Glucocorticoid receptor-dependent disruption of a specific nucleosome on the mouse mammary tumor virus promoter is prevented by sodium butyrate.

Abstract

Our laboratory has previously developed cell lines derived from mouse NIH 3T3 fibroblasts and C127 mammary tumor cells that stably express mouse mammary tumor virus (MMTV) long terminal repeat fusion genes in bovine papillomavirus-based episomes. Glucocorticoid hormone strongly activates transcription from episomes and induces the disruption of a single nucleosome in an array of phased nucleosomes on the MMTV promoter. Sodium butyrate inhibits the glucocorticoid hormone-dependent development of a nuclease-hypersensitive site that is due to the displacement of this nucleosome, and inhibits induction of RNA transcripts from episomes. Saturation binding studies show that butyrate treatment does not significantly affect the amount or the hormone-binding affinity of the glucocorticoid receptor. In a transient transfection assay, glucocorticoid hormone can activate transcription from a MMTV long terminal repeat-driven luciferase gene construct equivalently in untreated and butyrate-treated cells, indicating that the soluble factors necessary for transactivation of the MMTV promoter are unaffected by butyrate. The differential effect of butyrate on the induction of stable chromatin templates and transiently expressed plasmids suggests that butyrate prevents nucleosome displacement and represses transcription by inducing a modification of chromatin.The organization ofgenes into nucleosomes and higher-order chromatin structure has important implications for transcriptional regulation (for reviews see refs. 1-4). One example of this functional role of chromatin is illustrated by the regulation of mouse mammary tumor virus (MMTV) transcription by glucocorticoid hormone. A phased array of six nucleosomes is positioned on the MMTV long terminal repeat (LTR) in bovine papillomavirus (BPV)-based episomes (5) and single integrated copies (6). As an early step in the activation of the MMTV promoter by glucocorticoid hormone, the hormone-activated glucocorticoid receptor binds to glucocorticoid regulatory elements (GREs) on one of these nucleosomes (Nuc-B), initiating a process whereby Nuc-B is displaced (see Fig. 1). In vivo exonuclease III footprinting experiments have demonstrated that a consequence of this receptor-dependent chromatin reorganization is the binding of the transcription factors NF1 and TFIID to their cognate recognition sites on the MMTV promoter (7). As the apparent concentrations and specific DNA-binding activities of NF1 and TFIID are identical in extracts from hormone-treated and untreated cells (8), it has been proposed that these factors are excluded from their recognition sites by the specific positioning of Nuc-B (9).The disruption of precisely positioned nucleosomes has been shown to occur in several other systems during transcriptional activation in vivo. A yeast acid phosphatase gene (PHOS) has an array of phased nucleosomes covering its promoter when it is repressed by high Pi (10,11 transcription by regulating access of RNA polymerase and/ or transcription factors. By using DNA restriction ...

Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation

“…The data represent the results of three independent experiments. (c) Histones were extracted from cells treated for the times indicated with TSA and subjected to Western blotting with antibodies against acetylated H3 and H4 (Bresnick et al, 1990;Bartsch et al, 1996;Lambert and Nordeen, 1998), there is also a strong inhibition of basal (control) transcription, an observation also reported by Bartsch et al (1996). Transcription of the b-actin gene was not significantly changed by these TSA treatments ( Figure 1a and data not shown).…”

“…Previous studies examining regulation of the MMTV promoter by HDAC inhibitors found that, depending on a variety of conditions, butyrate or TSA could activate or repress the MMTV promoter in organized chromatin (Bresnick et al, 1990;Bartsch et al, 1996;Lambert and Nordeen 1998;Myers et al, 1998;Wilson et al, 2002). Repression was observed in seven distinct cell lines with two exceptions.…”

“…This promoter, when assembled into organized chromatin, can be repressed by the HDAC inhibitors trichostatin A (TSA) or sodium butyrate at concentrations which cause the accumulation of acetylated histones (Bresnick et al, 1990;Bartsch et al, 1996;Lambert and Nordeen 1998;Myers et al, 1998;Wilson et al, 2002). We have investigated the mechanism by which TSA represses the MMTV promoter in organized chromatin.…”

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

“…The 3617 cell line was stably transfected with a Flag-tagged BRG1-K-R to generate the 5555 cell line. The 1365.1 cell line is derived from NIH 3T3 mouse fibroblast cells by stable transformation with a multicopy episome and contains multiple tandem copies of a stably integrated MMTV-LTR array (Cordingley et al, 1987;Bresnick et al, 1990). Human adrenal carcinoma cells (SW13) are deficient in BRG1, BRM, and GR expression and were obtained from the American Type Culture Collection (ATCC, Manassas, VA).…”

Glucocorticoid receptor-mediated chromatin remodeling in vivo

“…Acetylation of core histones regulates transcription of many promoters, including MMTV (Struhl, 1998). Sodium butyrate, which induces histone hyperacetylation, inhibits glucocorticoid hormone-dependent nucleosome disruption and promoter activation from an MMTV template assembled as chromatin (Bresnick et al, 1990). However, glucocorticoid activation from a transiently introduced MMTV template was una ected.…”