Abstract 2954: Low level methylation (1-20%) or methylation seeds is a distinct epigenetic regulator of gene expression and is a strong predictor of methylation gains in AML and MDS patients
Authors: Madireddi Priyanka , Jelinek Jaroslav , Lee Justin , Cesaroni Matteo , Rodrigues Rodrigo , Zhang Yan , Issa Jean-Pierre 
Published: August 2015

DNA methylation is an important epigenetic regulator of tumour suppressor genes in several cancers and is an established target for therapy. Genome wide studies indicate distinct set of genes hypermethylated among different types of cancer. Identifying why only certain genes are predisposed to hypermethylation in a cancer type will help us develop better epigenetic therapies. To understand this important aspect, we studied methylation in tissues from healthy individuals and cancer patients. Study of methylation in normal tissues identified a distinct methylation category that strongly regulates gene expression and predisposes to hypermethylation in cancer patients. We studied methylation at SmaI (CCCGGG) sites using Digital Restriction Enzyme Analysis of Methylation (DREAM) and validated our results using ENCODE Reduced Representation Bisulphite Sequencing (RRBS) data. Study of methylation using DREAM in normal tissues identified methylation seeds or low (but non zero) methylation (1-20%) at a subset of promoter CpG islands. These seeded promoters were distinct from unmethylated (0-1%) promoters and covered around 448, 702 and 1103 genes in testis, liver and WBC, respectively. Seeded genes were enriched in developmental and differentiation pathways unrelated to that tissue, while unmethylated genes were enriched for housekeeping functions. It is well known that hypermethylation of promoter CpG islands inhibit gene expression but effects of very low levels of methylation have not been studied genome wide. Hence we analyzed ENCODE RNA-seq data for above 3 tissues. Surprisingly, correlation studies between methylation and gene expression identified that both seeded (1-20%) and methylated (20-100%) genes correlated with significantly reduced gene expression compared to unmethylated genes (0-1%). This data suggested that even very low methylation significantly decreases gene expression. To directly test this, we cloned different mammalian promoters into luciferase expression vectors, methylated them in vitro (to 10-20%) and then transfected them into 293T cells. As expected, all the seeded (10-20%) promoter constructs showed significantly reduced luciferase activity establishing that methylation seeds directly decrease gene expression. Importantly, comparison of methylation in WBC using DREAM between normal controls (N = 13), AML (N = 108) or MDS (N = 26) patients identified that seeded CpG sites in normal were 17 fold more frequently hypermethylated (&gt;40% difference) in AML patients than unmethylated CpG sites. (MDS = 80 fold more frequently). This indicates that having low methylation in normal tissues predisposes to gain methylation in cancer. To our knowledge, this is the first genome wide study identifying that very low levels of methylation can affect gene expression and is an important contributor of hypermethylation in leukemias.

Citation Format: Priyanka Madireddi, Jaroslav Jelinek, Justin Lee, Matteo Cesaroni, Rodrigo Rodrigues, Yan Zhang, Jean-Pierre Issa. Low level methylation (1-20%) or methylation seeds is a distinct epigenetic regulator of gene expression and is a strong predictor of methylation gains in AML and MDS patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2954. doi:10.1158/1538-7445.AM2015-2954