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Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency - PubMed

  • ️Fri Jan 01 2021

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Yongzheng Li et al. Genome Biol. 2021.

Abstract

Background: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined.

Results: We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired.

Conclusion: Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

Keywords: Chromatin structure; Replication origin; STORM; Super-resolution imaging; Topologically associating domain (TAD); Transcription.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1

Super-resolution imaging of RFi and TADs in the S phase. a Representative STORM images of TAD1 and TAD2 labeled by Oligopaint probes (green) and RFi labeled metabolically for different durations (purple) (the “Methods” section). TAD1 and TAD2 were chosen based on the replication timing profile and Hi-C interaction heatmap of HeLa cells (Additional file 1: Figure S1). TAD1: an early replicating domain (Chr1:16911932-17714928). TAD2: a middle replicating domain (Chr1:17722716-18846245). Metabolic labeling of DNA replication was performed by supplying EdU to the cell upon release into the S phase for 10 min, 15 min, and 60 min (purple). The areas inside the yellow squares are shown at higher magnification to the right of each nucleus. Portions of the two signals that overlap are shown in white. b Barycenter distances between the TAD and its spatially associated RFi (the “Methods” section) in a. Horizontal lines and error bars represent the mean values ± s.d. of three or more independent biological replicates (n = 16 cells). c Representative STORM images of RFi labeled metabolically for different durations in two consecutive cell cycles. Consecutive metabolic labeling of DNA replication was performed by supplying BrdU (green) to the cell upon release into the S phase in the first cell cycle, followed by supplying EdU (purple) to the cell upon release into the S phase in the second cell cycle (for indicated durations). The areas inside the yellow squares are shown at higher magnification below each nucleus. d Box plot of barycenter distances between BrdU and EdU-labeled RFi in c (data were pooled from n = 10 cells). Center line, median; box limits, 25% and 75% of the entire population; whiskers, observations within 1.5× the interquartile range of the box limits. Significance was analyzed by an un-paired two sample parametric t test. ****P < 0.0001, ***P < 0.0005, **P < 0.01, *P < 0.05, N.S. not significant. 3D results are shown in Fig. S2&S5

Fig. 2
Fig. 2

Spatial distribution of replication origins relative to the TADs in the G1 and G1/S phases. a A scheme of replication in TAD1 and TAD2. The top profile represents the replication landscape obtained by OK-seq. (−0.776–0.78) was the threshold of OK-seq [40]. The middle black peaks represent the dynamic replication profile, which was obtained by 10-min BrdU pulse labeling at 0 min, 30 min, 3 h, and 6 h into the S phase. (0–50) or (0–150) is the range of normalized BrdU-seq data. The gray bars represent the TAD boundaries in the RDs. The small red bars at the bottom represent the ORC1 and H2A.Z binding sites indicating the potential replication origins. Representative high-efficiency and low-efficiency replication origins defined by the BrdU-seq data and the OK-seq profile are marked with vertical rectangles. Yellow rectangle: high-efficiency replication origin (ORI1) at the TAD boundary. Red rectangles: high-efficiency replication origins in TAD1 (ORI2 and ORI3) and TAD2 (ORI6 and ORI7). Black rectangles: low-efficiency replication origins in TAD1 (ORI4 and ORI5). b Representative STORM images of TADs (green) and their origins (purple) labeled by FISH with oligoprobes in the G1 and G1/S phases. Upper, TADs and origins labeled at the G1/S transition. Lower, TADs and origins labeled at approximately 5 h into the G1 phase. Portions of the two signals that overlap are shown in white. The corresponding conventional images are shown in the inset. c Barycenter distances between origins and TADs in b (n ≥ 10 cells). To reduce the number of groups, the barycenter distance of each ORI was measured separately and displayed as four groups. d Radii of TAD1 and TAD2 in the G1 or G1/S phase (n ≥ 10 cells). For lines and statistics in c and d, see the description in the legend of Fig. 1. 3D results are shown in Fig. S11

Fig. 3
Fig. 3

The spatial distribution of replication origins within the TADs at the G1/S transition is dependent on CTCF, cohesin, and transcription. a Representative STORM images of origins (purple) in TAD1 (green) after treatment of cells with the indicated siRNAs. Conventional images indicate the concentration of CTCF (cyan) or cohesin (yellow) in the nucleus. b Left panel, efficiency of RNAi indicated by fluorescence of CTCF or cohesion. Right panel, barycenter distances between high-efficiency or low-efficiency origins in TAD1 after treatment of cells with the indicated siRNAs. Portions of the two signals that overlap are shown in white. c Representative STORM images of origins (purple) in TAD1 (green). Left: no DRB. Right: with DRB. d Barycenter distances between high-efficiency/low-efficiency origins and TAD1 with or without DRB treatment. e Radius of TAD1 treated with or without DRB. For lines and statistics in b, d, and e, see the description in the legend of Fig. 1 (n ≥ 10 cells). 3D results are shown in Fig. S13

Fig. 4
Fig. 4

Spatial distributions of CTCF, MCM2, and PCNA relative to the early replicating TADs in the G1 and G1/S phases. a–c Representative STORM images of CTCF, MCM2, and PCNA labeled by immunostaining (purple) and metabolically labeled TADs (green). Cells were fixed and labeled in the mid-G1 phase (upper) or G1/S phase (lower). The areas inside the yellow squares are shown at higher magnification next to each nucleus. Portions of the two signals that overlap are shown in white. d Barycenter distances between CTCF, MCM2, or PCNA with the TADs in the mid-G1 phase or G1/S phase. e Radii of CTCF, MCM2, or PCNA foci in the mid-G1 phase or G1/S phase. For lines and statistics in d and e, see the description in the legend of Fig. 1 (n = 10 cells)

Fig. 5
Fig. 5

“Chromatin Re-organization Induced Selective Initiation” (CRISI) model for selective initiation of DNA replication origins. a In the early G1 phase, the spatial distributions of potential replication origins (gray ribbons) are relatively even in the TAD. The TAD comprises several chromatin loops (blue) organized by CTCF and cohesin at the loop anchors (green rings). PCNA clusters (yellow balls) surrounding the TAD are bound to the nuclear matrix (hazed light blue straws). b, c With transcription proceeding, the chromatin loops undergo structural re-organization along with chromatin domain de-compaction in the G1 phase, exposing a subset of the origins to the periphery of the TAD (pink ribbons). Note that the origin at the sequence boundary of the TAD remains at the TAD periphery in the G1 phase. These peripheral origins are more accessible to the surrounding PCNA clusters and thus become high-efficiency origins for the initiation of DNA replication at the periphery of the TAD. The areas inside the black squares in a and b are shown at higher magnification above

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