BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

BACKGROUND: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. RESULTS: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. CONCLUSIONS: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

An updated catalog of CTCF variants associated with neurodevelopmental disorder phenotypes.

Introduction: CTCF-related disorder (CRD) is a neurodevelopmental disorder (NDD) caused by monoallelic pathogenic variants in CTCF. The first CTCF variants in CRD cases were documented in 2013. To date, 76 CTCF variants have been further described in the literature. In recent years, due to the increased application of next-generation sequencing (NGS), growing numbers of CTCF variants are being identified, and multiple genotype-phenotype databases cataloging such variants are emerging. Methods: In this study, we aimed to expand the genotypic spectrum of CRD, by cataloging NDD phenotypes associated with reported CTCF variants. Here, we systematically reviewed all known CTCF variants reported in case studies and large-scale exome sequencing cohorts. We also conducted a meta-analysis using public variant data from genotype-phenotype databases to identify additional CTCF variants, which we then curated and annotated. Results: From this combined approach, we report an additional 86 CTCF variants associated with NDD phenotypes that have not yet been described in the literature. Furthermore, we describe and explain inconsistencies in the quality of reported variants, which impairs the reuse of data for research of NDDs and other pathologies. Discussion: From this integrated analysis, we provide a comprehensive and annotated catalog of all currently known CTCF mutations associated with NDD phenotypes, to aid diagnostic applications, as well as translational and basic research.

The combined action of CTCF and its testis-specific paralog BORIS is essential for spermatogenesis.

CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf+/+Boris+/+, Ctcf+/-Boris+/+, and Ctcf+/+Boris-/- males are fertile, Ctcf+/-Boris-/- (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are "toxic" to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.

CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

BORIS Expression in Ovarian Cancer Precursor Cells Alters the CTCF Cistrome and Enhances Invasiveness through GALNT14.

High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecologic cancer-related death. We have previously shown that CTCFL (also known as BORIS, Brother of the Regulator of Imprinted Sites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed BORIS in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC. BORIS-expressing cells exhibited increased motility and invasion, and BORIS expression was associated with alterations in several cancer-associated gene expression networks, including fatty acid metabolism, TNF signaling, cell migration, and ECM-receptor interactions. Importantly, GALNT14, a glycosyltransferase gene implicated in cancer cell migration and invasion, was highly induced by BORIS, and GALNT14 knockdown significantly abrogated BORIS-induced cell motility and invasion. In addition, in silico analyses provided evidence for BORIS and GALNT14 coexpression in several cancers. Finally, ChIP-seq demonstrated that expression of BORIS was associated with de novo and enhanced binding of CTCF at hundreds of loci, many of which correlated with activation of transcription at target genes, including GALNT14. Taken together, our data indicate that BORIS may promote cell motility and invasion in HGSC via upregulation of GALNT14, and suggests BORIS as a potential therapeutic target in this malignancy. IMPLICATIONS: These studies provide evidence that aberrant expression of BORIS may play a role in the progression to HGSC by enhancing the migratory and invasive properties of FTSEC.

CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer.

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/-) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/- MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/- MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF's role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

Discovering a binary CTCF code with a little help from BORIS.

CCCTC-binding factor (CTCF) is a conserved, essential regulator of chromatin architecture containing a unique array of 11 zinc fingers (ZFs). Gene duplication and sequence divergence during early amniote evolution generated the CTCF paralog Brother Of the Regulator of Imprinted Sites (BORIS), which has a DNA binding specificity identical to that of CTCF but divergent N- and C-termini. While healthy somatic tissues express only CTCF, CTCF and BORIS are normally co-expressed in meiotic and post-meiotic germ cells, and aberrant activation of BORIS occurs in tumors and some cancer cell lines. This has led to a model in which CTCF and BORIS compete for binding to some but not all genomic target sites; however, regulation of CTCF and BORIS genomic co-occupancy is not well understood. We recently addressed this issue, finding evidence for two major classes of CTCF target sequences, some of which contain single CTCF target sites (1xCTSes) and others containing two adjacent CTCF motifs (2xCTSes). The functional and chromatin structural features of 2xCTSes are distinct from those of 1xCTS-containing regions bound by a CTCF monomer. We suggest that these previously overlooked classes of CTCF binding regions may have different roles in regulating diverse chromatin-based phenomena, and may impact our understanding of heritable epigenetic regulation in cancer cells and normal germ cells.

