Characterization of the repression of glucocorticoid-induced transcription of the mouse mammary tumor virus through negative regulatory element 1.

Abstract

The physiological effects of steroid hormones are mediated by their cognate receptors which regulate expression of target genes in response to binding of their steroid ligand. The mouse mammary tumor virus (MMTV) promoter is a widely used system for studying the effects of steroid hormones due to its strong activation by several classes of steroids. MMTV induces mammary tumors in female mice following lactation. T-cell lymphomas are also induced at much lower frequency and all cases of lymphoma that have been examined have deletions spanning a region of the long terminal repeat (LTR) from -421 to -364. This region has a transcriptional negative regulatory element called NRE1. Characterization of NRE1 revealed nuclear factors binding to the double-stranded (ds) form, as well as each of the single strands (up, lo - upper and lower strands respectively). A truncated element (MT) was also recognized but only the ds form. Kinetic studies showed binding occurred to the ds, up, to and MT with t1/2s of 11, 1.5, 3 and 4 min respectively. The off-rates (t1/2) were determined to be 60 min, 30 min, 12 h and 4 h respectively. Four factors were observed to bind NRE1 in southwestern analysis, while only one was observed binding to MT, that corresponded in mobility to the smallest NRE1 binding factor. The four factors appeared to cross-react with an octamer motif. The dsNRE1 binding factor was confirmed to recognize an octamer motif in EMSA. The dsNRE1 binding factor was identified as the Ku autoantigen heterodimer. It was demonstrated to bind directly to NRE1 with an affinity of Kd = 0.84 +/- 0.24 mum. Ku also bound upNRE1 with an affinity of K d = 3.5 +/- 1.3 mM. In kinetic analyses of purified Ku binding to upNRE1 the on-rate was determined to be t1/2 = 1.6 min, while the off rate was t1/2 = 68 min. Transient transfection assays using MMTV reporters either containing or lacking NRE1 were performed. Using cell lines not expressing functional Ku or its associated factor DNA-PK it was shown that both factors were required for the NRE1 mediated repression of glucocorticoid-induced transcription of MMTV. DNA-PK was shown to specifically phosphorylate a myc-tagged glucocorticoid receptor (GR) on serine 527 (5527) in vitro. A second phosphorylation site was determined to result from the addition of the myc tag, while a native receptor was phosphorylated only on S527. Transient transfections using 5527 mutants of GR demonstrated that substitution of S527 for alanine abrogated NRE1 mediated repression. The same effect was observed for glutamic acid and aspartic acid substitutions. We therefore conclude that binding of DNA-PK to NRE1 through its DNA binding subunit, Ku, allows it to phosphorylate GR on serine 527 resulting in a repression of glucocorticoid-induced transcription.