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Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program - PubMed

Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program

Vishnu Dileep et al. Genome Res. 2015 Aug.

Abstract

Mammalian genomes are partitioned into domains that replicate in a defined temporal order. These domains can replicate at similar times in all cell types (constitutive) or at cell type-specific times (developmental). Genome-wide chromatin conformation capture (Hi-C) has revealed sub-megabase topologically associating domains (TADs), which are the structural counterparts of replication domains. Hi-C also segregates inter-TAD contacts into defined 3D spatial compartments that align precisely to genome-wide replication timing profiles. Determinants of the replication-timing program are re-established during early G1 phase of each cell cycle and lost in G2 phase, but it is not known when TAD structure and inter-TAD contacts are re-established after their elimination during mitosis. Here, we use multiplexed 4C-seq to study dynamic changes in chromatin organization during early G1. We find that both establishment of TADs and their compartmentalization occur during early G1, within the same time frame as establishment of the replication-timing program. Once established, this 3D organization is preserved either after withdrawal into quiescence or for the remainder of interphase including G2 phase, implying 3D structure is not sufficient to maintain replication timing. Finally, we find that developmental domains are less well compartmentalized than constitutive domains and display chromatin properties that distinguish them from early and late constitutive domains. Overall, this study uncovers a strong connection between chromatin re-organization during G1, establishment of replication timing, and its developmental control.

© 2015 Dileep et al.; Published by Cold Spring Harbor Laboratory Press.

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Figures

Figure 1.
Figure 1.

Establishment of interphase chromatin compartments during early G1. (A) Validation of 4C by 3D FISH. Cumulative frequency curves and box plots of distances (in microns) between FISH signals from a pair of regions with low 4C contact count (red, n = 51) or high 4C contact count (green, n = 57). (B) Schematic diagram of spatial distribution of early (red) and late (green) RDs within the nucleus before and after the establishment of a replication-timing program at the TDP in mouse C127 cells. Time points chosen for 4C after release from mitosis are indicated with black arrows. (No) Nucleolus. (C,D) “Contact count”: smoothed 4C data (dark gray) overlaid with the replication timing (RT) profile (red/green is early/late replicating) for a late replicating bait Ch8.B53 (C) and an early replicating bait Ch8.B87 (D). Both baits were analyzed at 0.5 h (top) and 4 h (bottom) after mitosis. “P-value”: negative log10 of contact P-values (Methods) colored in red or green for early or late replicating regions, respectively. Between 0.5 and 4 h after mitosis, contacts between bait and other intra-chromosomal regions become more significant and focused to those with similar RT as the bait. (E) Distribution of replication timing (RT) values for the strongest (top 5% highest z-scores) contacts with either the early replicating (Ch8.B87, red) or the late replicating (Ch8.B53, green) bait. Positive values of RT indicate early replication, negative values of RT indicate late replication, and zero indicates middle replication. The higher the magnitude the earlier or later the region replicates. For reference, the RT distribution of the whole Chr 8 is shown in gray. Contacts at 0.5 h did not have statistical significance, whereas 4 h had significant contacts to regions with the same RT as the bait (Supplemental Fig. 7A).

Figure 2.
Figure 2.

Interphase chromatin compartments are established coincident with TDP. (A) Representative 4C contact count plots for baits positioned in early replicating (red) and late replicating (green) domains for the entire chromosome. 4C profiles are overlaid with C127 replication timing data (black). (B) Distribution of replication timing values for the strongest (top 5% highest z-scores) contacts with early replicating (red) and late replicating (green) baits across Chr 8 and Chr 16. For reference, the RT distributions of the whole chromosomes 8 and 16 are shown in gray. Contacts before the TDP did not have statistical significance, whereas significant contacts begin to appear coincident with the TDP and become more compartmentalized after the TDP (Supplemental Fig. 7A). (C) Loess (local polynomial regression fitting) fitted decay of 4C contact frequency with distance from the bait (decay curve) averaged for all baits from Chr 8 and Chr 16. (D) FISH-based measurement of 3D proximity normalized to the size of the nucleus (relative distance) (Methods) for two late loci (Supplemental Fig. 4) at different points after release from mitosis (nocodazole-free synchrony). There is a significant increase in 3D proximity (P < 0.001792) between before 1 h and after 2 h, which is coincident with the TDP. N = 91, 82, 57, 184, 67 for 30 min, 1, 2, 3 and 4 h, respectively. Mitosis is not included in the time course due to the inability to normalize to the state of condensation of mitotic chromatin (see Supplemental Methods).

