Inference of emergent spatio-temporal processes from single-cell sequencing reveals feedback between de novo DNA methylation and chromatin condensates

SummaryRecent breakthroughs in single-cell genomics allow probing molecular states of cells with unprecedented detail along the sequence of the DNA. Biological function relies, however, on emergent processes in the three-dimensional space of the nucleus, such as droplet formation through phase separation. Here, we use single-cell multi-omics sequencing to develop a theoretical framework to rigorously map epigenome profiling along the DNA sequence onto a description of the emergent spatial dynamics in the nucleus. Drawing on scNMT-seq multi-omics sequencing in vitro and in vivo we exemplify our approach in the context of exit from pluripotency and global de novo methylation of the genome. We show how DNA methylation patterns of the embryonic genome are established through the interplay between spatially correlated DNA methylation and topological changes to the DNA. This feedback leads to the predicted formation of 30-40nm sized condensates of methylated DNA and determines genome-scale DNA methylation rates. We verify these findings with orthogonal single cell multi-omics data that combine the methylome with HiC measurements. Notably, this scale of chromatin organization has recently been described by super-resolution microscopy. Using this framework, we identify local methylation correlations in gene bodies that precede transcriptional changes at the exit from pluripotency. Our work provides a general framework of how mechanistic insights into emergent processes underlying cell fate decisions can be gained by the combination of single-cell multi-omics and methods from theoretical physics that have not been applied in the context of genomics before.HighlightsWe develop methodology to infer collective spatio-temporal processes in the physical space of the nucleus from single-cell methylome sequencing experiments.We show that DNA methylation relies on a feedback between de novo methylation and nanoscale changes in DNA topology, leading to the formation of methylation condensates.Chromatin condensates at this scale have recently been described by high-resolution microscopy but have remained without mechanistic explanation.Using this framework, we identify changes in the distribution of DNA methylation marks in gene bodies that precede gene silencing at the exit from pluripotency.

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Genome-scale oscillations in DNA methylation during exit from pluripotency

10.1101/338822 ◽

2018 ◽

Author(s):

Steffen Rulands ◽

Heather J Lee ◽

Stephen J Clark ◽

Christof Angermueller ◽

Sébastien A Smallwood ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Epigenetic Mark ◽

List Type ◽

Dna Hypomethylation ◽

Cell Fate Decisions ◽

Oscillatory Dynamics ◽

Genome Scale

SummaryPluripotency is accompanied by the erasure of parental epigenetic memory with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, coexpression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modelling, we show that this variability is associated with coherent, genome-scale, oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide fresh insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.HighlightsCo-expression of DNMT3s and TETs drive genome-scale oscillations of DNA methylationOscillation amplitude is greatest at a CpG density characteristic of enhancersCell synchronisation reveals oscillation period and link with primary transcriptsMultiomic single-cell profiling provides evidence for oscillatory dynamics in vivo

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Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification

10.1101/519207 ◽

2019 ◽

Cited By ~ 5

Author(s):

Ricard Argelaguet ◽

Hisham Mohammed ◽

Stephen J Clark ◽

L Carine Stapel ◽

Christel Krueger ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Cell Fate ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

List Type ◽

Germ Layer ◽

Epigenetic Landscape ◽

Germ Layers ◽

Cell Fate Decisions

AbstractFormation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers.HighlightsFirst map of mouse gastrulation using comprehensive single cell triple-omic analysis.Exit from pluripotency is associated with a global repressive epigenetic landscape, driven by a sharp gain of DNA methylation and a gradual decrease of chromatin accessibility.DNA methylation and chromatin accessibility changes in enhancers, but not in promoters, are associated with germ layer formation.Mesoderm and endoderm enhancers become open and demethylated upon lineage commitment.Ectoderm enhancers are primed in the early epiblast and protected from the global repressive dynamics, supporting a default model of ectoderm commitment in vivo.

