Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumor

Abstract

Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.

Figure 1. A large hypermethylated domain on chromosome 5q31 in Wilms' tumour.

(A) The MeDIP-chip tumour/normal (T/N) signal ratio shown for 2 representative WTs identifies hypermethylation of multiple gene promoters across a domain spanning 800 kb on chromosome 5q31.3 (red box). (B) The MeDIP-chip profile of PCDHA@, PCDHB@ and PCDHG@ in a representative WT. A minority of genes escaping hypermethylation are indicated (black arrowheads). (C) DNA methylation assayed using COBRA. Percentage methylation was analysed by gel densitometry for each gene, and is represented by horizontal bars (black  =  percentage methylated, white  =  percentage unmethylated). Data from 6 normal tissues (4 fetal kidneys [FK], postnatal kidney [NK] and fetal brain [FB]) and 9 Wilms' tumours are shown. Non-PCDH genes flanking the domain and between PCDHB@ and PCDHG@ are also shown. (D) Unsupervised hierarchical clustering of COBRA methylation data from normal and tumour tissues (T), HEK293 and WiT49 cell lines. (E) Bisulfite sequencing of PCDHB3, PCDHB8, SLC25A2, PCDHGA3, PCDHGA6 and PCDHGB4 5′ CGIs in normal fetal kidney (FK) and 2 Wilms' tumours, T43 & T57.

Figure 2. Silencing of PCDH expression in Wilms' tumour.

(A) Expression levels of genes across the locus in 9 tumours (Texp), relative to the mean of 4 normal fetal kidney samples (N exp). Grey bars are used for genes showing tumour-specific hypermethylation, white bars for unmethylated genes and black bars for the constitutively methylated SLC25A2 gene. (B) Expression levels of 5q31 transcripts correspond with DNA methylation status in WiT49 cells. Expression of unmethylated genes (white bars), constitutively methylated genes (black bar) and hypermethylated PCDHs (grey bars) are shown. (C) Suppression of methylated and unmethylated PCDHs within the chromosome 5q31 LRES. Gene expression levels relative to the house-keeping gene TBP are shown. Horizontal black line, median value; box, interquartile range; whiskers, data range excluding outliers; black dots, outliers (defined as those data points greater than range multiplied by inter-quartile range beyond the box). Grey boxes give samples shown to be hypermethylated, open boxes represent unmethylated samples.

Figure 3. Hypermethylation across the chromosome 5q31 LRES is associated with specific histone modifications.

Bar charts (left) show ChIP–quantitative PCR measuring relative levels of specific histone modifications at individual gene loci across the 5q31 locus in WiT49 cells. (A) H3Ac ChIP, (B) H3K4me2 ChIP, (C) H3K9me2 ChIP, all expressed relative to input DNA. Scatter plots (right) show the relationship between relative gene expression levels and histone modifications at each gene. The x-axis shows specific histone levels relative to input DNA, and the y-axis shows mRNA expression in WiT49 cells (Texp) relative to average mRNA expression in 4 fetal kidney samples (Nexp). Grey bars/datapoints signify genes hypermethylated in WiT49, open bars/datapoints show unmethylated genes, and the black bar/datapoint shows constitutively methylated SLC25A2.

Figure 4. Developmental expression patterns of PCDHs.

(A) PCDH transcript levels in mouse developmental tissues. Quantitative real-time expression analysis, relative to Tbp, in placenta (P), and E17.5 mouse fetal liver (Li), spleen (S), brain (B), and kidney (K) (E17.5, postnatal 0.5 week, 1 week, and 3 week) using assays specific for the constant region exons of Pcdha@ and Pcdhg@, and individual Pcdh transcripts. Expression of Wt1 and Cdh1 are also shown (black bars). (B) Immunoblotting of human fetal tissue proteins with pan γ-PCDH antibody or actin for loading control. Samples are kidney (K), liver (Li), lung (Lu), spleen (S), brain (B), and gut (G). (C) Gene expression changes accompanying epithelial differentiation of rat metanephric mesenchyme following Lif, Fgf2, and Tgfα treatment. Quantitative real-time expression analysis, relative to Tbp, is shown for freshly dissected rat metanephric mesenchyme (MM) and differentiating mesenchyme (DM). (D) Immunohistochemical analysis of 1-day postnatal murine kidney with antibodies towards Wt1 and γ-PCDHs. Blastema (b), primitive glomeruli (pg), and ureteric buds (u) are labelled. The control panel shows a section where the primary antibody has been omitted. Bar  = 50 µm.

Figure 5. PCDH effects on Wnt signalling.

(A) Enhanced β-catenin/TCF activity following γ-PCDH knockdown induced by PCDHG@ constant region targeting siRNA, measured with Super8xTOPFLASH reporter (TOP). Super8xFOPFLASH (FOP) reporter is a negative control. RLU, relative luciferase units. Gamma-PCDH and β-catenin knockdowns are verified by immunoblotting (IB) in the inset, which also demonstrates increased cellular β-catenin accompanying γ-PCDH knockdown. (B) Quantitative real-time expression analysis, relative to TBP, showing induction of the Wnt pathway target genes CCND1, CMYC, and PAX8 accompanying γ-PCDH knockdown; altered expression of WT1 is also shown. (C) Repression of β-catenin/TCF reporter activity accompanying PCDH expression in WiT49, HCT116, and Wnt3a-conditioned medium treated HEK293 (HEK293+CM) cells. A plasmid containing a cDNA encoding dominant-negative TCF4 (TCF4-DN) is also shown as a positive control. Cells were co-transfected with PCDH expression vectors and Super8xTOPFLASH (TOP) or Super8xFOPFLASH (FOP) and luciferase activity measured. RLU, relative luciferase units.

Figure 6. Growth inhibition by PCDHs.

(A) Suppression of WiT49, HCT116, and HEK293 cell colony formation following ectopic expression of PCDH cDNAs. After selection and staining, plates were photographed and colony counts determined for each transfection. Representative plates (left) and mean colony counts (right) are shown, Verification of PCDH protein expression after transfection, together with tubulin as a loading control is shown below the histograms. (B) Inhibition of anchorage-independent growth of HCT116 cells by PCDHs, cells were plated in triplicate and colonies formed after 10–14 days were photographed and counted within 10 random fields. Representative fields are shown (left) together with colony forming efficiency (CFE), expressed as percentage of colonies >50 µm diameter (right). Cell-based assays were repeated at least twice, and representative data are shown.