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CCAAT/Enhancer Binding Protein Delta (C/EBPD) gene transcription is highly induced in G0 growth arrested mammary epithelial cells and “loss of function” alterations in C/EBPD have been reported in human breast cancer. This work investigated three aspects of C/EBPD the mechanisms of transcriptional regulation of C/EBPD gene, genes directly regulated by C/EBPD, and the phenotypes of loss of C/EBPD function. In chapter 2, we showed that C/EBPD promoter is maintained in a constitutively “open” chromatin conformation and preloaded with transcriptional activators (Sp1 and CREB) and pre-initiation complex components (TBP and RNA Pol II). However its activation is dependent on binding of pSTAT3 under growth arrest plus Oncostatin M treatment, which results in recruitment of coactivators (NCoA/SRC1 and CBP/p300), phosphorylation of CREB and RNA Pol II (pPolII) and active transcription of C/EBPD gene. We also demonstrated that C/EBPD 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/EBPD promoter-bound Miz-1. In chapter 3, we used the ChIP-chip technique to globally screen for C/EBPD protein binding genomic targets. Human 12k CGI microarray was hybridized with DNA recovered from C/EBPD-v5 ChIP assays under contact inhibition condition of MCF-12A cells. We identified 289 C/EBPD-v5 bound loci and 102 of them are linked to protein coding genes. 14 candidate genes were validated to be regulated by C/EBPD-v5 using conventional ChIP analysis and reverse transcription PCR assays. We reported the first time that C/EBPD plays a role on contact inhibition by regulating extracellular adhesion proteins and their downstream signaling components. We also identified 12 tumor suppressors and growth inhibitors in this study as novel C/EBPD direct targets suggesting C/EBPD’s role on tumor suppression. In chapter 4, we characterized the phenotypes of C/EBPD -/- MEFs related to the potential tumor suppression role of C/EBPD. C/EBPD -/- MEFs showed decrease in contact inhibition and cell-cell adherence at high cell density due to lacking C/EBPD expression. In addition the loss of C/EBPD enhances cell migration, which implies to C/EBPD’s role on inhibiting tumor metastasis. The work of dissertation opens new avenues of research of C/EBPD related to cancer development. |
