Spatio-temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cells

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 05/2005; 95(1):74-82. DOI: 10.1002/jcb.20395
Source: PubMed


The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains.

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Available from: David M Gilbert
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    • "Thus, Tet enzymatic activity may be associated with active chromatin formation and/or maintenance in ESCs. Active chromosomal regions generally replicate in early S phase of the cell cycle, while inactive chromosomal regions, including constitutive and facultative heterochromatin, replicate in late S. One exception to this is perinuclear heterochromatin, which replicates in mid S (Nakayasu and Berezney 1989; Panning and Gilbert 2005). Based on this, active chromosomal regions can be determined by the chromosomal replication bands called R-bands as regions that are negative for 5-bromodeoxyuridine (BrdU) labeling during late S to metaphase. "
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    ABSTRACT: DNA cytosine methylation (5mC) is indispensable for a number of cellular processes, including retrotransposon silencing, genomic imprinting, and X chromosome inactivation in mammalian development. Recent studies have focused on 5-hydroxymethylcytosine (5hmC), a new epigenetic mark or intermediate in the DNA demethylation pathway. However, 5hmC itself has no role in pluripotency maintenance in mouse embryonic stem cells (ESCs) lacking Dnmt1, 3a, and 3b. Here, we demonstrated that 5hmC accumulated on euchromatic chromosomal bands that were marked with di- and tri-methylated histone H3 at lysine 4 (H3K4me2/3) in mouse ESCs. By contrast, heterochromatin enriched with H3K9me3, including mouse chromosomal G-bands, pericentric repeats, human satellite 2 and 3, and inactive X chromosomes, was not enriched with 5hmC. Therefore, enzymes that hydroxylate the methyl group of 5mC belonging to the Tet family might be excluded from inactive chromatin, which may restrict 5mC to 5hmC conversion in euchromatin to prevent nonselective de novo DNA methylation. Electronic supplementary material The online version of this article (doi:10.1007/s10577-012-9317-9) contains supplementary material, which is available to authorized users.
    Full-text · Article · Oct 2012 · Chromosome Research
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    • "If the emerging cell was to commit before cell division, it would abrogate the possibility of self-renewal. Another interesting nuance of this model relates to the fact that the length of G1 phase is very short in mouse ESCs (Panning and Gilbert, 2005). In addition, chromatin is very dispersed relative to differentiated cells (Hiratani et al., 2010), and there are a larger number of smaller early-and late-replicating domains (Hiratani et al., 2008). "
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    ABSTRACT: Recent findings suggest that large-scale remodeling of three dimensional (3D) chromatin architecture occurs during a brief period in early G1 phase termed the replication timing decision point (TDP). In this speculative article, I suggest that the TDP may represent an as yet unappreciated window of opportunity for extracellular cues to influence 3D architecture during stem cell fate decisions. I also describe several testable predictions of this hypothesis.
    Full-text · Article · Nov 2010 · The Journal of Cell Biology
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    • "Unfortunately , DNA-FISH, even when carried out at a larger scale, would still only query a fraction of the genome. However, in both ESCs and differentiated cell types, DNA synthesis takes place almost exclusively in the interior of the nucleus throughout the first half of S-phase, and then dramatically transitions to the periphery in middle (mid) S-phase (Panning and Gilbert 2005; Wu et al. Genome-wide dynamics of replication timing "
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    ABSTRACT: Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.
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