Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Engineering of effector domains for targeted DNA methylation with reduced off-target effects

ABSTRACT: Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing of selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the dCas9-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L effector domain allowing a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A variants containing mutations, which reduced their endogenous DNA binding strength. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to the future applications of this approach.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142181 | GEO | 2020/01/14

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

DNA methylation changes in Dnmt3L-deficient mouse embryonic stem cells (mESCs)

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.

2018-10-04 | GSE116489 | GEO

Combinational effect of the ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells [RNA-seq]

Project description:DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3-Like (DNMT3L) form functional heterotetramers and ATRX-DNMT3-DNMT3L (ADD) domains, shared by both DNMT3A and DNMT3L, recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0) to deposit DNA methylation properly in mammalian germ cells. However, the combinational and differential role of ADD domains of DNMT3A and DNMT3L in vivo has not been fully defined. Here we analyze both female and male germ cells derived from mouse with amino-acid substitutions in ADD domains of DNMT3A and/or DNMT3L that impair their domain function. Loss of either DNMT3A-ADD or DNMT3L-ADD domain function shows moderate reduction of global CG methylation level, but in different degree, in both germ cells. However, when both DNMT3A and DNMT3L lost their ADD domain functions, reduction of the global CG methylation level is much more severe and comparable with that of Dnmt3a/3L knockout germ cells. In contrast, such double mutant germ cells have thousands of genomic regions where non-CG methylation is aberrantly accumulated. These results highlight a critical role of the combinational function of ADD domains for the robust CG and non-CG methylation in germ cells.

2024-03-18 | GSE238226 | GEO

Combinational effect of the ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells [WGBS]

2024-03-18 | GSE238227 | GEO

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [MeDIP-seq]

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. Examination of 5mC in shGFP and shDnmt3L ESC by MeDIP-Seq

2013-09-27 | E-GEOD-44642 | biostudies-arrayexpress

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [ChIP-seq]

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 methyltransferases 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. Examination of Ezh2 ChIP-Seq in shGFP and shDnmt3L ESC. Examination of Dnmt3L ChIP-Seq in shGFP and shSuz12 ESC.

2013-09-27 | E-GEOD-49178 | biostudies-arrayexpress

Ectopic expression of DNA methyltransferases DNMT3A2 and DNMT3L leads to aberrant hypermethylation and postnatal lethality in mice

Project description:DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage-specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage-specific DNA methylation. How such lineage- and gene-specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild-type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, mono-allelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.

2019-02-15 | GSE126566 | GEO

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [Expression array]

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

2013-09-27 | E-GEOD-44643 | biostudies-arrayexpress

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [ChIP-seq]

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 methyltransferases 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.

2013-09-27 | GSE49178 | GEO

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [Expression array]

2013-09-27 | GSE44643 | GEO

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [MeDIP-seq]

2013-09-27 | GSE44642 | GEO