scholarly journals High Resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

2019 ◽  
Author(s):  
Peiyao A. Zhao ◽  
Takayo Sasaki ◽  
David M. Gilbert
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Cell Types ◽  
S Phase ◽  
List Type ◽  
Cell Type ◽  
Histone Marks ◽  
Cell Type Specific

ABSTRACTDNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. RT is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116 as well as F1 subspecies hybrid mouse embryonic stem cells and their neural progenitor derivatives. Strong enrichment of nascent DNA in fine temporal windows reveals a remarkable degree of cell to cell conservation in replication timing and patterns of replication genome-wide. We identify 5 patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation were found throughout S phase and resolved to ~50kb precision. Temporal transition regions were resolved into segments of uni-directional replication punctuated with small zones of inefficient initiation. Small and large valleys of convergent replication were consistent with either termination or broadly distributed initiation, respectively. RT correlated with chromatin compartment across all cell types but correlations of initiation time to chromatin domain boundaries and histone marks were cell type specific. Haplotype phasing revealed previously unappreciated regions of allele-specific and alleleindependent asynchronous replication. Allele-independent asynchrony was associated with large transcribed genes that resemble common fragile sites. Altogether, these data reveal a remarkably deterministic temporal choreography of DNA replication in mammalian cells.Highly homogeneous replication landscape between cells in a populationInitiation zones resolved within constant timing and timing transition regionsActive histone marks enriched within early initiation zones while enrichment of repressive marks is cell type specific.Transcribed long genes replicate asynchronously.

scholarly journals Genome-wide stability of the DNA replication program in single mammalian cells

2017 ◽  
Author(s):  
Saori Takahashi ◽  
Hisashi Miura ◽  
Takahiro Shibata ◽  
Koji Nagao ◽  
Katsuzumi Okumura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Developmental Plasticity ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Small Degree ◽  
3D Genome ◽  
Inactive X Chromosome ◽  
Genome Wide ◽  
Individual Mouse

ABSTRACTHere, we report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication timing heterogeneity was present, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization.

scholarly journals Accurate imputation of histone modifications using transcription

2020 ◽  
Author(s):  
Zhong Wang ◽  
Alexandra G. Chivu ◽  
Lauren A. Choate ◽  
Edward J. Rice ◽  
Donald C. Miller ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Cell Type ◽  
Active Chromatin ◽  
Histone Marks ◽  
Repressive Mark ◽  
Cell Type Specific ◽  
The Relationship ◽  
Machine Learning Tool

AbstractWe trained a sensitive machine learning tool to infer the distribution of histone marks using maps of nascent transcription. Transcription captured the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. The relationship between active histone marks and transcription was conserved in all cell types examined, allowing individual labs to annotate active functional elements in mammals with similar richness as major consortia. Using imputation as an interpretative tool uncovered cell-type specific differences in how the PRC2-dependent repressive mark, H3K27me3, corresponds to transcription, and revealed that transcription initiation requires both chromatin accessibility and an active chromatin environment demonstrating that initiation is less promiscuous than previously thought.

scholarly journals Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 196 ◽  
Author(s):  
Phoebe Oldach ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Replication Timing ◽  
S Phase ◽  
Genome Architecture ◽  
3D Genome ◽  
Synchronized Cells ◽  
Dna Replication Timing ◽  
Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

scholarly journals Joint inference and alignment of genome structures enables characterization of compartment-independent reorganization across cell types

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Lila Rieber ◽  
Shaun Mahony
Keyword(s):  
K562 Cells ◽  
Cell Types ◽  
Data Sets ◽  
Cell Type ◽  
Histone Marks ◽  
Joint Inference ◽  
3D Localization ◽  
Topologically Associated Domains ◽  
Cell Type Specific

Abstract Background Comparisons of Hi–C data sets between cell types and conditions have revealed differences in topologically associated domains (TADs) and A/B compartmentalization, which are correlated with differences in gene regulation. However, previous comparisons have focused on known forms of 3D organization while potentially neglecting other functionally relevant differences. We aimed to create a method to quantify all locus-specific differences between two Hi–C data sets. Results We developed MultiMDS to jointly infer and align 3D chromosomal structures from two Hi–C data sets, thereby enabling a new way to comprehensively quantify relocalization of genomic loci between cell types. We demonstrate this approach by comparing Hi–C data across a variety of cell types. We consistently find relocalization of loci with minimal difference in A/B compartment score. For example, we identify compartment-independent relocalizations between GM12878 and K562 cells that involve loci displaying enhancer-associated histone marks in one cell type and polycomb-associated histone marks in the other. Conclusions MultiMDS is the first tool to identify all loci that relocalize between two Hi–C data sets. Our method can identify 3D localization differences that are correlated with cell-type-specific regulatory activities and which cannot be identified using other methods.

