Abstract
BackgroundDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated).ResultsDNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development.ConclusionsTaken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.
Highlights
Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation
These findings demonstrate that DNA methyltransferase 3L (DNMT3L) overexpression during neurodevelopment recreates a facet of the genomewide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS
Stable DNMT3L Overexpression in SH‐SY5Y Cells SH-SY5Y cells were transfected with either myc-tagged human DNMT3L [4] and enhanced GFP via a piggyBac transposase [24] construct or a matched control construct without DNMT3L (Fig. 1a). Enhanced green fluorescent protein (eGFP)-positive cells were selected by flow cytometry and cultured again to enrich for a cell line with stable DNMT3L overexpression
Summary
Down syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. We stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated). Laufer et al Epigenetics & Chromatin (2021) 14:13 related to DNA methylation and have the potential to result in the hypermethylation pattern observed in DS [2]. Functional experimentation into the cause of hypermethylation has demonstrated a key role of the chromosome 21 encoded DNA methyltransferase DNMT3L at select genes [3]. While DNMT3L is catalytically inactive, it is a regulatory factor that binds to and stimulates the de novo methyltransferases DNMT3A and DNMT3B [4,5,6]. Members of the de novo methyltransferase family (DNMT3A,B,L) contain an ATRX–DNMT3–DNMT3L (ADD) domain that binds to the unmodified histone H3 tail (H3K4me0), to localize
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