Mus musculus
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PROVIDER: PRJNA311045 | ENA |
REPOSITORIES: ENA
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Project description:Multiplex bisulfite sequencing identifies differentially methylated regions in target genes in embryonic cardiomyocytes following knockdown of DNMT1 expression
| PRJNA321722 | ENA
Project description:Purpose: This study aims to identify the differentially methylated regions in target genes in embryonic cardiomyocytes following the knockdown of DNMT3a or DNMT3b expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT3a siRNA (#GS13435), DNMT3b siRNAs (#GS13436), or AllStars negative control siRNA (#1027281) at 0.012 pmol using lipofectamine RNAiMAX (n=3 per treatment). At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). DNA was extracted with the Quick-gDNA™ MicroPrep kit (Zymo Research, Irvine, CA). Genomic DNA was treated with sodium metabisulfite for bisulfite conversion. Target gene regions for fifteen target genes (two promoter regions per gene) were PCR-amplified from the bisulfite-converted DNA with ZymoTaq™ PreMix (Hot start DNA taq polymerase, ZYMO Research, Irvine, CA). The fifteen genes include Myh6, Myh7, Myh7b, Nppa, Nppb, Tnnc1, Tnni3, Tnnt2, Camta1, Camta2, Cdkn1A, Cdkn1B, Cdkn1C, Mef2c, and Mef2d. PCR products were cleaned with the ZR-96 DNA Clean & Concentrator™-5 kit (ZYMO Research). PCR products from the same genomic DNA sample were pooled at equal concentration ratio and made into an indexed sequencing library with the Nextera XT DNA Library Preparation kit and the Nextera XT Index Kit (Illumina, San Diego, CA). The nine Illumina-adapted libraries were pooled at equal molar ratio, spiked with 20% PhiX control libraries (Illumina), and sequenced with one 1x150 cycles run (v3) on a MiSeq sequencer (Illumina). The fastq files generated from MiSeq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. A reference genome with the amplicon sequences was built and bisulfite converted in silico with Bismark Bisulfite Mapper. The high quality sequence reads were aligned to the reference genome using Bowtie 2. Cytosine methylation counting was performed with the Bismark methylation extractor. Differential methylation was analyzed with the Methylkit package. CpG sites with false discovery rate (FDR) less than 0.05 and absolute percent methylation difference greater than 5% were considered as significant. Results: Following knockdown of DNMT3a for 72 h, differentially methylated regions were identified in the promoter CpG sites of Myh7, Myh7b, Tnni3, Tnnt2, Nppa, Nppb, Mef2c, Mef2d, and Cdkn1C. Following knockdown of DNMT3b expression for 72 h, differentially methylated regions were identified in the promoter CpG sites of Myh6, Myh7b, Tnni3, Tnnt2, Nppa, Nppb, Mef2d, Cdkn1A, and Cdkn1C.
2017-09-08 | GSE77621 | GEO
Project description:Cytosine methylation is an epigenetic mark that dictates cell fate and response to stimuli. The timing and establishment of methylation logic during kidney development remains unknown. DNA methyltransferase 3a and 3b are the enzymes capable of establishing de novo methylation. We generated mice with genetic deletion of Dnmt3a and Dnmt3b in nephron progenitor cells (Six2Cre Dnmt3a/3b) and kidney tubule cells (KspCre Dnmt3a/3b). We characterized KspCre Dnmt3a/3b mice at baseline and after injury. Unbiased omics profiling, such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing and RNA sequencing were performed on whole-kidney samples and isolated renal tubule cells. KspCre Dnmt3a/3b mice showed no obvious morphologic and functional alterations at baseline. Knockout animals exhibited increased resistance to cisplatin-induced kidney injury, but not to folic acid–induced fibrosis. Whole-genome bisulfite sequencing indicated that Dnmt3a and Dnmt3b play an important role in methylation of gene regulatory regions that act as fetal-specific enhancers in the developing kidney but are decommissioned in the mature kidney. Loss of Dnmt3a and Dnmt3b resulted in failure to silence developmental genes. We also found that fetal-enhancer regions methylated by Dnmt3a and Dnmt3b were enriched for kidney disease genetic risk loci. Methylation patterns of kidneys from patients with CKD showed defects similar to those in mice with Dnmt3a and Dnmt3b deletion. Our results indicate a potential locus-specific convergence of genetic, epigenetic, and developmental elements in kidney disease development.