Figure 3.
Figure 3.

TADs are formed coincident with the TDP. (A) 4C contact counts shown for 1 to 3 Mb to either side of early/late baits. Below the contact count plots is the directionality index (DI) (Dixon et al. 2012) calculated from Hi-C in mouse ESC and cortex cell lines, shown to indicate the downstream (dark red) or upstream (dark green) bias. The vertical solid black line indicates TAD boundaries in mouse ESCs, and the dotted black line indicates the position of the bait. The kinetics of TAD formation can be visualized by the appearance of a sharp drop in contact frequency at RD/TAD boundaries. (B) Formation of TADs measured as an increase in directionality bias of the contacts with time, for baits positioned at constitutive early (red) and late (green) RDs (Ch8.B26, Ch8.B44, Ch8.B118, Ch16.B30, Ch16.B46, Ch16.B48). The y-axis shows the percentage of DI relative to the maximum DI for each bait.

Figure 4.
Figure 4.

Chromatin organization in G0 (quiescence) and G2 are similar to post-TDP. (A) 4C contact counts for an early (red) and late (green) replicating region, displayed as in Figure 2A, comparing 4-h interphase time point to G0 and G2. (B) 4C contact counts shown for 1 to 3 Mb to either side of early/late baits shows conserved TADs and directionality bias (as in Fig. 3A) during G0 and G2 phase, similar to the 4-h time point.

Figure 5.
Figure 5.

Developmental domains are less compartmentalized than constitutive domains. (A) Degree of compartmentalization (Methods) measured for constitutive baits (C baits), in blue, vs. developmental baits (D baits), in brown. The difference in degree of compartmentalization between C baits and D baits did not have statistical significance (P = 0.7374, KS test); therefore, we performed genome-wide comparison using Hi-C. (B) Degree of compartmentalization for constitutive (C) regions vs. developmental domains (D) (50-kb windows) across three mouse cells types and four human cell types using Hi-C data. The difference in degree of compartmentalization between constitutive domains and developmental domains showed high statistical significance (P < 2.2 × 10−16, KS test). (C) Density contour plot (lighter colors indicate less density) with local regression fitting (loess) for degree of compartmentalization vs. absolute replication timing value for developmental (brown) vs. constitutive (blue) regions. (D) Plot comparing frequency of Hi-C contacts vs. difference in replication timing value for pairs of 50-kb windows using Hi-C data. The top 50 percent of contacts from the Hi-C data was used for the analysis. Blue and brown lines show local regression fitting of the data for constitutive and developmental regions.

Figure 6.
Figure 6.

Developmentally regulated regions have different organizational principles. (A) Table comparing chromatin features between constitutive early and late (CE, CL) and developmental RDs that are either early (DE) or late (DL) in the particular cell type analyzed for each respective chromatin feature (Supplemental Table 3). (B) Lamin B association for constitutive early/late and developmental early/late RDs. (C) Distribution of constitutive early/late and developmental early/late RDs within the six subcompartments defined by Hi-C data in GM12878 (Rao et al. 2014). (D) Segway/ChromHMM analysis of human lymphoblast (GM12878) and HeLa cells showing the enrichment of seven chromatin labels within constitutive early/late and developmental early/late RDs. (WE) Weak enhancer, (E) predicted enhancer, (R) predicted repressed regions, (PF) promoter flanking region, (T) predicted transcribed regions, (TSS) predicted promoter region including TSS, (CTCF) CTCF enriched element. Note that in all cases, the properties of developmental domains are less distinct and less dependent on their replication time than constitutive domains.

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