TGF-β1 Alters DNA Methylation Levels in Promoter and Enhancer Regions of the WT1 Gene in Human Podocytes.

BACKGROUND: Wilms' tumor 1 (WT1) is essential for normal podocyte function. Previous reports have shown that the WT1 promoter is often methylated in cancers, leading to transcriptional silencing. Transforming growth factor-β1 (TGF-β1) is reported to downregulate WT1 expression in podocytes. Based on the hypothesis that epigenetic modification plays a role in this process, we examined whether TGF-β1 affects the methylation status of WT1 regulatory regions.

METHODS: Conditional immortalized human podocytes were treated with TGF-β1. A human renal proximal tubular epithelial cell line (HK-2), which does not express WT1, was used as a control. The degree of DNA methylation of the WT1 promoter, 5' enhancer, intron 3 enhancer, and 3' enhancer was determined by quantitative methylation-specific PCR, bisulfate sequencing, and pyrosequencing.

RESULTS: Both WT1 mRNA and protein expression were reduced by long-term treatment with TGF-β1. The WT1 promoter was hypomethylated, and the 5' enhancer and intron 3 enhancer were substantially methylated in untreated podocytes. In contrast, in HK2 cells, the WT1 promoter was strongly methylated, and the 5' enhancer and intron 3 enhancer were less methylated than in untreated podocytes. TGF-β1 tended to increase WT1 promoter methylation, tended to decrease 5' enhancer methylation, and significantly decreased intron 3 enhancer methylation in podocytes. Methylation levels of the 3' enhancer did not differ among untreated cells, TGF-β1-treated podocytes, or HK2 cells.

CONCLUSIONS: Our data suggest that the methylation pattern of the WT1 promoter and enhancers in human podocytes are distinctive from those in HK2. Further, TGF-β1 alters the methylation levels of the WT1 promoter and enhancers in human podocytes. This modification may be relevant to the attenuation of WT1 by TGF-β1, which could contribute to podocyte injury. This article is protected by copyright. All rights reserved.