| National Cancer Institute | Laboratory of Receptor Biology and Gene Expression | National Institutes of Health |
| Signal Transduction Group |
Regulation of the MMTV Promoter by cAMP Signaling
Mouse mammary adenocarcinoma cells containing a stably-replicating form of an MMTV-CAT reporter gene are transfected with an MMTV beta-globin reporter construct and treated as shown in Fig. 1. RNA was isolated from the cells and subjected to S1 nuclease protection assay with probes specific for MMTV or actin transcripts. In the presence of the synthetic glucocorticoid, dexamethasone (Dex), both templates are activated. In the presence of both Dex and cAMP, the transfected MMTV template is synergistically activated, but the replicating template is repressed (compare lanes 3 and 5).
A series of in vivo chromatin experiments (Pennie et al., Mol. Cell Biol. 15: 2125-2134) have demonstrated that cAMP signaling blocks a distinct step in the mechanism by which the replicating MMTV template is activated by glucocorticoid receptor (GR) (Fig. 2). Remodeling of the B and C nucleosome regions,derepression, and GR-induced binding of NF1 are unaffected by cAMP signaling. However, the binding of Oct1 and the basal transcription machinery to the template are inhibited. Mutational analysis of transfected MMTV templates has shown that the Oct1 binding sites and the TATA box are both necessary for activated transcription. However, cAMP signaling does not repress activation of the transfected template so we conclude that the presence of ordered chromatin at the MMTV template imposes a different activation mechanism even after the template has been derepressed. Therefore the two templates are regulated in distinct ways.
Current studies are directed towards determining the targets, both intermediate and template-based, of the cAMP signaling pathway which leads to repression of MMTV transcription specifically in the context of ordered chromatin.
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