Myc interacts with Max and Miz1 to repress C/EBPdelta promoter activity and gene expression.
BACKGROUND
"Loss of function" alterations in CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPdelta gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPdelta exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing. We previously reported that C/EBPdelta 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/EBPalpha, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPdelta expression.


RESULTS
Myc represses C/EBPdelta 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/EBPdelta promoter in proliferating cells, when C/EBPdelta expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPdelta promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPdelta promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPdelta promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPdelta promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPdelta expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPdelta promoter activity.


CONCLUSIONS
Myc represses C/EBPdelta expression by associating with the C/EBPdelta proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPdelta promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPdelta, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
Si
Junling
J
Department of Veterinary Biosciences, Ohio State University College of Veterinary Medicine, Columbus, Ohio 43210, USA.
Yu
Xueyan
X
Zhang
Yingjie
Y
DeWille
James W
JW
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D051743:CCAAT-Enhancer-Binding Protein-delta / Q000096:biosynthesis* / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000235:genetics*; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D051741:Kruppel-Like Transcription Factors; C106465:MNT protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C107961:ZBTB17 protein, human; D051743:CCAAT-Enhancer-Binding Protein-delta