Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication

Abstract

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.

Keywords: CTCF; Dppa; ERCEs; chromatin interactions; genome architecture; replication timing; sub-nuclear compartment; super-enhancer; topologically associating domain.

Figure 1.. Internal segments contribute partially to early replication.

A. The Dppa2/4 domain in mESCs: Hi-C heatmap, LaminB1 DamID (LaminB1), nuclear RNA (NucRNA), reference genes (Genes), CCCTC-binding factor ChIP (CTCF; motif orientation in pink), SMC1 ChIP signal in ChIAPET datasets (SMC1), SMC1 ChIAPET identified loops (SMC1 loops, horizontal black bars), short nascent strand mapped replication origins (SNS origins), domain boundaries (grey vertical lines). B. Positions of deletions and inversions. C,D,E. RT profiles of corresponding deletions or inversions (red lines) vs. WT control (black lines), showing domain boundaries (yellow), deleted regions (white), inversions are indicated by (red arrows) and breakpoints (grey dashed lines). F. FACS plots of GFP-tagged CTCF depletion. G. RT profile in untreated (black and grey lines), and 2-day CTCF depleted samples (red and pink lines).

Figure 2.. Identification of Early Replication Control Elements (ERCEs).

A. Capture Hi-C heatmap centered on the Dppa2/4 domain. Arc plot shows significant interaction pairs, including a long-range interaction (green arrow). B. Chromatin features of the Dppa2/4 domain highlighting ERCEs (a, b, c). Site X and Y show similar marks but are not ERCEs. C. Virtual 4C profiles from the viewpoints of sites a, b or c. Bait regions are removed and red arcs show significant interactions. D. RT profile for abc triple deletion from two independent CRISPR clones, with mutant allele in red and WT allele in black. E. Individual or pairwise deletions of the three ERCEs, as in D.

Figure 3.. ERCEs control A/B compartmentalization and TAD strength.

A. Hi-C matrix and eigenvector for 2 clones (E2 ands A6) of the abc deletions. B. Eigenvector and RT distribution of 4C significant contacts from a bait within the Dppa2/4 domain in abc deletion or NPC differentiation. C. Capture Hi-C results of the Dppa2/4 region in WT (mESC & NPC), individual (100k & c), pairwise (bc & ab), abc (clones E2&A6), boundary (145k) and interior non-ERCE (18k) deletions, with directionality index (DI), domainogram of insulation scores vs. window sizes (heatmap key to left of mESCs), Dppa2/4 TAD ( blue arrow) and boundaries (red stars and black dashed lines).

Figure 4.. Transcription after ERCE deletion.

A. Bru-seq profiles for abc deleted and WT alleles with CAGE (Cap Analysis of Gene Expression), positions of genic segments 1–7 shared among all a, b, c deletions (panel c), sites a, b, and c are highlighted (red, yellow and blue, respectively) negative (green) and positive (orange) strand expression, WT (black) and mutant (red) RT profiles. B. Bru-seq profiles as in A. An alternative TSS (purple arrows) appears in Morc1 b deletion. (Remaining profiles found in Supplemental Figure 6) C. Transcription and RT changes of the shared genic segments, indicating gene expression changes without RT changes (red arrows) and RT delay without gene expression changes (black arrows). D. Transcription vs. RT changes of the shared genic segments in all mutants. E. Transcription vs. RT for all genes within developmental domains.

Figure 5.. ERCE-containing inversions.

A. WT capture Hi-C heatmap, DI and domainograms as in Figure 3, showing positions of inversions (bottom blue arrows), sites a, b and c (red, yellow and blue, respectively), and TAD boundaries (red stars). B,C. RT profile for the 680kb (B) and 435kb (D) inversions with WT (black) and inverted (red) alleles and inverted allele with WT coordinates (pink). D. Capture Hi-C of the 680kb inversion with linear distance after inversion, indicating the inverted region (blue box) and newly formed boundaries (red stars and red dashed lines). Eigenvector and CTCF ChIP-seq peaks (pink arrowheads indicate orientation) are inverted from WT data.

Figure 6.. Genome-wide prediction and validation of ERCEs

A. Overlap between predicted ERCEs and enhancers, super enhancers, P300 and OSN (OCT4, SOX2, NANOG) binding sites. Please note that the Venn diagrams are not drawn to scale. B. Western blot of YY1 protein depletion. C. RT profile in untreated (black and grey lines), and YY1 depleted samples (red and pink lines). Dppa2/4 domain is highlighted in yellow. D. Validation of predicted ERCEs at the Zfp42/Rex1 domain with Hi-C heatmap as in Fig, 1 with predicted ERCEs (d&e) highlighted in yellow and RT profiles of two independent CRISPR clones. E. RT of boundary containing deletions/inversions at the Epha4/Wnt6 locus in mESCs. Hi-C heatmap and chromatin features as in D.

Figure 7.. Model for ERCE function.

A. ERCEs interact strongly with predicted ERCEs outside the domain. Virtual 4C profiles (bait regions removed) from Capture Hi-C data viewpoints of site a, b, c and a predicted ERCE. B. Chromatin features of the predicted ERCE from A. C. ERCEs regulate RT, A/B compartmentalization, TAD architecture and gene transcription.