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Global reorganization of replication domains during embryonic stem cell differentiation.

Hiratani I, Ryba T, Itoh M, Yokochi T, Schwaiger M, Chang CW, Lyou Y, Townes TM, Schübeler D, Gilbert DM - PLoS Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.

ABSTRACT
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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Replication Timing and Transcription Changes during Differentiation(A and B) Average replication-timing ratios of replication domains were plotted against their “present” (i.e., transcriptionally active) gene density for ESCs (A) and NPCs (B). Pearson R2 values are shown.(C and D) Correlation between early replication and the probability of expression. Genes were ranked by their replication-timing ratio and divided into bins of 100 genes, the height of which represents the percentage of active (i.e., ”present”) genes within each bin. The width and position of each bin represents its range of replication-timing ratios. Logistic regression (inner line) and 95% confidence intervals (outer lines) reveal a clear correlation in both ESCs (C) and NPCs (D). By the Likelihood Ratio test (a goodness-of-fit test), the fitted model is significantly different (p < 2 × 10−16 for both ESCs and NPCs) from that of a  hypothesis in which replication timing has no correlation to transcription.(E) Box plots showing the fold changes in transcription [i.e., log2(NPC/ESC)] of LtoE, EtoL, LtoL, and EtoE genes. RefSeq genes with the 5% greatest replication timing changes were defined as EtoL and LtoE, whereas those with the least changes (lowest 20%) that maintained replication timing ratio of above 0.5 or below −0.5 were defined as EtoE and LtoL, respectively.(F) Percentage of 2-fold up- or down-regulated genes within LtoE, EtoL, LtoL, and EtoE domains defined in Figure 4G.(G) Summary of expression patterns of genes within LtoE, EtoL, LtoL, and EtoE domains. Definitions are as follows: “Up” and “Down,” above 2-fold up- and down-regulation, respectively; “Unchanged,” below 2-fold up-/down-regulation; “Unchanged Only,” domains with both active and silent genes that do not change by 2-fold; and “Silent Only,” domains with only silent genes in both states.(H) LINE-1 transposable elements are actively transcribed in ESCs but become inactive in NPCs as assayed by RNA-FISH. Mean and standard error of mean (SEM) of the number of RNA-FISH signals per nucleus is shown (n = 30 from two biological replicates). The p-value was obtained from a two-tailed t-test for comparison of two unpaired groups.
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pbio-0060245-g005: Replication Timing and Transcription Changes during Differentiation(A and B) Average replication-timing ratios of replication domains were plotted against their “present” (i.e., transcriptionally active) gene density for ESCs (A) and NPCs (B). Pearson R2 values are shown.(C and D) Correlation between early replication and the probability of expression. Genes were ranked by their replication-timing ratio and divided into bins of 100 genes, the height of which represents the percentage of active (i.e., ”present”) genes within each bin. The width and position of each bin represents its range of replication-timing ratios. Logistic regression (inner line) and 95% confidence intervals (outer lines) reveal a clear correlation in both ESCs (C) and NPCs (D). By the Likelihood Ratio test (a goodness-of-fit test), the fitted model is significantly different (p < 2 × 10−16 for both ESCs and NPCs) from that of a hypothesis in which replication timing has no correlation to transcription.(E) Box plots showing the fold changes in transcription [i.e., log2(NPC/ESC)] of LtoE, EtoL, LtoL, and EtoE genes. RefSeq genes with the 5% greatest replication timing changes were defined as EtoL and LtoE, whereas those with the least changes (lowest 20%) that maintained replication timing ratio of above 0.5 or below −0.5 were defined as EtoE and LtoL, respectively.(F) Percentage of 2-fold up- or down-regulated genes within LtoE, EtoL, LtoL, and EtoE domains defined in Figure 4G.(G) Summary of expression patterns of genes within LtoE, EtoL, LtoL, and EtoE domains. Definitions are as follows: “Up” and “Down,” above 2-fold up- and down-regulation, respectively; “Unchanged,” below 2-fold up-/down-regulation; “Unchanged Only,” domains with both active and silent genes that do not change by 2-fold; and “Silent Only,” domains with only silent genes in both states.(H) LINE-1 transposable elements are actively transcribed in ESCs but become inactive in NPCs as assayed by RNA-FISH. Mean and standard error of mean (SEM) of the number of RNA-FISH signals per nucleus is shown (n = 30 from two biological replicates). The p-value was obtained from a two-tailed t-test for comparison of two unpaired groups.

