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

CCAAT/enhancer binding protein delta (C/EBP delta) gene transcription is highly induced in Go growth arrested mammary epithelial cells and loss of function alterations in C/EBP delta have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBP delta 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 HCl 1 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activition (pSTAT3) and increased C/EBP delta expression. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBP delta promoter. ChIP assays performed under exponentially growing (C/EBP delta non-expressing) conditions demonstrated that the C/EBP delta 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/EBP delta 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/EBP delta proximal promoter. C/EBP delta 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/EBP delta transcription. Finally, C/EBP delta 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/EBP delta promoter-bound Miz-1. These results provide a molecular model of C/EBP delta transcriptional regulation under G(0) growth arrest conditions.