Oncogenic Activation of the Human Pygopus2 Promoter by E74-Like Factor-1

Abstract

Pygopus is a component of the T-cell factor/B-catenin transcriptional complex essential for activation of Wnt target genes and is also required for cell regulation in the absence of Wnt signaling. Human Pygopus2 (hPygo2) is overexpressed in a high proportion of breast and epithelial ovarian malignant tumors and is required for the growth of several cell lines derived from these carcinomas. The mechanisms regulating hPygo2 gene activation, however, are unknown. Here, we have determined cis-and trans-interacting factors responsible for hPygo2 expression in cancer. The minimal region required for a maximal 109-fold activation of the hPygo2 promoter in MCF-7 breast cancer cells is 48 bp upstream of the start of transcription. Within 25 bp of the transcriptional start, there are two overlapping tandem Ets transcription factor -binding sites, which are critical for hPygo2 promoter activity. In vitro DNA pull-down assays and proteomic analyses identified the Ets family members Elk-1 and E74-like factor-1 (Elf-1) as potential hPygo2 promoter binding factors, whereas in vivo chromatin immunoprecipitation assays verified that only Elf-1 specifically bound to the hPygo2 promoter in MCF-7 cells. Modulation of elf-1 in MCF-7 cells by silencing via RNA interference or overexpression caused a corresponding decrease or increase, respectively, in hPygo2 promoter activity. Overexpression of Elf-1 in HeLa cells, in which Elf-1 is expressed at a lower level than in MCF-7 cells, caused a 4-fold increase in endogenous hPygo2 mRNA levels. These results provide new evidence that Elf-1 is involved in transcriptional activation of hPygo2. Like hPygo2, previous studies implicated Elf-1 in breast and ovarian cancer and our present findings suggest that the oncogenic requirement of hPygo2 is fulfilled, in part, by Elf-1. (Mol Cancer Res 2008;6(2):259 -66)

Human Papilloma Virus (HPV) E7-Mediated Attenuation of Retinoblastoma (Rb) Induces hPygopus2 Expression via Elf-1 in Cervical Cancer

“…cDNA samples were subjected to real-time quantitative PCR using RT2 SYBR green master mix (SA Biosciences; ref. 7) and analyzed by the relative quantitative comparative threshold cycle (DDC t ) method (3). Primer sequences are listed in Supplementary Table S5.…”

“…Chromatin immunoprecipitation (ChIP) assays were conducted as previously described (7). Cross-linked cells were and sonicated to produce 500 bp genomic DNA fragments.…”

“…pCS2þ was used as the empty vector control for Elf-1 plasmids. hPygo2 promoter constructs: pGL3-1494 antisense, pGL3-1494, pGL3-48, and pGL3-basic were used previously (7). Site-directed mutagenesis to generate the dominant-active (DA) Rb binding-deficient Elf-1 mutant, pCS2 þ -DA-Elf-1, using sequences in Wang and colleagues (21), and the Etsbinding site mutant, pGL3-48 mutEBS was conducted using the QuickChange Kit (Stratagene; primers listed in Supplementary Table S5).…”

“…Our previous work showed that the oncogenic v-Ets family transcription factor E74-like factor 1 (Elf-1) bound to the hPygo2 promoter and could promote its expression in MCF-7 breast cancer cells (7). We therefore used RNAi to determine whether Elf-1 was involved in hPygo2-dependent cervical cancer cell proliferation.…”

“…7), an E26 transformation-specific (ETS) family member. ETS factors have well-established roles in carcinogenesis, regulating oncogenes and tumor suppressors involved in apoptosis, angiogenesis, invasion, and metastasis (8).…”