Abstract 2981: Hypoxia-sensitive dynamic methylation of a CpG island in Intron 1 of the WT1 gene regulates expression of a long noncoding RNA that controls WT1 expression in myeloid leukemia cells.

The WT1 gene encodes a zinc finger transcription factor that plays a role in both transcriptional and post-transcriptional regulation of mRNA expression. WT1 is overexpressed in a variety of solid tumors, as well as in both acute and chronic myeloid leukemias. The expression of WT1 is under tight control in the developing kidney as well as in hematopoietic stem/progenitor cells. WT1 expression is also regulated by hypoxia. A detailed understanding of the mechanisms regulating WT1 expression is, however, lacking. There is a CpG island located in Intron 1 of the WT1 gene, and we investigated whether WT1 expression might therefore be epigenetically regulated. We found that WT1 expression in myeloid leukemia cell lines correlates with hypomethylation of the Intron 1 CpG island which leads to expression of an antisense long noncoding RNA (lncRNA) transcribed from a promoter within Intron 1. Leukemia cell lines that express WT1 also demonstrate a Histone H3 methylation pattern associated with transcriptionally active chromatin, while lines that do not express WT1 have a pattern associated with transcriptionally inactive chromatin. Making WT1-negative cell lines hypoxic leads to demethylation of the Intron 1 CpG island, a change in histone methylation from predominantly H3K9 methylation to predominantly H3K4 methylation, expression of the antisense lncRNA, and expression of WT1. Prevention of HIF1 induction using shRNA blocks CpG demethylation, the changes in Histone H3 methylation, expression of the antisense lncRNA, and WT1 expression. An shRNA that blocks expression of the lncRNA in WT1-positive cell lines and blocks induction of the antisense lncRNA also blocks WT1 expression, demonstrating that the lncRNA is necessary for WT1 expression. Finally, in primary acute myeloid leukemia samples, WT1 expression correlates directly with hypomethylation of the Intron 1 CpG island, and hypoxia causes demethylation of the CpG island as well as induction of WT1 expression. We conclude that WT1 expression is regulated by an antisense lncRNA transcribed from a promoter in Intron 1, and that this promoter is regulated by methylation of the CpG island in this intron. Furthermore, hypoxia leads to demethylation of this CpG island, allowing transcription of the lncRNA, which results in the expression of WT1. We propose a novel mechanism of hypoxia-mediated regulation of gene expression: stabilization of HIF1 leads to demethylation of a specific CpG island and a change in histone methylation of the surrounding region, allowing expression of an antisense lncRNA that is necessary for upregulation of WT1 mRNA expression. Future work will investigate whether this novel mechanism of hypoxia-mediated regulation of gene expression affects other genes as well as the specific mechanism by which the antisense lncRNA controls WT1 expression. Citation Format: David M. Loeb, Gregory McCarty. Hypoxia-sensitive dynamic methylation of a CpG island in Intron 1 of the WT1 gene regulates expression of a long noncoding RNA that controls WT1 expression in myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2981. doi:10.1158/1538-7445.AM2013-2981