Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

Abstract

The majority of long noncoding RNAs (lncRNAs) is still poorly characterized with respect to function, interactions with protein-coding genes, and mechanisms that regulate their expression. As for protein-coding RNAs, epigenetic deregulation of lncRNA expression by alterations in DNA methylation might contribute to carcinogenesis. To provide genome-wide information on lncRNAs aberrantly methylated in breast cancer we profiled tumors of the C3(1) SV40TAg mouse model by MCIp-seq (Methylated CpG Immunoprecipitation followed by sequencing). This approach detected 69 lncRNAs differentially methylated between tumor tissue and normal mammary glands, with 26 located in antisense orientation of a protein-coding gene. One of the hypomethylated lncRNAs, 1810019D21Rik (now called Esrp2-antisense (as)) was identified in proximity to the epithelial splicing regulatory protein 2 (Esrp2) that is significantly elevated in C3(1) tumors. ESRPs were shown previously to have a dual role in carcinogenesis. Both gain and loss have been associated with poor prognosis in human cancers, but the mechanisms regulating expression are not known. In-depth analyses indicate that coordinate overexpression of Esrp2 and Esrp2-as inversely correlates with DNA methylation. Luciferase reporter gene assays support co-expression of Esrp2 and the major short Esrp2-as variant from a bidirectional promoter, and transcriptional regulation by methylation of a proximal enhancer. Ultimately, this enhancer-based regulatory mechanism provides a novel explanation for tissue-specific expression differences and upregulation of Esrp2 during carcinogenesis. Knockdown of Esrp2-as reduced Esrp2 protein levels without affecting mRNA expression and resulted in an altered transcriptional profile associated with extracellular matrix (ECM), cell motility and reduced proliferation, whereas overexpression enhanced proliferation. Our findings not only hold true for the murine tumor model, but led to the identification of an unannotated human homolog of Esrp2-as which is significantly upregulated in human breast cancer and associated with poor prognosis.