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Bdh2 Deficiency Promotes Endoderm-Biased Early Differentiation of Mouse Embryonic Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.655145 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuting Fu ◽

Fangyuan Liu ◽

Shuo Cao ◽

Jia Zhang ◽

Huizhi Wang ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Iron Homeostasis ◽

Embryonic Stem ◽

Cell Fate Decisions ◽

Rate Limiting Step ◽

Fate Decision

3-hydroxybutyrate dehydrogenase-2 (Bdh2), a short-chain dehydrogenase, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore, playing a key role in iron homeostasis, energy metabolism and apoptosis. However, the function of Bdh2 in embryonic stem cells (ESCs) remains unknown. To gain insights into the role of Bdh2 on pluripotency and cell fate decisions of mouse ESCs, we generated Bdh2 homozygous knockout lines for both mouse advanced embryonic stem cell (ASC) and ESC using CRISPR/Cas9 genome editing technology. Bdh2 deficiency in both ASCs and ESCs had no effect on expression of core pluripotent transcription factors and alkaline phosphatase activity, suggesting dispensability of Bdh2 for self-renewal and pluripotency of ESCs. Interestingly, cells with Bdh2 deficiency exhibited potency of endoderm differentiation in vitro; with upregulated endoderm associated genes revealed by RNA-seq and RT-qPCR. We further demonstrate that Bdh2 loss inhibited expression of multiple methyltransferases (DNMTs) at both RNA and protein level, suggesting that Bdh2 may be essentially required to maintain DNA methylation in ASCs and ESCs. Overall, this study provides valuable data and resources for understanding how Bdh2 regulate earliest cell fate decision and DNA methylation in ASCs/ESCs.

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Understanding cell fate control by continuous single-cell quantification

Blood ◽

10.1182/blood-2018-09-835397 ◽

2019 ◽

Vol 133 (13) ◽

pp. 1406-1414 ◽

Cited By ~ 6

Author(s):

Dirk Loeffler ◽

Timm Schroeder

Keyword(s):

Single Cell ◽

Cell Fate ◽

Cell Imaging ◽

Population Based ◽

Molecular Processes ◽

Cell Fate Decisions ◽

Single Cell Imaging

Abstract Cells and the molecular processes underlying their behavior are highly dynamic. Understanding these dynamic biological processes requires noninvasive continuous quantitative single-cell observations, instead of population-based average or single-cell snapshot analysis. Ideally, single-cell dynamics are measured long-term in vivo; however, despite progress in recent years, technical limitations still prevent such studies. On the other hand, in vitro studies have proven to be useful for answering long-standing questions. Although technically still demanding, long-term single-cell imaging and tracking in vitro have become valuable tools to elucidate dynamic molecular processes and mechanisms, especially in rare and heterogeneous populations. Here, we review how continuous quantitative single-cell imaging of hematopoietic cells has been used to solve decades-long controversies. Because aberrant cell fate decisions are at the heart of tissue degeneration and disease, we argue that studying their molecular dynamics using quantitative single-cell imaging will also improve our understanding of these processes and lead to new strategies for therapies.

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The vitamin D receptor in cancer

Proceedings of The Nutrition Society ◽

10.1017/s0029665108006964 ◽

2008 ◽

Vol 67 (2) ◽

pp. 115-127 ◽

Cited By ~ 80

Author(s):

James Thorne ◽

Moray J. Campbell

Keyword(s):

Vitamin D ◽

Cell Fate ◽

Vitamin D Receptor ◽

De Novo ◽

Lithocholic Acid ◽

Cell Types ◽

Cell Fate Decisions ◽

Wide Range

Over the last 25 years roles have been established for vitamin D receptor (VDR) in influencing cell proliferation and differentiation. For example, murine knock-out approaches have revealed a role for the VDR in controlling mammary gland growth and function. These actions appear widespread, as the enzymes responsible for 1α,25-dihydroxycholecalciferol generation and degradation, and the VDR itself, are all functionally present in a wide range of epithelial and haematopoietic cell types. These findings, combined with epidemiological and functional data, support the concept that local, autocrine and paracrine VDR signalling exerts control over cell-fate decisions in multiple cell types. Furthermore, the recent identification of bile acid lithocholic acid as a VDR ligand underscores the environmental sensing role for the VDR.In vitroandin vivodissection of VDR signalling in cancers (e.g. breast, prostate and colon) supports a role for targeting the VDR in either chemoprevention or chemotherapy settings. As with other potential therapeutics, it has become clear that cancer cells displayde novoand acquired genetic and epigenetic mechanisms of resistance to these actions. Consequently, a range of experimental and clinical options are being developed to bring about more targeted actions, overcome resistance and enhance the efficacy of VDR-centred therapeutics.

