CCAAT/Enhancer Binding Protein-delta (C/EBP-delta) regulates cell growth, migration and differentiation

<p>Abstract</p> <p>Background</p> <p>CCAAT/enhancer binding protein-delta (C/EBP-delta) is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. C/EBP family members regulate cell growth and differentiation and "loss of function" alterations in C/EBPs have been reported in a variety of human cancers. C/EBP-delta gene expression is upregulated by G₀growth arrest, IL-6 family cytokines and endotoxin treatments. C/EBP-delta exhibits properties of a tumor suppressor gene, including reduced expression and promoter methylation-induced silencing in transformed cell lines and primary tumors. In addition, C/EBP-delta gene expression is repressed by c-Myc, an oncogene that is over-expressed in a wide range of human cancers. "ChIP-chip" studies demonstrated that C/EBP-delta functions as a transcriptional activator of target genes that function in intracellular signal transduction, transcription, DNA binding/repair, cell cycle control, cell adhesion, and apoptosis. Despite progress in determining the biochemical functions of C/EBP-delta, the specific cellular defects that are induced by C/EBP-delta "loss of function" alterations are poorly understood. This study investigated the impact of C/EBP-delta "loss of function" alterations on growth arrest, migration/invasion and differentiation in nontransformed mouse mammary epithelial cells (MECs) and primary mouse embryo fibroblasts (MEFs).</p> <p>Results</p> <p>C/EBP-delta siRNA transfected MECs exhibited ~90% reduction in C/EBP-delta mRNA and protein levels. C/EBP-delta siRNA treatment resulted in defective growth arrest as demonstrated by persistently elevated BrdU labeling, ³H-thymidine incorporation and cyclin D1 levels in response to growth arrest treatments. C/EBP-delta siRNA treatment also resulted in increased migration/invasion and defective differentiation. C/EBP-delta knockout MEFs exhibited defective growth arrest and increased proliferation/migration. Re-introduction of C/EBP-delta expression restored the growth arrest response of C/EBP-delta knockout MEFs. Finally, deletion of the C/EBP-delta DNA binding domain or the C/EBP-delta bZIP domain resulted in the loss of C/EBP-delta growth inhibition in clonogenic assays.</p> <p>Conclusions</p> <p>This study demonstrates that C/EBP-delta functions in the regulation of critical cell fate determining programs such as growth arrest, migration, and differentiation. These results support the tumor suppressor function of C/EBP-delta and identify potential mechanisms in which "loss of function" alterations in C/EBP-delta could promote cell transformation and tumorigenesis.</p

Myc interacts with Max and Miz1 to repress C/EBPδ promoter activity and gene expression

<p>Abstract</p> <p>Background</p> <p>"Loss of function" alterations in CCAAT/Enhancer Binding Proteinδ (C/EBPδ) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPδ gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPδ exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing.</p> <p>We previously reported that C/EBPδ expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPα, p15^{<it>INK</it>4}, p21^{<it>CIP</it>1}, p27^{<it>KIP</it>1}and p57^{<it>KIP</it>2}. This study investigated the mechanisms underlying Myc repression of C/EBPδ expression.</p> <p>Results</p> <p>Myc represses C/EBPδ promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPδ promoter in proliferating cells, when C/EBPδ expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPδ promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPδ promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPδ promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPδ promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPδ expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPδ promoter activity.</p> <p>Conclusion</p> <p>Myc represses C/EBPδ expression by associating with the C/EBPδ proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPδ promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPδ, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.</p

Proteasome-mediated CCAAT/enhancer-binding protein delta (C/EBPdelta) degradation is ubiquitin-independent

C/EBPδ (CCAAT/enhancer-binding protein δ) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPδ gene transcription is highly induced in G 0 growth-arrested mammary epithelial cells but the C/EBPδ protein exhibits a t ½ of only ∼ 120 min. The goal of the present study was to investigate the role of C/EBPδ modification by ubiquitin and C/EBPδ proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPδ ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPδ ubiquitination is not required for proteasome-mediated C/EBPδ degradation and the presence of ubiquitin does not increase C/EBPδ degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPδ protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPδ ubiquitination we treated G 0 growth-arrested mammary epithelial cells with DNA-damage-and oxidativestress-inducing agents and found that C/EBPδ ubiquitination is induced in response to H 2 O 2 . However, C/EBPδ protein stability is not influenced by H 2 O 2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPδ is ubiquitin-independent

Proteasome-mediated CCAAT/enhancer-binding protein δ (C/EBPδ) degradation is ubiquitin-independent

C/EBPδ (CCAAT/enhancer-binding protein δ) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPδ gene transcription is highly induced in G0 growth-arrested mammary epithelial cells but the C/EBPδ protein exhibits a t1/2 of only ∼120 min. The goal of the present study was to investigate the role of C/EBPδ modification by ubiquitin and C/EBPδ proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPδ ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPδ ubiquitination is not required for proteasome-mediated C/EBPδ degradation and the presence of ubiquitin does not increase C/EBPδ degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPδ protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPδ ubiquitination we treated G0 growth-arrested mammary epithelial cells with DNA-damage- and oxidative-stress-inducing agents and found that C/EBPδ ubiquitination is induced in response to H2O2. However, C/EBPδ protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPδ is ubiquitin-independent