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. 2003 Sep 1;17(17):2094-107.
doi: 10.1101/gad.1110703. Epub 2003 Aug 15.

Identification of a DNA-binding Site and Transcriptional Target for the EWS-WT1(+KTS) Oncoprotein

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Free PMC article

Identification of a DNA-binding Site and Transcriptional Target for the EWS-WT1(+KTS) Oncoprotein

Paul A Reynolds et al. Genes Dev. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Induction of LRRC15 by EWS–WT1(+KTS). (A) Schematic representation of EWS, WT1, and the EWS–WT1 translocation, fusing the N-terminal domain (NTD) of EWS (exons 1–7) to the last three zinc fingers of WT1 (exons 8–10). The KTS alternative splice inserts three amino acids (lysine, threonine, and serine) between zinc fingers 3 and 4, and is retained in the translocation product. (B) cDNA subtraction results after induction of EWS–WT1(+KTS). Of 62 clones initially found to be differentially expressed upon primary hybridization screening, 32 were confirmed to be induced by Northern blot analysis. These represented only two genes: LRRC15 and EWS–WT1 itself. (C) Northern blot analysis of U2OS cells demonstrating induction of endogenous LRRC15 mRNA, 12 h after inducible expression of EWS–WT1(+KTS), but not EWS–WT1(–KTS), WT1(+KTS), or WT1(–KTS). Blot was hybridized with probes for LRRC15, WT1 (detecting both WT1 and EWS–WT1), and GAPDH (loading control). (D) RNA in situ hybridization analysis of LRRC15 in two primary DSRCT samples (magnification, 80×). Adjacent sections were stained with hematoxylin and eosin (H&E), showing nests of tumor cells surrounded by reactive stroma. LRRC15 expression is restricted to tumor cells. No staining was observed with a control (LRRC15 sense) probe.
Figure 3.
Figure 3.
Identification of EWS–WT1(+KTS) responsive element. (A) Schematic representation of the LRRC15 locus (current release of http://genome.ucsc.edu and release 12.31.1 of http://www.ensembl.org). The location of the HC63 fragment, the transcriptional start site of LRRC15, and neighboring genes (GP5, GenBank accession no. Z23091; CPN2, GenBank accession no. J05158; and ATY3, GenBank accession no. AJ306929) are shown. (B) Activation of LRRC15 upstream sequences by EWS–WT1(+KTS). Luciferase activity, relative to vector-transfected cells, was measured in U2OS cells, 48 h after cotransfection of reporter constructs (0.2 μg) and either EWS–WT1(+KTS) or EWS–WT1(–KTS) expression plasmids (1 μg). H6 denotes the pool of eight HindIII-digested fragments derived from BAC 573k19, which consistently showed transactivation by EWS–WT1(+KTS) (out of 36 pools tested); HC6 is the individual clone (2-kb HindIII fragment; AC108676, 74101–76107) within the pool that was found to be induced by EWS–WT1(+KTS); HC62 is a 720-bp BglII/HindIII digest fragment from HC6 (AC108676, 75379–76107); HC63 is a 142-bp fragment of HC62. Transfection efficiency was standardized by using a cotransfected reporter (Renilla luciferase), and equal amounts of CMV promoter were present in each transfection. Standard deviations were derived from three independent experiments. (C) EMSA analysis of HC63 after incubation with the zinc finger domains of EWS–WT1(+KTS), EWS–WT1(–KTS), or WT1(+KTS). End-labeled probes were incubated with 200 ng of the respective GST fusion protein or GST alone. Addition of unlabeled probe at 100-fold molar excess is shown to demonstrate competitive binding. Supershifting of the protein–DNA complex is shown by using anti-WT1 antibody(C19) or a control antibody(Sp1). Migration of free probe is shown (brackets). (D) Chromatin immunoprecipitation (ChIP) analysis to demonstrate in vivo binding of EWS–WT1(+KTS) to HC63. Chromatin was extracted from U2OS cells with tetracycline-regulated expression of either EWS–WT1(+KTS) (top) or EWS–WT1(–KTS) (bottom) after growth in the absence (12 h) of tetracycline; was formaldehyde cross-linked; and was immunoprecipitated by using antibody C19 (directed against the WT1 zinc finger domain), anti-HA (against the HA epitope), anti-histone H3 antibody(positive control), rabbit preimmune serum (mock), or no antibody. Multiplex PCR was performed by using primers specific for HC63 together with β-actin (internal standard); progressive dilutions of total chromatin were also amplified to demonstrate the linearity of multiplex PCR amplification (input).
Figure 2.
Figure 2.
Structure and expression of LRRC15. (A) Sequence alignments of LRRC15 protein from human, rat, and mouse and of the related human GP5, using the ClustalW program. Domains of LRRC15 are underlined with colored bars: A signal peptide (red) is followed by a characteristic leucine-rich repeat (LRR) N-terminal flanking domain (yellow), 15 LRRs (green), a C-terminal flanking domain (yellow), one transmembrane domain (blue), and a short cytoplasmic domain. Within each LRR, a number of positions are highly conserved (bold). (B) Localization of HA-tagged LRRC15 in HT1080 cells. Cells were grown on vitronectin-coated slides and stained with antibody against the HA-epitope (green). Phalloidin staining marks the distribution of F-actin (red) with regions of overlap at the leading edge of migrating cells evident on the merged image (yellow). (C) Western blot analysis of cellular extracts from HT1080 cells expressing HA-tagged LRRC15. Denatured protein (lane 1) was incubated with PNGase F (lane 2); or PNGase F, Sialidase A, and endo-O-glycosidase (lane 3); or PNGase F, Sialidase A and endo-O-glycosidase, β(1–4) galactosidase, and glucosaminidase (lane 4).
