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

CCAAT/enhancer binding proteinδ (C/EBPδ) gene transcription is highly induced in G0 growth arrested mammary epithelial cells and “loss of function” alterations in C/EBPδ have been reported in human breast cancer. To gain a better understanding of the positive and negative factors that control C/EBPδ 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 HC11 mouse mammary epithelial cells. Growth arrest treatments result in increased STAT3 activation (pSTAT3) and increased C/EBPδ expression. Co‐immunoprecipitation and chromatin immunoprecipitation (ChIP) assays demonstrated that pSTAT3 and Sp1 interact and bind to the transcriptionally active C/EBPδ promoter. ChIP assays performed under exponentially growing (C/EBPδ non‐expressing) conditions demonstrated that the C/EBPδ promoter is preloaded with transcriptional activators (Sp1 and CREB) and transcriptional machinery components (TBP and RNA Pol II). In contrast, under G0 growth arrest (C/EBPδ 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/EBPδ proximal promoter. C/EBPδ 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/EBPδ transcription. Finally, C/EBPδ 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/EBPδ promoter‐bound Miz‐1. These results provide a molecular model of C/EBPδ transcriptional regulation under G0 growth arrest conditions. J. Cell. Biochem. 102: 1256–1270, 2007. © 2007 Wiley‐Liss, Inc.

[1]  R. Kingston,et al.  Purification and characterization of mSin3A-containing Brg1 and hBrm chromatin remodeling complexes. , 2001, Genes & development.

[2]  J. Dewille,et al.  CCAAT/enhancer binding protein δ (C/EBPδ) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post‐transcriptional, and post‐translational control , 2004, Journal of cellular biochemistry.

[3]  S. Gasser,et al.  Long-range silencing and position effects at telomeres and centromeres: parallels and differences , 2003, Cellular and Molecular Life Sciences CMLS.

[4]  R. Gibbons.  Histone modifying and chromatin remodelling enzymes in cancer and dysplastic syndromes. , 2005, Human molecular genetics.

[5]  M. Eilers,et al.  Transcriptional regulation and transformation by Myc proteins , 2005, Nature Reviews Molecular Cell Biology.

[6]  A. Jacobsson,et al.  Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region. , 1995, European journal of biochemistry.

[7]  R. Tjian,et al.  Transcriptional coactivator complexes. , 2001, Annual review of biochemistry.

[8]  R. Roeder,et al.  Transcriptional regulation and the role of diverse coactivators in animal cells , 2005, FEBS letters.

[9]  J. Darnell,et al.  Stat3 as an Oncogene , 1999, Cell.

[10]  T. Kouzarides,et al.  Myc represses transcription through recruitment of DNA methyltransferase corepressor , 2005, The EMBO journal.

[11]  J. Dewille,et al.  C/EBPδ is a downstream mediator of IL‐6 induced growth inhibition of prostate cancer cells , 2005 .

[12]  C. Vinson,et al.  Classification of Human B-ZIP Proteins Based on Dimerization Properties , 2002, Molecular and Cellular Biology.

[13]  Carl W. Miller,et al.  C/EBP-β, C/EBP-δ, PU.1, AML1 genes: mutational analysis in 381 samples of hematopoietic and solid malignancies , 2002 .

[14]  Matthew W. Strobeck,et al.  Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs CD44 expression. , 2002 .

[15]  D. Levens,et al.  Marking of active genes on mitotic chromosomes , 1997, Nature.

[16]  Danny Reinberg,et al.  Elongation by RNA polymerase II: the short and long of it. , 2004, Genes & development.

[17]  Daniel C Sanford,et al.  C/EBPdelta is a downstream mediator of IL-6 induced growth inhibition of prostate cancer cells. , 2005, The Prostate.

[18]  A. Gartel,et al.  Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  S. Henikoff,et al.  Human centromeric chromatin is a dynamic chromosomal domain that can spread over noncentromeric DNA. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Weis,et al.  Interleukin‐6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1 , 2005, International journal of cancer.

[21]  J. O’Rourke,et al.  Signal Transducer and Activator of Transcription 3 Activates CCAAT Enhancer-binding Protein δ Gene Transcription in G0Growth-arrested Mouse Mammary Epithelial Cells and in Involuting Mouse Mammary Gland* , 2000, The Journal of Biological Chemistry.

[22]  S. Sif.  ATP‐dependent nucleosome remodeling complexes: Enzymes tailored to deal with chromatin , 2004, Journal of cellular biochemistry.

[23]  J. O’Rourke,et al.  Transcriptional Regulation of C/EBPδ in G0 Growth-Arrested Mouse Mammary Epithelial Cells , 1999 .

[24]  C. Mayhew,et al.  Mechanism of hsp70i Gene Bookmarking , 2005, Science.

