JAC , a direct target of oncogenic transcription factor Jun, is involved in cell transformation and tumorigenesis

Abstract

Using subtractive hybridization techniques, we have isolated a gene termed JAC that is strongly and specifically activated in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore, JAC is highly expressed in cell lines derived from jun-induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb JAC mRNA is induced rapidly upon activation of the oncogenic v-jun allele. Nucleotide sequence analysis and transcriptional mapping revealed that the JAC gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian highsulfur keratin-associated proteins. The promoter region of JAC contains a consensus (5-TGACTCA-3) and a nonconsensus (5-TGAGTAA-3) AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. Mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of JAC in avian fibroblasts leads to anchorageindependent growth, strongly suggesting that deregulation of JAC is an essential event in jun-induced cell transformation and tumorigenesis.jun oncogene ͉ signal transduction ͉ gene expression ͉ protein-DNA interaction ͉ transcriptional control

WS5, a direct target of oncogenic transcription factor Myc, is related to human melanoma glycoprotein genes and has oncogenic potential

“…Cell culture and transformation of CEF or QEF was carrired out as described (Bister et al, 1977;Oberst et al, 1999;Bister, 1995, 1998;Bader et al, 2000;Hartl et al, 2001). The following established lines of transformed QEF (Bister et al, 1977;Hartl et al, 2003) were used: VJ, transformed by ASV17 v-jun; Q8, transformed by MC29 v-myc; R(-)3, transformed by RSV v-src; and QT6, derived from a methylcholanthrene-induced fibrosarcoma.…”

“…For construction of RCAS-WS5, a cDNA fragment containing the entire WS5 coding region was ligated into the ClaI site of the RCAS-BP vector. DNA transfection and colony assays of transfected cells in 0.3%(w/v) agarose were done as described (Bister et al, 1977;Oberst et al, 1999;Hartl et al, 2001). …”

“…WS5, a direct transcriptional target of Myc F Reiter et al DNA cloning and nucleic acid analysis cDNA and genomic DNA library construction, subtractive hybridization, DNA labeling, subcloning, Northern and Southern analyses, and nucleotide sequence analysis were performed as described Bister, 1995, 1998;Hartl et al, 2001Hartl et al, , 2003. Screening of a Q8-specific cDNA library with the 919-bp insert of a clone isolated from a Q8-QEF subtractive cDNA library and encompassing the 3 0 -end of WS5 cDNA (Oberst et al, 1999) led to the isolation of a 1398-bp 3 0 cDNA clone.…”

“…In vitro transcription/translation reactions were done as described (Hartl et al, 2001). To construct pBS-WS5, the ClaI fragment of pA-WS5 containing the WS5 coding region was inserted into the ClaI site of pBluescript II SK( þ ) (Stratagene Inc., Amsterdam, Netherlands).…”

“…A 13mer peptide (NH 2 -SGESSPLLRANAV-COOH) corresponding to the C-terminal residues of the WS5 protein was used to generate a rabbit polyclonal WS5-specific antiserum. Immunoprecipitations of [ 35 S]methionine-labeled proteins from cell lysates or from in vitro transcription/translation reactions, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described (Hartl et al, 2001(Hartl et al, , 2003. Co-immunoprecipitation of Myc-C/EBP protein complexes from lysates of [ 35 S]methionine-labeled cells using antisera directed against a C-terminal recombinant v-Myc protein (see below), or against recombinant C/EBPa or C/ EBPb proteins (provided by K-H Klempnauer; Mink et al, 1999) was as described (Hartl et al, 2003).…”