The mouse C/EBPδ gene promoter is regulated by STAT3 and Sp1 transcriptional activators, chromatin remodeling and c‐Myc repression

Abstract

CCAAT/enhancer binding proteindelta (C/EBPdelta) gene transcription is highly induced in G(0) growth arrested mammary epithelial cells and "loss of function" alterations in C/EBPdelta have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBPdelta gene expression we investigated the role of transcriptional activators, coactivators, repressors, histone modifications, chromatin remodeling and basal transcriptional machinery components in growing and growth arrested HC11 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activation (pSTAT3) and increased C/EBPdelta expression. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBPdelta promoter. ChIP assays performed under exponentially growing (C/EBPdelta non-expressing) conditions demonstrated that the C/EBPdelta promoter is preloaded with transcriptional activators (Sp1 and CREB) and transcriptional machinery components (TBP and RNA Pol II). In contrast, under G(0) growth arrest (C/EBPdelta expressing) conditions ChIP analysis detected pSTAT3, Sp1, NCoA/SRC1, CBP/p300, pCREB, TBP, and serine 2 phosphorylated Pol II (pPol II) in association with the C/EBPdelta proximal promoter. C/EBPdelta promoter-associated histone post-translational modification analysis revealed histone H3 and H4 acetylation and methylation patterns consistent with a constitutively "open" chromatin conformation. Chromatin remodeling experiments demonstrated that BRG1, the ATPase component of the SWI/SNF chromatin remodeling complex, is required for C/EBPdelta transcription. Finally, C/EBPdelta expression is repressed in proliferating mammary epithelial cells by c-Myc via a mechanism that involves the binding of c-Myc:Max dimers to C/EBPdelta promoter-bound Miz-1. These results provide a molecular model of C/EBPdelta transcriptional regulation under G(0) growth arrest conditions.