Figure 4.
Figure 4.
Characterization of EWS–WT1(+KTS) responsive element E(KTS)RE. (A) EMSA analysis of two fragments within HC63 that demonstrate binding by EWS–WT1(+KTS): HC63-1 (30 bp) and HC63-2 (30 bp). End-labeled probes were incubated with 200 ng of the zinc finger domains of EWS–WT1(+KTS), EWS–WT1(–KTS), or GST alone. Migration of free probe is shown (brackets). The two panels are derived from the same gel. (B) Identification of essential residues for EWS–WT1(+KTS) binding within HC63-1. EMSA of EWS–WT1(+KTS) protein (+) versus free probe (–) is compared by using probes containing a substitution at each nucleotide that constitute the 6-bp minimal binding domain, which we call E(KTS)RE-1. All nonadenine bases were changed to adenine; adenine bases were changed to thymine and compared with binding to wild-type sequence (WT). Numerical positions correspond to the E(KTS)RE sequence. Equal amounts of probe and protein were added in all cases. Migration of free probe is shown (brackets). The two panels are derived from the same gel. (C) Comparable binding of EWS–WT1(+KTS) to E(KTS)RE containing either a guanine or adenine at position 6. This nucleotide is the only divergence between the binding sequence identified in fragment HC63-1 and HC63-2. EMSA lanes derived from the same gel are shown, with equal amounts of probe and protein (+) added in all cases. (D) Competition of unlabeled oligonucleotide with the guanine-to-adenine substitution at E(KTS)RE position 6 for binding to end-labeled HC63-2 (100-fold excess competitor). In contrast, the G2A substitution fails to compete in EMSA, as does a nonspecific oligonucleotide derived from HC63 (nonsp). (E) Minimal binding sequence for EWS–WT1(+KTS). The sequences derived independently from HC63-1 [E(KTS)RE1] and HC63-2 [E(KTS)RE2] are shown. These sites differ at position 6, where equivalent binding is observed with either adenine or guanine, but not with thymine. The E(KTS)RE sequence does not constitute a subset of the DNA-binding consensus derived for the related zinc fingers of WT1(–KTS) (WTE, 5′-GCGTGGGAG-3′) or EWS–WT1(–KTS) [E-WRE, 5′-(G/C)(C/G)(G/C)TGGGGG-3′]. (F) Schematic representation of the promoter-less pGL3 basic reporter, containing the two E(KTS)RE binding sites within HC63 (construct A). Triple substitutions of G1, G2, G4 to A were engineered in E(KTS)RE1 (construct B), or in both E(KTS)RE1 and E(KTS)RE2 (construct C). (G) Relative luciferase activity, 48 h after transfection of mutant reporter constructs A through C (0.2 μg), along with EWS–WT1(+KTS), EWS–WT1(–KTS), or vector (1 μg), into U2OS cells. Transfection efficiency was standardized by using a cotransfected reporter (Renilla luciferase), and equal amounts of CMV promoter were present in each transfection. Standard deviations were derived from three independent experiments.
Figure 5.
Figure 5.
Characterization of zinc finger binding to E(KTS)RE. (A) EMSA analysis of combinations of WT1-derived zinc finger proteins. Binding is shown by using HC63-1, which contains the EWS–WT1(+KTS) binding sequence E(KTS)RE, and using consensus sequences previously identified for EWS–WT1(–KTS) (called E-WRE1) or for WT1(–KTS) (called WTE). A loading control for expression of zinc finger proteins is shown by using Western blotting with anti-GST antibody(αGST). (B) Relative binding affinity of combinations of WT1-derived zinc finger proteins for the E(KTS)RE sequence. Results from multiple EMSA experiments using HC63-1 probe are represented schematically.
Figure 6.
Figure 6.
Role of LRRC15 in cellular invasion. (A) RNA in situ hybridization analysis of Lrrc15 in mouse placenta (magnification, 80×). Adjacent section is stained with H&E showing cytotrophoblast layer (cy), stroma (s), and maternal decidua (md). Lrrc15 expression is restricted to cytotrophoblast cells. No staining is observed with a control (Lrrc15 sense) probe. (B, top) Effect of LRRC15 expression on matrigel invasion by Hs467T breast cancer cells. Quantitative real-time RT–PCR (TaqMan) analysis of LRRC15 transcript from Hs467T cells, 72 h after treatment with specific siRNA duplexes, nonspecific duplexes, or untreated (mock). The expression of GAPDH was used to normalize for variances in input cDNA. (Bottom) Results of transwell migration assays are shown for matrigel-coated plates (correlated with cellular invasion) or for uncoated plastic (correlated with cellular migration). Hs467T cells treated with LRRC15 siRNA or nonspecific siRNA, or mock-treated controls are compared with the highly invasive HT1080 cells and the noninvasive NIH 3T3 cells. Standard deviations are derived from three independent experiments.

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