Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution

Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.National Institutes of Health (U.S.) (Grant HD 045022

Identification of Novel Imprinted Differentially Methylated Regions by Global Analysis of Human-Parthenogenetic-Induced Pluripotent Stem Cells

Parental imprinting is an epigenetic phenomenon by which genes are expressed in a monoallelic fashion, according to their parent of origin. DNA methylation is considered the hallmark mechanism regulating parental imprinting. To identify imprinted differentially methylated regions (DMRs), we compared the DNA methylation status between multiple normal and parthenogenetic human pluripotent stem cells (PSCs) by performing reduced representation bisulfite sequencing. Our analysis identified over 20 previously unknown imprinted DMRs in addition to the known DMRs. These include DMRs in loci associated with human disorders, and a class of intergenic DMRs that do not seem to be related to gene expression. Furthermore, the study showed some DMRs to be unstable, liable to differentiation or reprogramming. A comprehensive comparison between mouse and human DMRs identified almost half of the imprinted DMRs to be species specific. Taken together, our data map novel DMRs in the human genome, their evolutionary conservation, and relation to gene expression

Editing DNA Methylation in the Mammalian Genome

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.National Institutes of Health (U.S.) (Grant HD045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant GM114864

Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.Simons Foundation (Grant SFLIFE 286977)National Institutes of Health (U.S.) (Grant RO1-CA084198)National Science Foundation (U.S.). Graduate Research FellowshipJerome and Florence Brill Graduate Student Fellowshi

Parent-of-Origin DNA Methylation Dynamics during Mouse Development

Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development, suggesting that imprints are faithfully maintained in embryos and adults. To test this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single-cell resolution. Biallelic gain or loss of IG-DMR methylation occurred in a small fraction of mouse embryonic stem cells, significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation, causing substantial and consistent tissue- and cell-type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis, resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance

Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts

Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5′ RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes

Mouse embryo model derived exclusively from embryonic stem cells undergoes neurulation and heart development.

Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure

Balanced gene dosage control rather than parental origin underpins genomic imprinting.

Mammalian parental imprinting represents an exquisite form of epigenetic control regulating the parent-specific monoallelic expression of genes in clusters. While imprinting perturbations are widely associated with developmental abnormalities, the intricate regional interplay between imprinted genes makes interpreting the contribution of gene dosage effects to phenotypes a challenging task. Using mouse models with distinct deletions in an intergenic region controlling imprinting across the Dlk1-Dio3 domain, we link changes in genetic and epigenetic states to allelic-expression and phenotypic outcome in vivo. This determined how hierarchical interactions between regulatory elements orchestrate robust parent-specific expression, with implications for non-imprinted gene regulation. Strikingly, flipping imprinting on the parental chromosomes by crossing genotypes of complete and partial intergenic element deletions rescues the lethality of each deletion on its own. Our work indicates that parental origin of an epigenetic state is irrelevant as long as appropriate balanced gene expression is established and maintained at imprinted loci

Editing DNA methylation in the mammalian genome

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.NIH [HD045022, R37-CA084198, HG002668, GM114864]; Damon Runyon Cancer Foundation Postdoctoral Fellowship; NARSAD Young Investigator Fellowship; Helen Hay Whitney Foundation Postdoctoral Fellowship; HARMONIA grant from the National Science Centre in Poland [UMO-2014/14/M/NZ1/00010]; Human Frontier Science Program postdoctoral fellowshipSCI(E)[email protected]+16