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Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109475118 ◽

2021 ◽

Vol 118 (38) ◽

pp. e2109475118

Author(s):

Masaki Kinoshita ◽

Meng Amy Li ◽

Michael Barber ◽

William Mansfield ◽

Sabine Dietmann ◽

...

Keyword(s):

Dna Methylation ◽

Cell Fate ◽

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Dna Methyltransferases ◽

Bhlh Transcription Factor ◽

Mammalian Development

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

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Defining the Emerging Blood System During Development at Single-Cell Resolution

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.660350 ◽

2021 ◽

Vol 9 ◽

Author(s):

Göran Karlsson ◽

Mikael N. E. Sommarin ◽

Charlotta Böiers

Keyword(s):

Single Cell ◽

Cell Fate ◽

Blood Disorders ◽

Microfluidic Platforms ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Wide Range ◽

Targeted Gene Expression ◽

Definition Of

Developmental hematopoiesis differs from adult and is far less described. In the developing embryo, waves of lineage-restricted blood precede the ultimate emergence of definitive hematopoietic stem cells (dHSCs) capable of maintaining hematopoiesis throughout life. During the last two decades, the advent of single-cell genomics has provided tools to circumvent previously impeding characteristics of embryonic hematopoiesis, such as cell heterogeneity and rare cell states, allowing for definition of lineage trajectories, cellular hierarchies, and cell-type specification. The field has rapidly advanced from microfluidic platforms and targeted gene expression analysis, to high throughput unbiased single-cell transcriptomic profiling, single-cell chromatin analysis, and cell tracing—offering a plethora of tools to resolve important questions within hematopoietic development. Here, we describe how these technologies have been implemented to address a wide range of aspects of embryonic hematopoiesis ranging from the gene regulatory network of dHSC formation via endothelial to hematopoietic transition (EHT) and how EHT can be recapitulated in vitro, to hematopoietic trajectories and cell fate decisions. Together, these studies have important relevance for regenerative medicine and for our understanding of genetic blood disorders and childhood leukemias.

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Moderate DNA methylation changes associated with nitrogen remobilization and leaf senescence in Arabidopsis

10.1101/2021.09.17.460744 ◽

2021 ◽

Author(s):

Emil Vatov ◽

Ulrike Zentgraf ◽

Uwe Ludewig

Keyword(s):

Dna Methylation ◽

Transcription Factor ◽

Leaf Senescence ◽

De Novo ◽

Transcription Factor Binding ◽

List Type ◽

Nitrogen Remobilization ◽

Factor Binding ◽

Genetic Components

SummaryThe lifespan of plants and tissues is restricted by environmental and genetic components. Following the transition to reproductive growth, leaf senescence ceases cellular life in monocarpic plants to remobilize nutrients to storage organs.We observed altered leaf to seed ratios, faster senescence progression and enhanced nitrogen remobilization from the leaves in two methylation mutants (ros1 and the triple dmr1/2 cmt3 knockout).DNA methylation in wild type Col-0 leaves initially moderately declined with progressing leaf senescence, predominantly in the CG context, while the ultimate phase of leaf discoloration was associated with moderate de novo methylation of cytosines, primarily in the CHH context.Relatively few differentially methylated regions, including one in the ROS1 promoter linked to the down-regulation of ROS1, were present, but these were unrelated to known senescence-associated genes.Differential methylation patterns were identified in transcription factor binding sites, such as the W-boxes that are targeted by WRKYs, which impaired transcription factor binding when methylated in vitro.Mutants that are defective in DNA methylation showed distinct nitrogen remobilization, which was associated with altered patterns of leaf senescence progression. But moderate methylome changes during leaf senescence were not specifically associated with up-regulated genes during senescence.

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