Corrigendum: Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells.

This corrects the article DOI: 10.1038/srep41279.

Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells.

Despite sharing the same sequence specificity in vitro and in vivo, CCCTC-binding factor (CTCF) and its paralog brother of the regulator of imprinted sites (BORIS) are simultaneously expressed in germ cells. Recently, ChIP-seq analysis revealed two classes of CTCF/BORIS-bound regions: single CTCF target sites (1xCTSes) that are bound by CTCF alone (CTCF-only) or double CTCF target sites (2xCTSes) simultaneously bound by CTCF and BORIS (CTCF&BORIS) or BORIS alone (BORIS-only) in germ cells and in BORIS-positive somatic cancer cells. BORIS-bound regions (CTCF&BORIS and BORIS-only sites) are, on average, enriched for RNA polymerase II (RNAPII) binding and histone retention in mature spermatozoa relative to CTCF-only sites, but little else is known about them. We show that subsets of CTCF&BORIS and BORIS-only sites are occupied by several testis-specific transcriptional regulators (TSTRs) and associated with highly expressed germ cell-specific genes and histone retention in mature spermatozoa. We also demonstrate a physical interaction between BORIS and one of the analyzed TSTRs, TATA-binding protein (TBP)-associated factor 7-like (TAF7L). Our data suggest that CTCF and BORIS cooperate with additional TSTRs to regulate gene expression in developing male gametes and histone retention in mature spermatozoa, potentially priming certain regions of the genome for rapid activation following fertilization.

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions.

BACKGROUND: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation. RESULTS: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. CONCLUSIONS: We discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells.

Chromatin architecture reorganization during stem cell differentiation.

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer.

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

A map of the cis-regulatory sequences in the mouse genome.

The laboratory mouse is the most widely used mammalian model organism in biomedical research. The 2.6 × 10(9) bases of the mouse genome possess a high degree of conservation with the human genome, so a thorough annotation of the mouse genome will be of significant value to understanding the function of the human genome. So far, most of the functional sequences in the mouse genome have yet to be found, and the cis-regulatory sequences in particular are still poorly annotated. Comparative genomics has been a powerful tool for the discovery of these sequences, but on its own it cannot resolve their temporal and spatial functions. Recently, ChIP-Seq has been developed to identify cis-regulatory elements in the genomes of several organisms including humans, Drosophila melanogaster and Caenorhabditis elegans. Here we apply the same experimental approach to a diverse set of 19 tissues and cell types in the mouse to produce a map of nearly 300,000 murine cis-regulatory sequences. The annotated sequences add up to 11% of the mouse genome, and include more than 70% of conserved non-coding sequences. We define tissue-specific enhancers and identify potential transcription factors regulating gene expression in each tissue or cell type. Finally, we show that much of the mouse genome is organized into domains of coordinately regulated enhancers and promoters. Our results provide a resource for the annotation of functional elements in the mammalian genome and for the study of mechanisms regulating tissue-specific gene expression.

Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos.

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency.

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4me1 and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

Histone modifications at human enhancers reflect global cell-type-specific gene expression.

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.

Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.

Interspecies comparative genomic hybridization (I-CGH): a new twist to study animal tumor models.

Animal models of human diseases are widely used to address questions of tumor development. Selection of a particular animal model depends upon a variety of factors, among them: animal cost, species lifespan and hardiness; availability of biomolecular and genetic tools for that species; and evolutionary distance from humans. In spite of the growth in genomic data in the past several years, many animal models cannot yet be studied extensively due to gaps in genetic mapping, sequencing and functional analyses. Thus, alternative molecular genetic approaches are needed. We have designed an interspecies comparative genomic hybridization approach to analyze genetic changes in radiation-induced brain tumors in the non-human primate, Macaca mulatta. Using homologies between the primate and human genomes, we adapted widely-available CGH techniques to generate cytogenetic profiles of malignant gliomas in four monkey tumors. Losses and gains were projected onto the corresponding homologous chromosomal regions in the human genome, thus directly translating the status of the monkey gliomas into human gene content. This represents a novel method of comparative interspecies cytogenetic mapping that permits simultaneous analysis of genomic imbalances of unknown sequences in disparate species and correlation with potential or known human disease-related genes.

Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1.

Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms' tumourigenesis.

We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis.