title: Identification of target genes for EWS/ATF-1 chimeric transcription factor.
abstract: Chromatin immunoprecipitation is a useful technique to detect in vivo direct interaction between any transcription factor and its binding site on genomic DNA. We applied this skill to identify the direct target gene for EWS/ATF-1 by coupling with a GFP reporter assay. This novel approach isolated 62 of cloned DNA fragments responding upon EWS/ATF-1 expression and 16 of 62 clones included putative ATF-1 binding sites. Further analysis revealed that six of the cloned fragments included possible regulatory regions of ATM, GPP34, ARNT2, NKX6.1, NYD-SP28 and POSH. Most of these clones upregulated reporter activity by overexpression of EWS/ATF-1, suggesting that putative ATF-1 binding sites in these clones are functional elements for ATF-1 in vivo. Consistently, endogenous expression of these genes was upregulated by EWS/ATF-1. Interestingly, the clone containing the promoter region of POSH, which is known to be a strong inducer of apoptosis, repressed reporter activity by overexpression of EWS/ATF-1. Correspondingly, EWS/ATF-1 expression decreased endogenous POSH expression, suggesting that six isolated genes may be involved in direct regulation by EWS/ATF-1. Moreover, induction of POSH brought apoptotic cell death to KAS, the clear cell sarcoma (CCS) cell line, suggesting that repressed expression of POSH in CCS may be relevant to the normal signaling pathway in apoptosis.
authors.affiliation: Department of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Jishage
authors.forename: Takashi
authors.initials: T
authors.lastname: Fujino
authors.forename: Yukari
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kuroda
authors.forename: Takuro
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D051697:Activating Transcription Factor 1; D000328:Adult; D017209:Apoptosis; D001483:Base Sequence; D004247:DNA; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D034802:RNA-Binding Protein EWS / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D051697:Activating Transcription Factor 1; D004268:DNA-Binding Proteins; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA