis mobility shift assays were used to demonstrate specffic binding of a nuclear protein to this region of the promoter. A third mutationsensitive region contains a binding site for nuclear factor 1 (NF-1) located between positions-77 and-63. Sitedirected mutations in the NF-1-binding site which increase the apparent affinity of NF-1 for the promoter in vitro do not decrease the hormone dependence of transcription, suggesting that transcriptional activation mediated by steroid hormone-receptor complexes cannot be explained by facilitation or stabilization of the interaction of promoter sequences with NF-1 and consistent with the idea that binding of NF-1 is not rate determining in transcription from the mouse mammary tumor virus promoter. None of the promoter mutations functionally separates basal from glucocorticoid-induced transcription, suggesting that hormone induction does not make the promoter independent of any of the DNA-binding factors required for its basal activity.

Organization of chromatin and histone modifications at a transcription site

“…This differential effect was enhanced upon the hormone induction of transcription but was still detected to a lesser degree without hormone ( Fig. 5 a), which is consistent with basal transcription from the MMTV promoter (Toohey et al, 1990) and with our unpublished observations of RNA FISH accumulation at the array in the absence of hormone.…”

Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo.

“…In most cells, expression of this promoter is highly dependent on induction by steroid hormones, in particular glucocorticoids (Parks et al, 1976) and progestins (Cato et al, 1986). Induction is mediated by several sequence elements, including a hormoneresponsive region (HRR) with four receptor binding sites (HREs) (Chandler et al, 1983;Scheidereit et al, 1983), a NF-I binding site (Nowock et al, 1985;Miksicek et al, 1987;Buetti et al, 1989;Bruggemeier et al, 1990), and two octamer motifs (Toohey et al, 1990;Bruggemeier et al, 1991;Buetti, 1994). Mutation of the NF-I binding site has a dramatic influence on hormonal induction, suggesting a functional synergism between receptors and NF-I (Miksicek et al, 1987;Buetti et al, 1989;Briggemeier et al, 1990).…”

Functional redundancy of octamer elements in the mouse mammary tumor virus promoter

“…Plasmids pLSwt and pLS(-64/-57) have been previously described (4). These plasmids contain MMTV LTR sequences between 363 (RsaI) and + 133 (Pvull) ( Fig.…”

“…Promoters of genes transcribed by RNA polymerase II are, in general, composed of multiple elements, each of which serves as a binding site for a specific, trans-acting transcription factor (for a review, see (1)(2)(3)). Consistent with this general organization, the promoter of the retrovirus mouse mammary tumor virus (MMTV) contains at least four distinct elements that have been defined by mutational analysis and identified as sites recognized by sequence-specific DNA binding proteins (4,5). These elements include a binding site for the transcription factor nuclear factor-I (NF-1) from 77 to 63, three sequences related to the octamer element which are almost completely contained within two exact 10 bp repeats between 60 and 39, the TATA box at about 30, and an element located 3' of the transcription initiation site between +4 and + 10, which we have shown is a binding site for a protein we have termed initiation site binding protein (ISBP) ( Fig.…”

“…The basal promoter of MMTV must in some way respond to the positive and negative signals of the HRE and NRE. Mutations in any of the sequence elements of the MMTV promoter affect both basal and steroid hormone-induced transcription, and hormone induction does not make transcription independent of any of the DNA-binding transcription factors required for basal promoter activity (4).…”