The fundamental role of genetic changes in cancer progression is now unquestioned. These aberrations are being cataloged at an unprecedented pace through the application of high-throughput genomic tools (Stratton et al., 2009
). In contrast, the extent to which epigenetic events and chromatin environments contribute to cellular transformation remains controversial. The genomic instability present in many cancers complicates the study of DNA methylation and chromatin. Moreover, the most relevant self-renewing cell populations are frequently obscured by tumor heterogeneity. Nonetheless, there is increasing evidence that aberrant DNA methylation and chromatin regulation profoundly contribute to specific types of cancer (Feinberg et al., 2006
; Jones and Baylin, 2007
). The advent of new epigenomic tools provides an opportunity to investigate their contributions broadly (Barski et al., 2007
; Lister et al., 2009
; Mikkelsen et al., 2007
). Pediatric cancers represent attractive models to study as their relatively normal genomic background facilitates epigenomic characterization and suggests that epigenetic factors may make play particularly critical roles in pathogenesis.
Wilms tumor is characterized by a multipotent “triphasic” histology that includes an undifferentiated ‘blastemal’ component and varying amounts of epithelial and stromal elements (Rivera and Haber, 2005
). These tumors can also be associated with developmental abnormalities, including persistent embryonic tissue known as nephrogenic rests, and are thus believed to be intimately connected to kidney organogenesis.
Genetic abnormalities described in Wilms tumor involve genes that regulate the metanephric mesenchyme, a kidney-specific stem cell population that resembles blastemal tumor cells and gives rise to most epithelia in adult kidneys. The tumor suppressor WT1
has been linked to survival and differentiation of these cells (Call et al., 1990
; Gessler et al., 1990
; Kreidberg et al., 1993
; Moore et al. 1999
), and the activation of β-catenin is a crucial step in epithelialization (Koesters et al., 1999
). Similarly, the recently identified tumor suppressor WTX
is expressed in kidney stem cells and has been linked to Wnt signaling and WT1 transcriptional control (Major et al., 2007
; Rivera et al., 2007
; Rivera et al., 2009
). Yet known mutations account for less than 50% of Wilms tumors, leaving a majority without any known causal genetic alteration.
Two aspects of Wilms tumor suggest that epigenetic alterations also play critical roles in pathogenesis. First, the classical imprinted gene IGF2
, normally expressed only from the paternal allele, frequently exhibits bi-allelic expression in sporadic Wilms tumors (Ogawa et al., 1993
imprinting is also lost in Beckwith-Wiedeman, an overgrowth syndrome associated with an elevated risk of Wilms tumors (Weksberg et al., 1993
). Second, similarities in gene expression between Wilms tumor and fetal kidney raise the possibility that pathways active in organ-specific stem cells may be shared by the tumor (Li et al., 2002
). This relationship is further supported by the occasional spontaneous regression of nephrogenic rests, which may reflect the re-activation of kidney differentiation pathways (Beckwith et al., 1990
Whole genome analysis of chromatin state is now feasible by combining chromatin immunoprecipitation (ChIP) with sequencing – ‘ChIP-Seq’ (Barski et al., 2007
; Mikkelsen et al., 2007
). Of particular interest are specific histone modifications that relate closely to transcriptional programs, cellular state and epigenetic processes (Kouzarides, 2007
). Maps of histone H3 trimethylated at lysine 4 (K4me3), lysine 36 (K36me3) or lysine 27 (K27me3) identify promoters, transcripts or sites of Polycomb repression, respectively (Barski et al., 2007
; Li et al., 2007
; Mikkelsen et al., 2007
). In embryonic stem (ES) cells, ‘bivalent domains’ with overlapping K27me3 and K4me3 are associated with developmental genes that are presently silent, but poised for activation upon differentiation (Azuara et al., 2006
; Bernstein et al., 2006
). Bivalent domains have been proposed to predispose gene promoters to DNA methylation in cancer (Ohm et al., 2007
). However, the global role of such marks in cancer has not been explored. Such an analysis could provide insight into the developmental state of tumor cells and how they relate to non-malignant counterparts.
Here, we present a whole genome analysis of chromatin in primary Wilms tumors. We focused on Wilms as an initial model because (i) resected tumors provide a ready source of homogeneous, undifferentiated ‘blastemal’ cells which resemble embryonic renal tissue and may represent a model of tumor stem cells (Rivera and Haber, 2005
) and (ii) Wilms cells exhibit relatively normal genetic backgrounds with few copy number alterations or known mutations, thus facilitating and highlighting the importance of epigenomic analysis (Rivera et al., 2007
). We mapped K4me3, K27me3 and K36me3 in Wilms tumors, normal kidneys, and fetal kidneys, and compared the maps to analogous data for human ES cells. These chromatin data were integrated with published transcript profiles, and complemented by mutation and copy number analyses.
The data reveal an interconnected network of genes that appear to drive Wilms tumor phenotype and proliferation. Many of these genes correspond to known regulators of kidney development, but some may be novel master regulators of this process. The maps also point to critical roles for Polycomb repression in both fully silenced and bivalent patterns, an important feature of ES cell biology that is recapitulated in Wilms tumor. For example, the p16 tumor suppressor is repressed by Polycomb in a manner reminiscent of normal stem cells, but distinct from many adult tumors. Similarly, markers of epithelial differentiation are maintained in a bivalent, poised state that may signal a latent differentiation potential akin to a normal renal stem cell. In summary, in depth analysis of Wilms tumor chromatin points to a transformed phenotype that is sustained through the precise control of developmental and proliferative mechanisms shared with early kidney precursors and ES cells.