Mentions: Genes that are transcribed are generally early replicating, whereas genes that are late replicating are almost always silent; however, exceptions to this rule have been described [16,47,48]. Importantly, no study has comprehensively examined the changes in gene expression as they relate to changes in replication timing. To address this issue, we analyzed the steady-state levels of annotated gene transcripts before and after differentiation to NPCs using Affymetrix GeneChips. Regardless of whether levels, density, or number of active genes were examined, either at the level of domains (Figure 5A and 5B) or individual genes (Figure 5C and 5D), both differentiation states clearly showed a similar positive correlation between early replication and transcription. Consistent with previous findings across a portion of the Drosophila genome [25], this positive correlation was greater when integrated over large regions (approximately 600 kb for ESCs and NPCs vs. 180 kb in Drosophila; not shown). The maintenance of this statistical relationship during differentiation can be accounted for by the directionality of transcriptional changes within each domain (Figure 5E and 5F). At the level of individual genes, LtoE genes were mostly up-regulated, whereas EtoL genes showed a weak tendency to be down-regulated. At the level of domains, among those domains that contained at least one RefSeq gene (National Center for Biotechnology Information [NCBI] most well annotated; http://www.ncbi.nlm.nih.gov/RefSeq/), the majority of LtoE domains contained only up-regulated genes, whereas EtoL domains contain mostly down-regulated or unchanged genes (Figure 5G). Note that because most genes remain early replicating, the overall trend in gene expression changes is dominated by this EtoE group, with a similar amount of genes activated or repressed during this period.


Global reorganization of replication domains during embryonic stem cell differentiation.

Hiratani I, Ryba T, Itoh M, Yokochi T, Schwaiger M, Chang CW, Lyou Y, Townes TM, Schübeler D, Gilbert DM - PLoS Biol. (2008)

Replication Timing and Transcription Changes during Differentiation(A and B) Average replication-timing ratios of replication domains were plotted against their “present” (i.e., transcriptionally active) gene density for ESCs (A) and NPCs (B). Pearson R2 values are shown.(C and D) Correlation between early replication and the probability of expression. Genes were ranked by their replication-timing ratio and divided into bins of 100 genes, the height of which represents the percentage of active (i.e., ”present”) genes within each bin. The width and position of each bin represents its range of replication-timing ratios. Logistic regression (inner line) and 95% confidence intervals (outer lines) reveal a clear correlation in both ESCs (C) and NPCs (D). By the Likelihood Ratio test (a goodness-of-fit test), the fitted model is significantly different (p < 2 × 10−16 for both ESCs and NPCs) from that of a  hypothesis in which replication timing has no correlation to transcription.(E) Box plots showing the fold changes in transcription [i.e., log2(NPC/ESC)] of LtoE, EtoL, LtoL, and EtoE genes. RefSeq genes with the 5% greatest replication timing changes were defined as EtoL and LtoE, whereas those with the least changes (lowest 20%) that maintained replication timing ratio of above 0.5 or below −0.5 were defined as EtoE and LtoL, respectively.(F) Percentage of 2-fold up- or down-regulated genes within LtoE, EtoL, LtoL, and EtoE domains defined in Figure 4G.(G) Summary of expression patterns of genes within LtoE, EtoL, LtoL, and EtoE domains. Definitions are as follows: “Up” and “Down,” above 2-fold up- and down-regulation, respectively; “Unchanged,” below 2-fold up-/down-regulation; “Unchanged Only,” domains with both active and silent genes that do not change by 2-fold; and “Silent Only,” domains with only silent genes in both states.(H) LINE-1 transposable elements are actively transcribed in ESCs but become inactive in NPCs as assayed by RNA-FISH. Mean and standard error of mean (SEM) of the number of RNA-FISH signals per nucleus is shown (n = 30 from two biological replicates). The p-value was obtained from a two-tailed t-test for comparison of two unpaired groups.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2561079&req=5