scholarly journals Mammalian non-CG methylations are conserved and cell-type specific and may have been involved in the evolution of transposon elements

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Weilong Guo ◽  
Michael Q. Zhang ◽  
Hong Wu
Keyword(s):  
Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Biological Significance ◽  
Prediction Method ◽  
Cell Types ◽  
Evolutionary Development ◽  
Cell Type ◽  
Brain Neurons ◽  
Cell Type Specific

Abstract Although non-CG methylations are abundant in several mammalian cell types, their biological significance is sparsely characterized. We gathered 51 human and mouse DNA methylomes from brain neurons, embryonic stem cells and induced pluripotent stem cells, primordial germ cells and oocytes. We utilized an unbiased sub-motif prediction method and reported CW as the representative non-CG methylation context, which is distinct from CC methylation in terms of sequence context and genomic distribution. A two-dimensional comparison of non-CG methylations across cell types and species was performed. Unambiguous studies of sequence preferences and genomic region enrichment showed that CW methylation is cell-type specific and is also conserved between humans and mice. In brain neurons, it was found that active long interspersed nuclear element-1 (LINE-1) lacked CW methylations but not CG methylations. Coincidentally, both human Alu and mouse B1 elements preferred high CW methylations at specific loci during their respective evolutionary development. Last, the strand-specific distributions of CW methylations in introns and long interspersed nuclear elements are also cell-type specific and conserved. In summary, our results illustrate that CW methylations are highly conserved among species, are dynamically regulated in each cell type, and are potentially involved in the evolution of transposon elements.

scholarly journals Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects underlying impaired adult neurogenesis

2018 ◽  
Author(s):  
Vera Zywitza ◽  
Aristotelis Misios ◽  
Lena Bunatyan ◽  
Thomas E. Willnow ◽  
Nikolaus Rajewsky
Keyword(s):  
Single Cell ◽  
Adult Neurogenesis ◽  
Subventricular Zone ◽  
Single Cells ◽  
Cell Types ◽  
Neural Cell ◽  
Adult Brain ◽  
List Type ◽  
Cell Type ◽  
Cell Type Specific

SUMMARYNeural stem cells (NSCs) contribute to plasticity and repair of the adult brain. Niches harboring NSCs are crucial for regulating stem cell self-renewal and differentiation. We used single-cell RNA profiling to generate an unbiased molecular atlas of all cell types in the largest neurogenic niche of the adult mouse brain, the subventricular zone (SVZ). We characterized > 20 neural and non-neural cell types and gained insights into the dynamics of neurogenesis by predicting future cell states based on computational analysis of RNA kinetics. Furthermore, we apply our single-cell approach to mice lacking LRP2, an endocytic receptor required for SVZ maintenance. The number of NSCs and proliferating progenitors was significantly reduced. Moreover, Wnt and BMP4 signaling was perturbed. We provide a valuable resource for adult neurogenesis, insights into SVZ neurogenesis regulation by LRP2, and a proof-of-principle demonstrating the power of single-cell RNA-seq in pinpointing neural cell type-specific functions in loss-of-function models.HIGHLIGHTSunbiased single-cell transcriptomics characterizes adult NSCs and their nichecell type-specific signatures and marker genes for 22 SVZ cell typesFree online tool to assess gene expression across 9,804 single cellscell type-specific dysfunctions underlying impaired adult neurogenesis

scholarly journals Differential Subnuclear Localization and Replication Timing of Histone H3 Lysine 9 Methylation States

2005 ◽  
Vol 16 (6) ◽  
pp. 2872-2881 ◽  
Author(s):  
Rong Wu ◽  
Anna V. Terry ◽  
Prim B. Singh ◽  
David M. Gilbert
Keyword(s):  
Mammalian Cells ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Replication Timing ◽  
Pericentric Heterochromatin ◽  
S Phase ◽  
Subnuclear Localization ◽  
Chromosome Domains ◽  
Mouse Cells ◽  
Additional Level

Mono-, di-, and trimethylation of specific histone residues adds an additional level of complexity to the range of histone modifications that may contribute to a histone code. However, it has not been clear whether different methylated states reside stably at different chromatin sites or whether they represent dynamic intermediates at the same chromatin sites. Here, we have used recently developed antibodies that are highly specific for mono-, di-, and trimethylated lysine 9 of histone H3 (MeK9H3) to examine the subnuclear localization and replication timing of chromatin containing these epigenetic marks in mammalian cells. Me1K9H3 was largely restricted to early replicating, small punctate domains in the nuclear interior. Me2K9H3 was the predominant MeK9 epitope at the nuclear and nucleolar periphery and colocalized with sites of DNA synthesis primarily in mid-S phase. Me3K9H3 decorated late-replicating pericentric heterochromatin in mouse cells and sites of DAPI-dense intranuclear heterochromatin in human and hamster cells that replicated throughout S phase. Disruption of the Suv39h1,2 or G9a methyltransferases in murine embryonic stem cells resulted in a redistribution of methyl epitopes, but did not alter the overall spatiotemporal replication program. These results demonstrate that mono-, di-, and trimethylated states of K9H3 largely occupy distinct chromosome domains.

