The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases

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A comparative encyclopedia of DNA elements in the mouse genome

© 2014 Macmillan Publishers Limited. All rights reserved.The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain

A comparative encyclopedia of DNA elements in the mouse genome - eScholarship

Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation

Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

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Embryonic stem cell potency fluctuates with endogenous retrovirus activity

Epigenetic inheritance concerns the mechanisms that ensure the transmission of epigenetic marks from mother to daughter cell. Chromatin modifications and nuclear organization are candidate epigenetic marks — whether they fulfil the criterion of heritability and what mechanisms ensure their propagation is an area of intensive research. Studies that concern the mechanism of DNA replication have provided a major framework for understanding genetic transmission through multiple cell cycles. Recent work has begun to gain insight into possible means to ensure the stable transmission of information beyond just DNA, and has led to the concept of epigenetic inheritance. Considering chromatin-based information, key candidates have arisen as epigenetic marks, including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Understanding the dynamics and stability of these marks through the cell cycle is crucial in maintaining a given chromatin state.

Epigenetic inheritance during the cell cycle

DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these “replicon clusters” coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call “replication domains,” separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

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Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

/pdf/g9a-selectively-represses-a-class-of-late-replicating-genes-34rt726mxl.pdf

G9a selectively represses a class of late-replicating genes at the nuclear periphery

Eukaryotic DNA replication is regulated at the level of large chromosomal domains (0.5–5 megabases in mammals) within which replicons are activated relatively synchronously. These domains replicate in a specific temporal order during S-phase and our genome-wide analyses of replication timing have demonstrated that this temporal order of domain replication is a stable property of specific cell types. We have developed ReplicationDomain http://www.replicationdomain.org
 as a web-based database for analysis of genome-wide replication timing maps (replication profiles) from various cell lines and species. This database also provides comparative information of transcriptional expression and is configured to display any genome-wide property (for instance, ChIP-Chip or ChIP-Seq data) via an interactive web interface. Our published microarray data sets are publicly available. Users may graphically display these data sets for a selected genomic region and download the data displayed as text files, or alternatively, download complete genome-wide data sets. Furthermore, we have implemented a user registration system that allows registered users to upload their own data sets. Upon uploading, registered users may choose to: (1) view their data sets privately without sharing; (2) share with other registered users; or (3) make their published or "in press" data sets publicly available, which can fulfill journal and funding agencies' requirements for data sharing. ReplicationDomain is a novel and powerful tool to facilitate the comparative visualization of replication timing in various cell types as well as other genome-wide chromatin features and is considerably faster and more convenient than existing browsers when viewing multi-megabase segments of chromosomes. Furthermore, the data upload function with the option of private viewing or sharing of data sets between registered users should be a valuable resource for the scientific community.

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ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication-related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein-protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S-phase.

Tomoki Yokochi

Papers

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

G9a selectively represses a class of late-replicating genes at the nuclear periphery

ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data

Replication Labeling with Halogenated Thymidine Analogs