Project description:In this study, we mapped modification of lysine 4 and lysine 27 of histone H3 genome-wide in a series of mouse embryonic stem cells (mESCs) varying in DNA methylation levels based on knock-out and reconstitution of DNA methyltransferases (DNMTs). We extend previous studies showing cross-talk between DNA methylation and histone modifications by examining a breadth of histone modifications, causal relationships, and direct effects. Our data shows a causal regulation of H3K27me3 at gene promoters as well as H3K27ac and H3K27me3 at tissue-specific enhancers. We also identify isoform differences between DNMT family members. This study provides a comprehensive resource for the study of the complex interplay between DNA methylation and histone modification landscape. RNA-seq performed on wild-type, Dnmt triple knock-out (Dnmt1/3a/3b; TKO), Dnmt double knock-out (Dnmt3a/3b; DKO), and respective reconstitution mouse embryonic stem cell lines
Project description:In this study, we mapped modification of lysine 4 and lysine 27 of histone H3 genome-wide in a series of mouse embryonic stem cells (mESCs) varying in DNA methylation levels based on knock-out and reconstitution of DNA methyltransferases (DNMTs). We extend previous studies showing cross-talk between DNA methylation and histone modifications by examining a breadth of histone modifications, causal relationships, and direct effects. Our data shows a causal regulation of H3K27me3 at gene promoters as well as H3K27ac and H3K27me3 at tissue-specific enhancers. We also identify isoform differences between DNMT family members. This study provides a comprehensive resource for the study of the complex interplay between DNA methylation and histone modification landscape. Reduced representation bisulfite sequencing (RRBS) performed on wild-type, Dnmt triple knock-out (Dnmt1/3a/3b; TKO), Dnmt double knock-out (Dnmt3a/3b; DKO), and respective reconstitution mouse embryonic stem cell lines.
Project description:In this study, we mapped modification of lysine 4 and lysine 27 of histone H3 genome-wide in a series of mouse embryonic stem cells (mESCs) varying in DNA methylation levels based on knock-out and reconstitution of DNA methyltransferases (DNMTs). We extend previous studies showing cross-talk between DNA methylation and histone modifications by examining a breadth of histone modifications, causal relationships, and direct effects. Our data shows a causal regulation of H3K27me3 at gene promoters as well as H3K27ac and H3K27me3 at tissue-specific enhancers. We also identify isoform differences between DNMT family members. This study provides a comprehensive resource for the study of the complex interplay between DNA methylation and histone modification landscape. Histone ChIP-seq of H3K4me3, H3K27me3, H3K4me1, and H3K27ac were performed on wild-type, Dnmt triple knock-out (Dnmt1/3a/3b; TKO), Dnmt double knock-out (Dnmt3a/3b; DKO), and respective reconstitution mouse embryonic stem cell lines
Project description:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Somatic patterns of DNA methylation are largely static, apart from focal dynamics at gene regulatory elements. To further advance our understanding of the role of DNA methylation in human development and disease, we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death, demonstrating that DNA methylation is essential for human ESCs cultured in standard conditions. In summary, our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets. RRBS methylation profiling of DNMT3A/3B DKO human ES cells
Project description:Nephron number is a major determinant of long-term renal function. We hypothesized a link between epigenetic regulation and nephron formation. In support of this hypothesis, expression analysis evidenced high levels of DNA methyltransferases Dnmt1 and Dnmt3a in the nephrogenic zone of the developing mouse kidney. Using targeted loss-of-function manipulations in mice, we show that deletion of Dnmt1 in nephron progenitor cells results in a marked hypoplasia and reduction of nephron number at birth. In contrast, deletion of Dnmt3a/3b in nephron progenitor cells or deletion of Dnmt1/3a/3b in differentiated renal cells did not lead to any overt kidney phenotype. Whole mount optical projection tomography and 3D-reconstructions uncovered a significant reduction of stem cell niches and progenitor cells in Dnmt1-deficient mice. Ultimately, RNA sequencing analysis revealed that Dnmt1 controls DNA transcription regulating progenitor renewal, identity and differentiation. In summary, this study establishes DNA methylation as key regulatory event of prenatal renal programming.
Project description:DNA methylation is the net result of deposition by DNA methyltransferases (DNMT1, 3A and 3B) and removal by the Ten-Eleven Translocation 1-3 (TET1-3) family of proteins and/or passive loss by replication. The relative contribution of the individual enzymes and pathways is only partially understood. Here we comprehensively analyzed and mathematically simulated the dynamics of DNA de-methylation during the reprogramming of the hypermethylated serum-cultured mouse embryonic stem cells (ESCs) to the hypomethylated 2i-cultured ground state of mESC. We show that DNA demethylation readily occurs in TET[1-/-, 2-/-] ESCs with similar kinetics as their WT littermates. Vitamin C activation of TET causes accelerated and more profound DNA demethylation without markedly affecting reprogramming kinetics. We developed a mathematical model that highly accurately predicts the global level of 5methyl- and 5hydroxymethylcytosine during the transition. Modeling and experimental validation show that the concentration of DNMT3A and DNMT3B determines the steady state level of global DNA methylation and absence of DNMT3A/B even in continued presence of DNMT1 results in gradual loss of 5mC. Taken together, DNMT1 alone is insufficient to maintain DNA methylation but requires the action of DNMT3A/3B that act as a “dimmer switches”.
Project description:DNA methylation is the net result of deposition by DNA methyltransferases (DNMT1, 3A and 3B) and removal by the Ten-Eleven Translocation 1-3 (TET1-3) family of proteins and/or passive loss by replication. The relative contribution of the individual enzymes and pathways is only partially understood. Here we comprehensively analyzed and mathematically simulated the dynamics of DNA de-methylation during the reprogramming of the hypermethylated serum-cultured mouse embryonic stem cells (ESCs) to the hypomethylated 2i-cultured ground state of mESC. We show that DNA demethylation readily occurs in TET[1-/-, 2-/-] ESCs with similar kinetics as their WT littermates. Vitamin C activation of TET causes accelerated and more profound DNA demethylation without markedly affecting reprogramming kinetics. We developed a mathematical model that highly accurately predicts the global level of 5methyl- and 5hydroxymethylcytosine during the transition. Modeling and experimental validation show that the concentration of DNMT3A and DNMT3B determines the steady state level of global DNA methylation and absence of DNMT3A/B even in continued presence of DNMT1 results in gradual loss of 5mC. Taken together, DNMT1 alone is insufficient to maintain DNA methylation but requires the action of DNMT3A/3B that act as a “dimmer switches”.