Identification and characterization of CCAAT/Enhancer Binding proteindelta (C/EBPdelta) target genes in G₀ growth arrested mammary epithelial cells

Background: CCAAT/Enhancer Binding Proteinδ (C/EBPδ) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPδ is highly expressed in G₀ growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPδ have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPδ expression has also been reported in breast cancer and acute myeloid leukemia (AML). C/EBPδ functions as a transcriptional activator, however, only a limited number of C/EBPδ target genes have been reported. As a result, the role of C/EBPδ in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPδ contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPδ target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K) Array gene chip ("ChIP-chip") assay and to assess the expression and potential functional roles of C/EBPδ target genes in growth control. Results: ChIP-chip assays identified ∼100 C/EBPδ target gene loci which were classified by gene ontology (GO) into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPδ target loci were bound by C/ EBPδ in G₀ growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPδ-inducible expression of 14/14 C/EBPδ target genes in G₀ growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPδ and selected C/EBPδ target genes was also demonstrated in contact-inhibited G₀ growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPδ in growth arrested human and murine MECs. Conclusion: C/EBPδ target genes were identified by a global gene array approach and classified into functional categories that are consistent with biological contexts in which C/EBPδ is induced, such as contact-mediated G₀ growth arrest, apoptosis, metabolism and inflammation. The identification and validation of C/EBPδ target genes provides new insights into the mechanistic role of C/EBPδ in mammary epithelial cell biology and sheds new light on the potential impact of "loss of function" alterations in C/EBPδ in tumorigenesis.