Hypoxia-Sensitive Epigenetic Regulation of an Antisense-Oriented lncRNA Controls WT1 Expression in Myeloid Leukemia Cells

Gregory Mccarty,David M Loeb,Ken Mills

doi:10.1371/journal.pone.0119837

Gregory Mccarty, David M Loeb + Show 1 more

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https://doi.org/10.1371/journal.pone.0119837

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Abstract

WT1 is a transcription factor expressed in hematopoietic stem cells and in most cases of myeloid leukemia. We investigated the roles of hypoxia and epigenetics in the regulation of WT1 expression in myeloid leukemia cells. WT1 expression correlates with hypomethylation of the CpG island in Intron 1, and pharmacologic demethylation of this CpG island induces WT1 mRNA expression. Hypoxia causes decreases in DNMT expression and activity and increased expression and activity of TET2 and TET3, resulting in demethylation of this CpG island and expression of WT1 mRNA. Demethylation of the CpG island, either from pharmacologic treatment or induction of hypoxia, results in transcription of an antisense-oriented lncRNA, and inhibiting lncRNA expression with shRNA blocks WT1 mRNA expression. These results reveal a novel model of hypoxia-mediated epigenetic gene regulation. In addition, this is the first report that TET2 and TET3, increasingly recognized as important epigenetic regulators of gene expression in stem cells and in cancer cells, can be regulated by hypoxia, providing a solid mechanistic link between hypoxia and epigenetic regulation of gene expression with important implications for the role of hypoxia in stem cell function.

Highlights

WT1 is a transcription factor containing 4 zinc fingers in a C-terminal DNA binding domain [1, 2]
We focused on the CpG island in Intron 1, which surrounds a cryptic promoter that regulates the expression of an antisense-oriented transcript which shows monoallelic expression in the developing kidney, and this region, termed the antisense regulatory region (ARR), is hypomethylated in Wilms’ tumors with biallelic WT1 expression [21]
To investigate the relationship between methylation of the CpG island in Intron 1 and WT1 expression, we evaluated 3 human myeloid leukemia cell lines—K562, U937, and HL60

Summary

Introduction

WT1 is a transcription factor containing 4 zinc fingers in a C-terminal DNA binding domain [1, 2]. Originally identified as a tumor suppressor gene in children with Wilms’ tumor, subsequent work has demonstrated that WT1 is overexpressed in a wide variety of tumor types, including acute myeloid leukemia (AML) [3, 4]. Expression of WT1 is tightly regulated during development of the kidney (the organ in which Wilms’ tumor arises) and during hematopoiesis. Aberrant expression of WT1 contributes to the development of tumors arising in organs that ordinarily express WT1 under tight developmental control. The prognostic significance of WT1 expression in AML remains controversial [5,6,7], its importance as a tumor antigen and marker of minimal residual disease is growing [8,9,10,11].

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