2020-03-03 | GSE134267 | GEO
Project description:RNA sequencing identifies differentially expressed genes in embryonic cardiomyocytes following knockdown of DNMT3a or DNMT3b expression
| PRJNA310690 | ENA
Project description:Purpose: This study aims to identify the differentially expressed genes in embryonic cardiomyocytes following the knockdown of DNMT3a or DNMT3b expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT3a siRNA (#GS13435), DNMT3b siRNAs (#GS13436), or AllStars negative control siRNA (#1027281) at 0.012 pmol using lipofectamine RNAiMAX (n=3 per treatment). At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA was isolated from the sorted cardiomyocytes with the RNAqueous®-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). RNA-Seq libraries were prepared with the NEBNext® mRNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (NEB, Ipswich, MA). The nine Illumina-adapted libraries were pooled at equal molar ratio and sequenced with one High Output 1x75 cycles run on a NextSeq500 sequencer (Illumina). The fastq files generated from RNA-Seq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. The remaining high quality RNA-Seq reads were mapped to the mouse genome (mm10) with the Tophat2 tool. Counting of RNA-seq reads were performed with HTSeq. Differential expression (DE) of genes between treatments was analyzed using two methods: R packages EdgeR and DEseq2, with Ensembl Mus_GRCm38.79.gtf as the reference annotation. Genes with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Unique DE genes were identified by combining the results generated from the two analytical methods. Results: DE analysis by DEseq2 and EdgeR software revealed that 524 genes were up-regulated, and 586 genes were down-regulated after knockdown of DNMT3a expression for 72 h. With the same analytical methods and criteria, the results of DE analysis showed that the knockdown of DNMT3b increased the expression of 655 genes and decreased the expression of 378 genes.
2017-09-08 | GSE77514 | GEO
Project description:DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity. Whole-genome bisulfite sequencing for Dnmt1,3a,3b-triple-KO ES cells expressing DNMT3A2 or DNMT3B1 and for Dnmt1,3a,3b,Setd2-KO ES cells expressing DNMT3B1
2015-01-20 | E-GEOD-57411 | biostudies-arrayexpress
Project description:Exploring the sequence specificity of methylation by DNMT3A and DNMT3B using bisulfite sequencing in E.coli and publicly available mouse and human datasets
2020-06-22 | E-MTAB-8406 | biostudies-arrayexpress
Project description:DNA methylation plays essential roles in mammalian development. During post-implantation development, de novo establishment of DNA methylation is accomplished by DNA methyltransferases, in particular DNMT3A and DNMT3B. At present, the distinct functions of these two enzymes remain largely elusive. To comprehensively identify the target sites for de novo DNA methylation by the DNMT3 enzymes, we took advantage of female mouse ES cells established in the presence of MEK and Gsk3 inhibitors, which lack most DNA methylation (2i/L ES cells), in combination with genetic ablation of Dnmt3a or Dnmt3b. We analyzed de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from Dnmt3 knockout (KO) 2i/L ES cells. Both Dnmt3a and Dnmt3b KO 2i-MEFs exhibited a modest but global reduction in CpG methylation, which was particularly notable on the X chromosome in Dnmt3b KO cells. Although most genes were methylated similarly in both knockouts, we identified 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b KO 2i-MEFs, respectively. Notably, Dnmt3a was exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes. Consistent with this, tissue-specific DNA methylation at PcG target developmental genes was substantially reduced in Dnmt3a KO embryos. Finally, we found that human patients with DNMT3A mutant acute myeloid leukemia (AML) or harboring DNMT3B mutation associated with immunodeficiency, centromere, and facial anomalies (ICF) syndrome exhibited reduced CpG methylation at regions that were hypomethylated in Dnmt3a or Dnmt3b KO 2i-MEFs, respectively. Collectively, our findings in DNA-hypomethylated 2i/L ES cells revealed a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.
2020-05-13 | GSE111172 | GEO
Project description:In early embryos, DNA methylation is remodelled to initiate the developmental program. For mostly unknown reasons, methylation marks are acquired unequally between embryonic and placental cells. To better understand this, we generated high-resolution maps of DNA methylation in mouse mid-gestation (E10.5) embryo and placenta. We uncovered specific subtypes of differentially methylated regions (DMRs) that contribute directly to the developmental asymmetry existing between mid-gestation embryo and placenta. We show that the asymmetry between embryonic and placental DNA methylation patterns occurs rapidly during the acquisition of marks in the post-implanted conceptus (E3.5-E6.5), and that these patterns are long-lasting across subtypes of DMRs throughout prenatal development and in somatic tissues. We reveal that at the peri-implantation stages, the de novo methyltransferase activity of DNMT3B is the main driver of methylation marks on asymmetric DMRs, and that DNMT3B can largely compensate for lack of DNMT3A in the epiblast and extraembryonic ectoderm, whereas DNMT3A can only partially palliate for the absence of DNMT3B. However, as development progresses and as DNMT3A becomes the principal de novo methyltransferase, the compensatory DNA methylation mechanism on DMRs becomes less effective.
2020-02-18 | GSE95610 | GEO
Project description:We demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome.
2017-08-22 | GSE97815 | GEO