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

Abstract

We have isolated a gene (WS5) that is specifically expressed at the mRNA and protein level in avian fibroblasts transformed by the v-myc oncogene of avian acute leukemia virus MC29. In a conditional cell transformation system, WS5 gene expression was tightly correlated with v-myc activation. The WS5 gene contains 11 exons, encoding a 733-amino acid protein with a transmembrane region and a polycystic kidney disease (PKD) domain. Near the transcriptional start site, the WS5 promoter contains a cluster of four binding sites for the Myc-Max complex and a binding site for transcription factor C/EBPa. Electrophoretic mobility shift assays and chromatin immunoprecipitation showed that Myc, Max and C/EBPa bind specifically to these sites. Functional promoter analyses revealed that both the Myc-binding site cluster and the C/EBPa-binding site are essential for strong transcriptional activation, and that Myc and C/EBPa synergistically activate the WS5 promoter. Ectopic expression of WS5 led to cell transformation documented by anchorage-independent growth. The human melanoma antigen Pmel17, a type I transmembrane glycoprotein, is the mammalian protein with the highest amino acid sequence identity (38%) to WS5. The Pmel17 gene is regulated by the MITF protein, a bHLHZip transcription factor with DNA binding specificities similar to those of Myc/Max. WS5 is also related to human glycoprotein GPNMB expressed in metastatic melanoma cells and implicated in the progression of brain and liver tumors.

v-Myc inhibits C/EBPβ activity by preventing C/EBPβ-induced phosphorylation of the co-activator p300

“…We have shown earlier that v-Myc suppresses the activity of C/EBPb and down-regulates C/EBPb target genes in myelomonocytic cells (Mink et al, 1996). The work described here shows that v-Myc interacts with C/EBPb, thereby confirming and extending earlier work which showed coprecipitation of Myc and C/EBPb from cell extracts (Tinel et al, 2003;Reiter et al, 2007). Furthermore, our work suggests that v-Myc inhibits C/EBPb activity by perturbing the cooperation of C/EBPb and p300.…”

“…The co-precipitation of proteins in untransfected cells was specific as shown by the control precipitation with nonimmune serum (last two lanes of Figure 1c). This experiment confirms earlier results obtained with v-Myc transformed quail fibroblasts (Reiter et al, 2007). S-methionine and subjected to sequential immunoprecipitation as described above.…”

Inhibition of Myc-induced cell transformation by brain acid-soluble protein 1 (BASP1)

“…Normal QEF or QEF/RCAS-BASP1 cells did not contain WS5 or Q83 mRNAs, whereas high levels were found in the Myc-transformed QEF/ RCAS-MC29 cells, in agreement with previous reports (19,22). Cells transfected with the bicistronic construct pRCAS-MC29-IRES-BASP1 contained no or low levels of WS5 or Q83 mRNAs, indicating an inhibitory effect of BASP1 on target gene activation by Myc (Fig.…”

“…We have previously established avian cell lines conditionally transformed by doxycycline-controlled v-myc alleles (18), displaying differential gene expression of putative Myc target genes like the strongly-activated WS5 gene that was fully characterized recently (19). One of the original partial cDNA clones (VCB2) isolated in these screens corresponded to a gene that was strongly suppressed in Myc-transformed quail embryo fibroblasts (QEFs) (18).…”

“…To test whether ectopic BASP1 interferes with the activation of direct Myc target genes like WS5 or Q83 (19,22), QEFs were transfected with pRCAS-MC29, pRCAS-MC29-IRES-BASP1, or pRCAS-BASP1. Normal QEF or QEF/RCAS-BASP1 cells did not contain WS5 or Q83 mRNAs, whereas high levels were found in the Myc-transformed QEF/ RCAS-MC29 cells, in agreement with previous reports (19,22).…”