Control of DNA replication timing in the 3D genome


The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.

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Fig. 1: Replication timing relationship to 3D chromatin structure.
Fig. 2: 3D chromatin structure and replication timing are dynamic during cell differentiation and during the cell cycle.
Fig. 3: Epigenetic versus sequence-specific regulation of replication timing.
Fig. 4: Cis- and trans-acting elements regulating replication timing.
Fig. 5: Proposed model of organization within the nucleus.


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This work was supported by National Institutes of Health (NIH) grants GM083337 and DK107965 to D.M.G. The authors thank M. Thayer for helpful discussions and critique of the manuscript.

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C.M. and D.M.G. have contributed equally to all stages of writing and revision of the article and display items; J.S. contributed to the initial draft of the main text.

Correspondence to David M. Gilbert.

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Constant timing regions

(CTRs). Regions of the chromatin containing one or more adjacent replication domains that are replicating at nearly the same time.


Units of replication comprising a replication origin and the DNA being replicated.

Timing transition regions

(TTRs). Regions of the chromatin between two replications domains replicating at different times.

Replication forks

Complexes comprising template DNA, oligonucleotide primers and proteins necessary for DNA replication. Two ‘sister’ replication forks are assembled at each replication origin and may be associated in 3D space.

Replication stress

Stalling of replication forks, which can be caused by chemical interference or radiation, or can be the result of a lack of nucleotides or proteins necessary for DNA replication.

Mismatch repair

(MMR). DNA repair mechanism occurring at the replication fork involved in the repair of base mismatch and small insertions/deletions occurring during DNA replication or recombination.


A genome-wide chromatin conformation capture technology that uses chromatin digestion, re-ligation of sequences in close proximity and sequencing to identify chromatin pairwise 3D chromatin interactions in the nucleus.

Principal component analysis

(PCA). Mathematical transformation applied on complex (with multiple variables) datasets, to extract values describing the data called PC1, PC2 and so forth, with PC1 the value best describing the data.


Long non-coding RNA that is expressed in female cells from only one X chromosome. Xist inactivates the X chromosome from which it is expressed, enabling dosage compensation (similar X-linked gene expression levels in male and female cells).

Asynchronous replication and autosomal RNAs

(ASARs). Long non-coding RNAs that are essential for the timely replication and condensation of the entire chromosome from which they are expressed.

Lamina-associated domains

(LADs). Domains of chromatin that come in close proximity to the nuclear lamina.


Domains between two adjacent lamina-associated domains.

Topologically associated domains

(TADs). Domains of chromatin enriched for 3D interactions within the domain as compared with between domains.

Correlative live and super-resolution microscopy

Combines the temporal resolution of time-lapse fluorescence microscopy with the spatial resolution of super-resolution microscopy.

CCCTC-binding factor

(CTCF). Protein highly conserved in eukaryotes that associates with chromatin to mediate transcription and chromatin insulation.


Protein complex composed of SMC1 and SMC3 that forms a large ring structure that accommodates two strands of chromatin. Cohesin has a key architectural role, forming chromatin loops and maintaining sister chromatids tied together after DNA replication.

Early replication control elements

(ERCEs). DNA sequences necessary for the early replication of entire replication domains.


Chromatin region rich in enhancers, with a high level of transcription associated factors and acetylated histones.

Chromatin conformation capture

Technologies that assess 3D interactions within chromatin. These technologies are based on the cutting and re-ligation of chromatin immobilized in intact nuclei and identification of pairwise interactions by quantitative PCR or sequencing.

Zygotic genome activation

(ZGA). Stage of embryonic development at which the transcription of the zygotic genome becomes activated.

Imprinted genes

Genes that are expressed only from one chromosome homologue, the choice of which is dependent on its parental origin. The mechanism behind genomic imprinting relies on DNA methylation.

β-Globin domain

A megabase-sized locus containing the β-globin gene, which is expressed only in erythroid cells. DNA replication properties of this domain have been extensively studied as it replicates early in erythroid cells and late in other cell types.

Ribosomal DNA

(rDNA). Part of the genome coding for ribosomal RNA. rDNA is constituted of several copies of the same genes, the expression of each varying depending on cell type.


Insertions or deletions in the genome of a cell or an individual.

Long interspersed nuclear element 1

(LINE1). Class of transposable elements estimated to be present at around 500,000 copies in the human and mouse genomes.

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Marchal, C., Sima, J. & Gilbert, D.M. Control of DNA replication timing in the 3D genome. Nat Rev Mol Cell Biol 20, 721–737 (2019).

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