scholarly journals Distinct transcriptomic cell types and neural circuits of the subiculum and prosubiculum along the dorsal-ventral axis

2019 ◽  
Author(s):  
Song-Lin Ding ◽  
Zizhen Yao ◽  
Karla E. Hirokawa ◽  
Thuc Nghi Nguyen ◽  
Lucas T. Graybuck ◽  
...  
Keyword(s):  
Neural Circuits ◽  
Cell Types ◽  
Brain Regions ◽  
Spatial Processing ◽  
List Type ◽  
Cell Type ◽  
Functional Studies ◽  
Hybridization Data ◽  
Cell Type Specific ◽  
Subcortical Regions

SummarySubicular region plays important roles in spatial processing and many cognitive functions and these were mainly attributed to subiculum (Sub) rather than prosubiculum (PS). Using single-cell RNA-sequencing (scRNA-seq) technique we have identified up to 27 distinct transcriptomic clusters/cell types, which were registered to anatomical sub-domains in Sub and PS. Based on reliable molecular markers derived from transcriptomic clustering and in situ hybridization data, the precise boundaries of Sub and PS have been consistently defined along the dorsoventral (DV) axis. Using these borders to evaluate Cre-line specificity and tracer injections, we have found bona fide Sub projections topographically to structures important for spatial processing and navigation. In contrast, PS along DV axis sends its outputs to widespread brain regions crucial for motivation, emotion, reward, stress, anxiety and fear. Brain-wide cell-type specific projections of Sub and PS have also been revealed using specific Cre-lines. These results reveal two molecularly and anatomically distinct circuits centered in Sub and PS, respectively, providing a consistent explanation to historical data and a clearer foundation for future functional studies.Highlights27 transcriptomic cell types identified in and spatially registered to “subicular” regions.Anatomic borders of “subicular” regions reliably determined along dorsal-ventral axis.Distinct cell types and circuits of full-length subiculum (Sub) and prosubiculum (PS).Brain-wide cell-type specific projections of Sub and PS revealed with specific Cre-lines.In BriefDing et al. show that mouse subiculum and prosubiculum are two distinct regions with differential transcriptomic cell types, subtypes, neural circuits and functional correlation. The former has obvious topographic projections to its main targets while the latter exhibits widespread projections to many subcortical regions associated with reward, emotion, stress and motivation.

scholarly journals scRNA-seq reveals ACE2 and TMPRSS2 expression in TROP2+ Liver Progenitor Cells: Implications in COVID-19 associated Liver Dysfunction

2020 ◽  
Author(s):  
Justine Jia Wen Seow ◽  
Rhea Pai ◽  
Archita Mishra ◽  
Edwin Shepherdson ◽  
Tony Kiat Hon Lim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Liver Dysfunction ◽  
Mammalian Cells ◽  
Fetal Liver ◽  
Cell Types ◽  
List Type ◽  
Cell Type ◽  
Mammalian Liver ◽  
Common Respiratory Virus ◽  
Transmembrane Serine Protease

SummaryThe recent pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 was first reported in China (December 2019) and now prevalent in ∼170 countries across the globe. Entry of SARS-CoV-2 into mammalian cells require the binding of viral Spike (S) proteins to the ACE2 (angiotensin converting enzyme 2) receptor. Once entered the S protein is primed by a specialised serine protease, TMPRSS2 (Transmembrane Serine Protease 2) in the host cell. Importantly, beside respiratory symptoms, consistent with other common respiratory virus infection when patients become viraemic, a significant number of COVID-19 patients also develop liver comorbidities. We explored if specific target cell-type in the mammalian liver, could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here we employed single-cell RNA-seq (scRNA-seq) to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We report the co-expression of ACE2 and TMPRSS2 in a TROP2+ liver progenitor population. Importantly, we fail to detect the expression of ACE2 in hepatocyte or any other liver (immune and stromal) cell types. These results indicated that in COVID-19 associated liver dysfunction and cell death, viral infection of TROP2+ progenitors in liver may significantly impaired liver regeneration and could lead to pathology.Highlights- EPCAM+ Liver progenitors co-express ACE2 and TMPRSS2- ACE2 and TMPRSS2 expression is highest in TROP2high progenitors- ACE2 and TMPRSS2 cells express cholangiocyte biased fate markers- ACE2 and TMPRSS2 positive cells are absent in human fetal liver

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