Frequent hypermethylation of a CTCF binding site influences Wilms tumor 1 expression in Wilms tumors.

The Wilms tumor 1 (WT1) gene plays an essential role in early development and differentiation of the urinary tract, particularly the kidneys. Aberrant transcriptional activity of WT1 is a key finding in the genesis of Wilms tumors (WTs). However, the mechanisms responsible for this alteration remain poorly understood. In the present study, we examined the methylation pattern of a putative CCCTC-binding factor (CTCF) binding site downstream of the WT1 gene as a potential cause of WT1 misregulation in 44 native WT specimens. We found that 16 WT cases exhibited a much higher WT1 expression compared to normal kidney tissue, and that the high mRNA expression of WT1 is strongly correlated with a high degree of DNA methylation of the CTCF binding site near the WT1 promoter. However, there was no correlation between the KTS+/KTS- splicing variants of WT1 and the methylation status of the CpGs of the CTCF binding site. Our results demonstrated an aberrant methylation pattern at a CTCF binding site downstream the WT1 gene, which is associated with an elevated WT1 transcriptional activity. Thus, methylation of the CTCF binding site may be partially responsible for the transcriptional activation of the WT1 locus and hypermethylation of this site may be an important oncogenic mechanism in the genesis of WT.

Selective methylation of CpGs at regulatory binding sites controls NNAT expression in Wilms tumors.

Aberrant expression of imprinted genes, such as those coding for the insulin-like growth factor 2 (IGF2) and neuronatin (NNAT), is a characteristic of a variety of embryonic neoplasms, including Wilms tumor (WT). In case of IGF2, it is generally accepted that loss of imprinting in a differentially methylated region of the IGF2/H19 locus results in biallelic expression and, thus, upregulation of the gene. In this study we examined methylation pattern at potential regulatory elements of the paternally expressed NNAT gene in a cohort of WT patients in order to further characterize the molecular mechanism causing overexpression of this regulatory gene. We demonstrate that transcriptional upregulation of NNAT in WT is grossly independent of the bladder cancer-associated protein (BLCAP) gene, an imprinted gene within the imprinted domain of the NNAT locus. However, expression of the BLCAP transcript isoform v2a formerly known to be selectively expressed from the paternal allele in brain was associated with high expression of NNAT. This contrasts the situation we found at the IGF2/H19 locus, which shows high overexpression of IGF2 and inversely correlated expression of the H19 gene in WT. An analysis of DNA methylation in two potential regulatory regions of the NNAT locus by pyrosequencing revealed significant hypomethylation of the tumors compared to normal kidney tissue. Interestingly, the difference in DNA methylation was highest at CpGs that were observed within three putative binding sites of the CCCTC-binding factor CTCF. Most importantly, hypomethylation of both NNAT regulatory regions is significantly associated with the upregulation of NNAT expression and the BLCAP_v2a transcript. Our data indicate that the methylation status of a not-yet-described regulatory element within the NNAT locus that contains four potential CTCF binding sites determines the expression level of NNAT and the nearby located BLCAP_v2a transcript, thereby suggesting a functional role in the aberrant upregulation of NNAT in WT.