STAT3-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the α2-macroglobulin gene

Abstract

We describe a detailed time course of the assembly and disassembly of a STAT3-dependent, glucocorticoid-supplemented enhanceosome for the α2-macroglobulin (α2-M) gene and compare this with a detailed time course of transcription of the gene by run-on analysis. The glucocorticoid receptor (GR) can associate with the enhanceosome without STAT3. Furthermore, the enhanceosome contains c-Jun/c-Fos and OCT-1 constitutively. All of these factors (GR, c-Jun, OCT-1) have transcription activation domains, but STAT3 is required for the observed transcriptional increase. The time course of enhanceosome occupation by GR and tyrosine-phosphorylated STAT3 shows that these transcription factors precede by ∼5-10 min the arrival of RNA polymerase II (Pol II). The enhanceosome remains assembled for ∼90 min in the continued presence of both inducers. When IL-6 and Dex are removed (after 30 min of treatment), the disappearance within an additional 30 min of the established enhanceosome indicates that renewal of STAT3 and GR binding must occur in the continued presence of IL-6+Dex. Compared with the total nuclear tyrosine-phosphorylated STAT3 capable of binding DNA, the chromatin-associated STAT3 resists dephosphorylation and appears to recycle to maintain the enhanceosome. Run-on transcription shows a lag after full enhanceosome occupation that can be largely but not completely explained by the ∼30 min transit time of Pol II across the α2-Mlocus.