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.

• Authors: Paul A Reynolds, Gromoslaw A Smolen, Rachel E Palmer, Dennis Sgroi, Vijay Yajnik, William L Gerald, Daniel A Haber
• Journal: Genes & development (Genes Dev) Vol. 17 Issue 17 Pg. 2094-107 (Sep 01 2003) ISSN: 0890-9369 [Print] United States
• PMID: 12923058 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• EWS1-WT1 fusion protein, human
• Oncogene Proteins, Fusion
• DNA

Topics

• Amino Acid Sequence
• Binding Sites (physiology)
• DNA (metabolism)
• Humans
• Molecular Sequence Data
• Oncogene Proteins, Fusion (metabolism)
• Peritoneal Neoplasms (genetics, metabolism)
• RNA Processing, Post-Transcriptional (physiology)