The genetic architecture of DNA replication timing in human pluripotent stem cells

2022

Nat Commun

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DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We develop an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes in silico flow cytometry, a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyze up to 2437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.

Section: Introductionmentioning

confidence: 89%

“…For all other cell lines, a profile for the specific cell line was used. For Illumina Platinum LCLs (GM12878, GM12891, and GM12892) 39 and hESCs (H1, H7, and H9) 8 , these data are previously published.…”

Section: Methodsmentioning

confidence: 99%

High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control

2022

Nat Commun

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“…Furthermore, some recent replication origin-mapping methods have indicated that replication origins are highly abundant and highly dispersed throughout the human genome 1,7 , suggesting that many sites may function as origins used in a subset of cell cycles. In contrast, highresolution measurements of hundreds of human replication timing profiles 8,9 , or replication timing across multiple S-phase fractions 10 , support initiation of replication from more localized genomic regions. While these replication-timing methods reveal genomic regions that reproducibly replicate at characteristic times during S phase, it remains contested whether these represent a conserved pattern across cells or reflect the average behavior of single cells.…”

Section: Introductionmentioning

confidence: 91%

High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control

2021

Preprint

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DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

“…In general, late replication appears to be associated with a more repressive chromatin state: late-replicating regions tend to localize to the nuclear periphery 2 , 3 and to broadly associate with the condensed “B” compartment in chromatin conformation capture assays 4 , 5 . Likewise, genes in late-replicating regions often have lower expression 6 , 7 , with corresponding histone methylation 8 , 9 and deacetylation 8 , 10 , than genes in early-replicating regions. Constitutive heterochromatin, which is gene-poor and highly-condensed, is often described to be late replicating 11 – 13 , although direct visualization by microscopy has classified five sequential nuclear localization patterns of nascently-replicated DNA, with euchromatic replication primarily occurring during the first wave 2 .…”

Section: Introductionmentioning

confidence: 99%

Telomere-to-telomere human DNA replication timing profiles

2022

Sci Rep

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The spatiotemporal organization of DNA replication produces a highly robust and reproducible replication timing profile. Sequencing-based methods for assaying replication timing genome-wide have become commonplace, but regions of high repeat content in the human genome have remained refractory to analysis. Here, we report the first nearly-gapless telomere-to-telomere replication timing profiles in human, using the T2T-CHM13 genome assembly and sequencing data for five cell lines. We find that replication timing can be successfully assayed in centromeres and large blocks of heterochromatin. Centromeric regions replicate in mid-to-late S-phase and contain replication-timing peaks at a similar density to other genomic regions, while distinct families of heterochromatic satellite DNA differ in their bias for replicating in late S-phase. The high degree of consistency in centromeric replication timing across chromosomes within each cell line prompts further investigation into the mechanisms dictating that some cell lines replicate their centromeres earlier than others, and what the consequences of this variation are.