Protein Kinase A-Dependent Derepression of the Human Prodynorphin Gene via Differential Binding to an Intragenic Silencer Element

Abstract

Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5 deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position ؉40 in the 5 untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

Transcriptional Repressor DREAM Interacts with Thyroid Transcription Factor-1 and Regulates Thyroglobulin Gene Expression

“…In keeping with previous reports (17,18), DREAM mRNA or protein was not detected in HeLa cells (Fig. 1, A and B).…”

“…DREAM protein in FRTL-5 cells are comparable to the levels in NB69 cells, the human neuroblastoma cell line where DREAM activity was first characterized (18). In keeping with previous reports (17,18), DREAM mRNA or protein was not detected in HeLa cells (Fig.…”

“…The Tg DRE sequence is: 5Ј-AAAGTGAGCCACTGCCCAGTCAAGTGTTCTTGA-3Ј. Oligonucleotides containing c-fosDRE or Sp1 have been previously described (17,18). For supershift experiments the extracts were incubated for 1 h at room temperature in the presence of 1 g of anti-Pax-8 (Santa Cruz Biotechnology), anti-TTF-1 (Biopat), or anti-DREAM (Ab 1013) antibodies and then incubated for 20 min with 80,000 cpm of probe in 20 l of final volume.…”

“…3. pBLCAT2, pTKDRECAT, pDRETKCAT, pHD1Luc, and pHD1mDRELuc have been described elsewhere (17,18). RSV-Luc and pRL-CMV were used to correct for transfection efficiency.…”

“…DREAM was identified through its binding to the downstream regulatory element (DRE) in the prodynorphin gene (17,18). Binding of DREAM to DRE sequences is regulated by the level of nuclear Ca 2ϩ (17), by the interaction with other nucleoproteins (19), and by the PI 3-kinase pathway (20).…”