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

Abstract

Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormoneinduced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway.The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been used extensively as a model for both steroid-induced transcription and the effects of chromatin structure on transcription. The MMTV promoter can be activated by glucocorticoids, progestins, androgens, and mineralocorticoids through their individual receptors (8,18,21,55). In chromatin, the MMTV LTR exists as an ordered array of six nucleosomes, one of which (Nuc-B) is associated with the promoter region containing binding sites for glucocorticoid receptor (GR) and nuclear factor 1 (NF1) (54). In the absence of activated GR, the MMTV chromatin template is found to be in a ''closed'' architecture, with the transcription factors NF1 and OTF1 largely occluded from their high-affinity binding sites (20,35). Upon treatment with glucocorticoids, the Nuc-B region undergoes a structural transition which results in an opening of chromatin structure, characterized by increased nuclease accessibility and the binding of NF1, OTF1, and the basal transcriptional machinery (4,20,35,54). These observations suggested a bimodal model of MMTV transcriptional activation (6), in which nucleoprotein structure limits the access of transcription factors to their sites on MMTV chromatin in the absence of hormone. This repression is ...

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

“…Cell line 1470.2 was derived from C127i mouse mammary adenocarcinoma cells through transformation with episomes containing the transforming (69%) fragment of the bovine papilloma virus (BPV) genome (Pennie et al, 1995). It contains multiple copies of MMTV-CAT (chloramphenicol acetyltransferase) transcription units in the context of BPV sequences.…”

“…Nuclei were isolated and digested with either SacI (10 U/mg DNA) or a combination of HaeIII (5 U/mg DNA) and l exonuclease (0.1 U/mg DNA) as described previously (Pennie et al, 1995). DNA was purified from digested nuclei by phenol/chloroform extraction and ethanol precipitation.…”

“…RNA isolation and S1 nuclease analysis RNA was isolated from 1470.2 cells as previously described (Pennie et al, 1995). Radiolabeled probes for S1 analysis were prepared by linear amplification of both MMTV and b-actin sequences using specific oligonucleotide primers and linearized template plasmids in the presence of a-[ 32 P]dATP.…”

“…Nuclei were isolated as described previously (Pennie et al, 1995) and frozen in liquid nitrogen. Run-on analysis was carried out as described (Pennie et al, 1995).…”

“…Radiolabeled probes for S1 analysis were prepared by linear amplification of both MMTV and b-actin sequences using specific oligonucleotide primers and linearized template plasmids in the presence of a-[ 32 P]dATP. S1 nuclease analysis was carried out as described previously (Pennie et al, 1995).…”