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. Keywords: genetic modification, DNA methylation
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:The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methylase that has been previously shown to cooperate with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESC) but its function in these cells is unknown. We here report that Dnmt3L is required for the differentiation of ESC into primordial germ cells (PGC) through activation of the homeotic gene Rhox5. By genome-wide analysis we found that Dnmt3L is a positive regulator of methylation at gene bodies of housekeeping genes and a negative regulator of methylation at promoters of bivalent genes. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyl transferases Dnmt3a and Dnmt3b to maintain low the methylation level at H3H27me3 regions. Thus in ESC, Dnmt3L counteracts the activity of de novo DNA methylases to keep low the level of DNA methylation at developmental gene promoters.
Project description:The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methylase that has been previously shown to cooperate with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESC) but its function in these cells is unknown. We here report that Dnmt3L is required for the differentiation of ESC into primordial germ cells (PGC) through activation of the homeotic gene Rhox5. By genome-wide analysis we found that Dnmt3L is a positive regulator of methylation at gene bodies of housekeeping genes and a negative regulator of methylation at promoters of bivalent genes. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyl transferases Dnmt3a and Dnmt3b to maintain low the methylation level at H3H27me3 regions. Thus in ESC, Dnmt3L counteracts the activity of de novo DNA methylases to keep low the level of DNA methylation at developmental gene promoters. Total RNA extracted from shGFP or shDnmt3L (three different) embryonic stem cells. A duplicate was performed for each point. Cells were transfected with shRNA and selected with Puromicin for 3 days before RNA extraction
Project description:We used RRBS to analyze DNA methylation in mESC lines deficient for maternal Dnmt3L (Dnmt3L mKO), zygotic Dnmt3L (Dnmt3L KO), and both maternal and zygotic Dnmt3L (Dnmt3L mzKO). Compared to wild-type (WT) mESCs, Dnmt3L mKO mESCs exhibit severe loss of methylation at imprinted loci but no changes in global DNA methylation, Dnmt3L KO mESCs exhibit moderate loss of methylation at many Dnmt3a target regions but do not affect methylation at imprinted loci, and Dnmt3L mzKO mESCs exhibit combined changes of mKO and KO cells, with severe loss of methylation at imprinted loci and moderate loss of methylation at Dnmt3a target regions.