Project description:Genome wide DNA methylation profiling of cultured fetal human neurons infected with lentivirus carrying DNMT3L or ZsGreen genes for 4days. The Illumina Infinium 850k Human MethylationEPIC BeadChip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in the infected cells. Samples included 3 control samples (ZsGreen) and 3 DNMT3L-overexpressing samples (DNMT3L).
Project description:The objective of this study is to test the gene expression changes caused by DNMT3L overexpression in human neurons. The total RNA of each sample was extracted from the lentivirus infected, early differentiated human neuroprogenitors with ZsGreen (n=3) or DNMT3L (n=3) overexpression by using TRIzol reagent. Then the RNA samples were processed for high throughput transcriptome sequencing on Illumina HiSeq 3000 platform. Results: among 57 905 cleaned RNAs, 7983 RNAs were differentially expressed in DNMT3L overexpressing neurons compared with control neurons, with q-value ≤ 0.05. After limited the cut-off by adding |fold change| ≥ 1.5, the numbers of differential expressed RNAs lowered to 1073, with more down-regulation (590) than up-regulation (483). The differential expressed genes distributed in all chromosomes, also showing the pattern of more down-regulation than up-regulation in every chromosome except chromosome 21. Functional annotation with DAVID revealed the top functional groups including type I interferon signaling pathway and RNA and protein processing.
Project description:During oogenesis, DNA methyltransferase 3-like (Dnmt3l) is required for the establishment of the maternal germline DNA methylation imprints that in the offspring, govern the parent-of-origin-specific expression of most known imprinted genes (Science 2001, 294:2536-9). Dnmt3l-deficient dams were crossed with wildtype sires to obtain Dnmt3l-/+ embryos that lack maternal methylation imprints. Gene expression was measured in Dnmt3l-/+ and wildtype embryos and is expected to differ for imprinted genes that are under the control of a maternal methylation mark. Experiment Overall Design: 1 normal control sample/array. 2 biologically replicate Dnmt3l-/+ samples/arrays.
Project description:During oogenesis, DNA methyltransferase 3-like (Dnmt3l) is required for the establishment of the maternal germline DNA methylation imprints that in the offspring, govern the parent-of-origin-specific expression of most known imprinted genes (Science 2001, 294:2536-9). Dnmt3l-deficient dams were crossed with wildtype sires to obtain Dnmt3l-/+ embryos that lack maternal methylation imprints. Gene expression was measured in Dnmt3l-/+ and wildtype embryos and is expected to differ for imprinted genes that are under the control of a maternal methylation mark. Keywords: genetic modification, DNA methylation
Project description:Background: 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. 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). Results: DNMT3L 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. Conclusions: Taken 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.