pbio-0060245-g005: Replication Timing and Transcription Changes during Differentiation(A and B) Average replication-timing ratios of replication domains were plotted against their “present” (i.e., transcriptionally active) gene density for ESCs (A) and NPCs (B). Pearson R2 values are shown.(C and D) Correlation between early replication and the probability of expression. Genes were ranked by their replication-timing ratio and divided into bins of 100 genes, the height of which represents the percentage of active (i.e., ”present”) genes within each bin. The width and position of each bin represents its range of replication-timing ratios. Logistic regression (inner line) and 95% confidence intervals (outer lines) reveal a clear correlation in both ESCs (C) and NPCs (D). By the Likelihood Ratio test (a goodness-of-fit test), the fitted model is significantly different (p < 2 × 10−16 for both ESCs and NPCs) from that of a hypothesis in which replication timing has no correlation to transcription.(E) Box plots showing the fold changes in transcription [i.e., log2(NPC/ESC)] of LtoE, EtoL, LtoL, and EtoE genes. RefSeq genes with the 5% greatest replication timing changes were defined as EtoL and LtoE, whereas those with the least changes (lowest 20%) that maintained replication timing ratio of above 0.5 or below −0.5 were defined as EtoE and LtoL, respectively.(F) Percentage of 2-fold up- or down-regulated genes within LtoE, EtoL, LtoL, and EtoE domains defined in Figure 4G.(G) Summary of expression patterns of genes within LtoE, EtoL, LtoL, and EtoE domains. Definitions are as follows: “Up” and “Down,” above 2-fold up- and down-regulation, respectively; “Unchanged,” below 2-fold up-/down-regulation; “Unchanged Only,” domains with both active and silent genes that do not change by 2-fold; and “Silent Only,” domains with only silent genes in both states.(H) LINE-1 transposable elements are actively transcribed in ESCs but become inactive in NPCs as assayed by RNA-FISH. Mean and standard error of mean (SEM) of the number of RNA-FISH signals per nucleus is shown (n = 30 from two biological replicates). The p-value was obtained from a two-tailed t-test for comparison of two unpaired groups.
Mentions: Genes that are transcribed are generally early replicating, whereas genes that are late replicating are almost always silent; however, exceptions to this rule have been described [16,47,48]. Importantly, no study has comprehensively examined the changes in gene expression as they relate to changes in replication timing. To address this issue, we analyzed the steady-state levels of annotated gene transcripts before and after differentiation to NPCs using Affymetrix GeneChips. Regardless of whether levels, density, or number of active genes were examined, either at the level of domains (Figure 5A and 5B) or individual genes (Figure 5C and 5D), both differentiation states clearly showed a similar positive correlation between early replication and transcription. Consistent with previous findings across a portion of the Drosophila genome [25], this positive correlation was greater when integrated over large regions (approximately 600 kb for ESCs and NPCs vs. 180 kb in Drosophila; not shown). The maintenance of this statistical relationship during differentiation can be accounted for by the directionality of transcriptional changes within each domain (Figure 5E and 5F). At the level of individual genes, LtoE genes were mostly up-regulated, whereas EtoL genes showed a weak tendency to be down-regulated. At the level of domains, among those domains that contained at least one RefSeq gene (National Center for Biotechnology Information [NCBI] most well annotated; http://www.ncbi.nlm.nih.gov/RefSeq/), the majority of LtoE domains contained only up-regulated genes, whereas EtoL domains contain mostly down-regulated or unchanged genes (Figure 5G). Note that because most genes remain early replicating, the overall trend in gene expression changes is dominated by this EtoE group, with a similar amount of genes activated or repressed during this period.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus