Identification of a DNA-binding site and transcriptional target for the EWS–WT1(+KTS) oncoprotein

Abstract

Desmoplastic small round cell tumor (DSRCT) is defined by a chimeric transcription factor, resulting from fusion of the N-terminal domain of the Ewing's sarcoma gene EWS to the three C-terminal zinc fingers of the Wilms' tumor suppressor WT1. Although DNA-binding sites have been defined for the uninterrupted WT1 zinc finger domains, the most prevalent isoforms of both WT1 and EWS-WT1 have an insertion of three amino acids [lysine, threonine, and serine (KTS)], which abrogates binding to known consensus sequences and transactivation of known target genes. Here, we used cDNA subtractive hybridization to identify an endogenous gene, LRRC15, which is specifically up-regulated after inducible expression of EWS-WT1(+KTS) in cancer cell lines, and is expressed within primary DSRCT cells. The chimeric protein binds in vitro and in vivo to a specific element upstream of LRRC15, leading to dramatic transcriptional activation. Mutagenesis studies define the optimal binding site of the (+KTS) isoform of EWS-WT1 as 5-GGAGG(A/G)-3. LRRC15 encodes a leucine-rich transmembrane protein, present at the leading edge of migrating cells, the expression of which in normal tissues is restricted to the invasive cytotrophoblast layer of the placenta; small interfering (siRNA)-mediated suppression of LRRC15 expression in breast cancer cells leads to abrogation of invasiveness in vitro. Together, these observations define the consequence of (KTS) insertion within WT1-derived zinc fingers, and identify a novel EWS-WT1 transcriptional target implicated in tumor invasiveness.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway

“…As for the downstream target of 17AA( þ )KTS( þ )WT1 isoform, we could not determine it. However, since this isoform has an insertion of three amino acids (lysine, threonine, and serine (KTS)), by which binding of 17AA( þ ) KTS( þ )WT1 isoform to a consensus sequence as that for 17AA( þ )KTS(À)WT1 isoform was abrogated, 17AA( þ )KTS( þ )WT1 isoform may transcriptionally regulate other gene(s) than one(s) regulated by 17AA( þ )KTS(À)WT1 isoforms (Reynolds et al, 2003). Comprehensive studies, including microarray analysis are being planned to identify the targets of 17AA( þ ) KTS( þ )WT1 isoform.…”

The Wilms’ tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron

“…However, although a transcriptional role for a chimeric Ewing's Sarcoma gene (EWS)-WT1(+KTS) protein has been described (Reynolds et al 2003), no clear function has been attributed to the native WT1(+KTS) protein, in spite of the fact that proper +KTS/−KTS ratios are crucial in the development of several organs (Hammes et al 2001;Davies et al 2004;Wagner et al 2005). In this study, we have shown for the first time that WT1(+KTS) functions at the post-transcriptional level to regulate RNA expression and, specifically, that the +KTS protein can function in conjunction with a cis-acting CTE to promote translation of an unspliced RNA with a retained intron.…”

The TFIID subunit TAF4 regulates keratinocyte proliferation and has cell-autonomous and non-cell-autonomous tumour suppressor activity in mouse epidermis

“…The most strongly upregulated gene was Lrrc15 [leucine rich repeat containing 15; also known as LRR-induced by ␤-amyloid (LIB)], which encodes a transmembrane protein of unknown function that localises to the leading edge of migrating cells (Reynolds et al, 2003;Satoh et al, 2002). It is interesting to note the upregulation of genes encoding ABL2, a tyrosine kinase implicated in cell motility (Woodring et al, 2003), ABI1 (ABLinteractor 1) and MAP4K4, a promigratory kinase (Collins et al, 2006).…”