Mouse embryonic stem cells (mESCs) cultured under serum/LIF conditions exhibit heterogeneous expression of pluripotency-associated factors that can be overcome by two inhibitors (2i) of the MEK and ...GSK3 pathways. Several studies have shown that the “ground state” induced by 2i is characterized by global hypomethylation and specific transcriptional profiles, but little is known about the contributing effectors. Here we show that 2i conditions rapidly alter the global binding landscape of OCT4, SOX2, and NANOG. The dynamic binding influences enhancer activity and shows enrichment for regulators linked to Wnt and Erk signaling. Epigenomic characterization provided limited insights to the immediate transcriptional dynamics, suggesting that these are likely more secondary effects. Likewise, loss of the PRC2 component EED to prevent H3K27me3 deposition had minimal effect on the transcriptome, implying that it is largely dispensable for continued repression of bivalent genes and de novo silencing in 2i.
Display omitted
•Early and widespread reconfiguration of OCT4, SOX2, and NANOG binding in 2i•Differential OSN binding correlates with enhancer activity in 2i•Dynamic targets show co-occurring motifs of factors linked to Wnt and Erk signaling•PRC2 appears dispensable for continued silencing and de novo repression in 2i
Meissner and colleagues provide a comprehensive characterization of the early OCT4, SOX2, and NANOG binding dynamics during the transition from serum/LIF to 2i. The target rewiring predominantly affects distal regulatory elements with a plausible link to the upstream signaling pathways. Notably, these events appear before any major epigenetic reprogramming events, which occur only later in the transition.
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases ...(DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG ...modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2R140Q in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2R140Q can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.
Display omitted
•Development of a doxycycline-inducible mouse model of IDH2R140Q•IDH2R140Q drives aberrant self-renewal and a block of differentiation in HSPCs•IDH2R140Q cooperates with Flt3ITD to drive acute leukemia in vivo•IDH2R140Q is essential for leukemia maintenance
Kats et al. show that mutant IDH2 drives self-renewal of HSPCs and cooperates with oncogenes FLT3, HoxA9, and Meis1a to drive initiation of leukemia in vivo.
DNA methylation plays an essential role in mammalian genomes and expression of the responsible enzymes is tightly controlled. Deregulation of the de novo DNA methyltransferase DNMT3B is frequently ...observed across cancer types, yet little is known about its ectopic genomic targets. Here, we used an inducible transgenic mouse model to delineate rules for abnormal DNMT3B targeting, as well as the constraints of its activity across different cell types. Our results explain the preferential susceptibility of certain CpG islands to aberrant methylation and point to transcriptional state and the associated chromatin landscape as the strongest predictors. Although DNA methylation and H3K27me3 are usually non-overlapping at CpG islands, H3K27me3 can transiently co-occur with DNMT3B-induced DNA methylation. Our genome-wide data combined with ultra-deep locus-specific bisulfite sequencing suggest a distributive activity of ectopically expressed Dnmt3b that leads to discordant CpG island hypermethylation and provides new insights for interpreting the cancer methylome.
Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by ...examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.
Display omitted
•Sequential epigenetic changes occur on the Xi and define reprogramming stages•XCR requires pluripotency gene induction, DNA demethylation, and Xist loss•Tet1, Tet2, and global 5hmC are dispensable for XCR•The reprogramming stage of any cell can be determined by epigenetic states
Tracking the steps of reactivation of the inactive X chromosome during reprogramming to pluripotency defines discrete stages of the reprogramming process and reveals that some aspects of pluripotency induction follow developmental sequences in reverse, but others, such as removal DNA methylation, occur in a different sequence.
Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. ...Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes, and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed-onset neuropsychiatric complications.
Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life.
The generation of genomic binding or accessibility data from massively parallel sequencing technologies such as ChIP-seq and DNase-seq continues to accelerate. Yet state-of-the-art computational ...approaches for the identification of DNA binding motifs often yield motifs of weak predictive power. Here we present a novel computational algorithm called MotifSpec, designed to find predictive motifs, in contrast to over-represented sequence elements. The key distinguishing feature of this algorithm is that it uses a dynamic search space and a learned threshold to find discriminative motifs in combination with the modeling of motifs using a full PWM (position weight matrix) rather than k-mer words or regular expressions. We demonstrate that our approach finds motifs corresponding to known binding specificities in several mammalian ChIP-seq datasets, and that our PWMs classify the ChIP-seq signals with accuracy comparable to, or marginally better than motifs from the best existing algorithms. In other datasets, our algorithm identifies novel motifs where other methods fail. Finally, we apply this algorithm to detect motifs from expression datasets in C. elegans using a dynamic expression similarity metric rather than fixed expression clusters, and find novel predictive motifs.
A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during ...reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.
Display omitted
•Female iPSCs are globally hypomethylated•Hypomethylation affects multiple genomic features including imprint control regions•Loss of X chromosome restores global methylation but not at imprint control regions•Methylation changes at enhancers in male and female reprogramming intermediates
Somatic cells can be reprogrammed to iPSCs, inducing reactivation of the inactive X chromosome. Using genome-scale DNA methylation analyses, Plath, Pasque, and colleagues show that iPSCs adopt sex-specific differences in global DNA methylation that correlate with the presence of two active X chromosomes. Upon culture, female iPSCs lose one of the two X chromosomes and adopt male-like DNA methylation.
Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal ...microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.
Display omitted
•PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus•PRC2 transcriptionally regulates the Gtl2-Rian-Mirg locus through DNAme at IG-DMR•IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus•PRC2 prevents de novo DNAme at IG-DMR for maternal Gtl2-Rian-Mirg locus expression
Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are both typically correlated with gene repression. Das et al. find that PRC2 prevents recruitment of Dnmt3s and DNAme at the IG-DMR element, thus allowing proper expression of the nearby maternal Gtl2-Rian-Mirg locus.
PDF