[25]  J. Dewille,et al.  Promoter methylation reduces C/EBPδ (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors , 2005, Breast Cancer Research and Treatment.

[26]  M. Nagao,et al.  Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit* , 1997, The Journal of Biological Chemistry.

[27]  E. Kalkhoven,et al.  CBP and p300: HATs for different occasions. , 2004, Biochemical pharmacology.

[28]  H. Gascan,et al.  Functional Interaction of STAT3 Transcription Factor with the Coactivator NcoA/SRC1a* , 2002, The Journal of Biological Chemistry.

[29]  T. Lee,et al.  Myc represses transcription of the growth arrest gene gas1. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Dewille,et al.  Oncostatin M Induces Growth Arrest of Mammary Epithelium via a CCAAT/enhancer-binding Protein δ-dependent Pathway1Supported in part by NIH Grant RR00136CA (to J. A. H.) and NIH Grants CA 57607 and P30CA16058 from the National Cancer Institute (to J. W. D.).1 , 2002 .

[31]  J. Dewille,et al.  Oncostatin M induces growth arrest of mammary epithelium via a CCAAT/enhancer-binding protein delta-dependent pathway. , 2002, Molecular cancer therapeutics.

[32]  N. Copeland,et al.  Mouse chromosomal location of the CCAAT/enhancer binding proteins C/EBPβ (Cebpb), C/EBPδ (Cebpd), and CRP1 (Cebpe) , 1995 .

[33]  Matthew W. Strobeck,et al.  Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs CD44 expression , 2002, Oncogene.

[34]  A. Gartel,et al.  Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes. , 2003, Experimental cell research.

[35]  Peter F. Johnson,et al.  Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors , 2005, Journal of Cell Science.

[36]  Y. Oshika,et al.  P-glycoprotein-mediated acquired multidrug resistance of human lung cancer cells in vivo. , 1996, British Journal of Cancer.

[37]  J. Dewille,et al.  CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines. , 2004, Journal of Cellular Biochemistry.

[38]  R. Jove,et al.  Stat3 regulates genes common to both wound healing and cancer , 2005, Oncogene.

[39]  J. Williams,et al.  Changes in RNA in relation to growth of the fibroblast. IV. Alterations in theproduction and processing of mRNA and rRNA in resting and growing cells , 1976, The Journal of cell biology.

[40]  Shuh Narumiya,et al.  Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins β and -δ in preadipocytes , 2001 .

[41]  J. Massagué,et al.  Repression of p15INK4b expression by Myc through association with Miz-1 , 2001, Nature Cell Biology.

[42]  Peter F. Johnson,et al.  Interleukin-6-Specific Activation of the C/EBPδ Gene in Hepatocytes Is Mediated by Stat3 and Sp1 , 1998, Molecular and Cellular Biology.

[43]  D. W. Kim,et al.  Repression of transcription of the p27Kip1 cyclin-dependent kinase inhibitor gene by c-Myc , 2001, Oncogene.

[44]  K. Sarge,et al.  Gene bookmarking: keeping the pages open. , 2005, Trends in biochemical sciences.

[45]  R. Kingston,et al.  Cooperation between Complexes that Regulate Chromatin Structure and Transcription , 2002, Cell.

[46]  J. Dewille,et al.  Detection of base sequence changes in the CEBPD gene in human breast cancer cell lines and primary breast cancer isolates. , 2003, Molecular and cellular probes.

[47]  E. Ainbinder,et al.  Mechanism of Rapid Transcriptional Induction of Tumor Necrosis Factor Alpha-Responsive Genes by NF-κB , 2002, Molecular and Cellular Biology.

[48]  Carl W. Miller,et al.  C/EBP-beta, C/EBP-delta, PU.1, AML1 genes: mutational analysis in 381 samples of hematopoietic and solid malignancies. , 2002, Leukemia research.

[49]  C. Dani,et al.  Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins beta and -delta in preadipocytes. , 2001, Molecular endocrinology.

[50]  M. Eilers,et al.  Transcriptional repression by Myc. , 2003, Trends in cell biology.

[51]  J. O’Rourke,et al.  Transcriptional regulation of C/EBPdelta in G(0) growth-arrested mouse mammary epithelial cells. , 1999, Biochemical and biophysical research communications.

[52]  J. O’Rourke,et al.  CCAAT/Enhancer-binding Protein-δ (C/EBP-δ) Is Induced in Growth-arrested Mouse Mammary Epithelial Cells* , 1997, The Journal of Biological Chemistry.

[53]  L. Penn,et al.  c-Myc represses the proximal promoters of GADD45a and GADD153 by a post-RNA polymerase II recruitment mechanism , 2004, Oncogene.

[54]  D. Ramji,et al.  CCAAT/enhancer-binding proteins: structure, function and regulation. , 2002, The Biochemical journal.