eLife Assessment

This manuscript provides valuable insight into how genome organization changes as cells progress through the cell cycle after mitotic exit. The conclusions are supported by solid, rigorous data, and the use of sorted unsynchronized cells rather than cells treated with drugs is a particular strength. Two sharp genome remodeling events are identified at G1-S and to a lesser extent, at S-G2 transitions. A discussion on the limitations of Hi-C and a broader interpretation of results in the context of other mechanistic models would strengthen the overall rigor.

Reviewer #1 (Public review):

This work convincingly shows that, rather than gradually "evolving" throughout interphase, global chromatin architecture undergoes unexpectedly sharp remodeling at G1-S (and to a lesser extent, S-G2) transitions. By applying "standard" Hi-C analyses on carefully sorted cells, the authors provide an excellent temporal view of how global chromatin architecture is changed throughout the cell cycle. They show a surprisingly abrupt increase in compartmentation strength (particularly interactions between the "active" A compartments) at G1-S transition, which is slightly weakened at S-G2 transition. Follow-up experiments show convincingly that the compartment "maturation" does not require the DNA synthesis accompanying S phase per se, but the authors have not identified the responsible factors (work for future publications). The possible biological ramifications of these architectural changes (setting up potential replication "factories", and/or facilitating transcription-replication conflict resolution, both more pertinent for the active A compartments, which are most affected) have been well discussed in the article, but still remain speculative at this stage.

My major criticism of this article is aimed more at the state of the field in general, rather than this specific article, but it should be discussed to give a more balanced view: what actually is a chromatin compartment? Chromosomal tracing and live tracking experiments have shown that the majority of "structures" identified from Hi-C experiments are statistical phenomena, with even "strong" interactions only being infrequent and transient. A-B compartments are "built up" from multiple very low-frequency "interactions", so ascribing causal effects for genome functions is even tougher. As a result, I have very little confidence in the results of the authors' polymer simulations and their inferred "peninsula" A compartment structures without any other supporting experimental data.

Specific minor points:

(1) A better explanation for how Figure 1E was generated is required, because this figure could be very misleading. Figure 1F and all other cis-decay plots (and the Hi-C maps themselves) show that the strongest interactions are always at smaller genomic separations, so why should there be more "heat" at the megabase ranges in Figure 1E?

(2) An ultra-high-resolution Hi-C study (Harris et al., Nat Commun, 2023) identified very small A and B compartments, including distinctions between gene promoters and gene bodies, raising further questions as to what the nature of a compartment really is beyond a statistical phenomenon. It is unreasonable to expect the authors to generate maps as deep as this prior study, but how much do their conclusions change according to the resolution of their compartment calling? The authors should include a balanced discussion on the "meaning" of A/B compartments.

Reviewer #2 (Public review):

Summary:

This manuscript by Choubani et al presents a technically strong analysis of A/B compartment dynamics across interphase using cell-cycle-resolved Hi-C. By combining the elegant Fucci-based staging system with in situ Hi-C, the authors achieve unusually fine temporal resolution across G1, S, and G2, particularly within the short G1 phase of mESCs. The central finding that A/B compartment strength increases abruptly at the G1/S transition, stabilizes during S phase, and subsequently weakens toward G2 challenges the prevailing view that compartmentalization strengthens monotonically throughout interphase. The authors further propose that this "compartment maturation" is triggered by S-phase entry but occurs independently of active DNA synthesis, and that it involves a consolidation and large-scale reorganization of A-compartment domains.

Strengths:

Overall, this is a thoughtfully executed study that will be of broad interest to the 3D genome community. The data are of high quality, and the analyses are extensive, albeit not completely novel. In particular, previous work (Nagano et al 2017 and Zhang et al 2019) has shown that compartments are re-established after mitosis and strengthened during early interphase, and single-cell Hi-C studies have reported changes in compartment association across S phase. In particular, Nagano et al show that DNA replication correlates with a build-up of compartments, similar to what is presented here, with the authors' conclusion that compartment strength peaks in early S. The idea that it weakens toward G2, rather than continuing to strengthen, appears to be novel and differs from the prevailing framing in the literature.

Weaknesses:

That said, several aspects of the conceptual framing and interpretation would also benefit from further clarification, and the mechanistic interpretation of the reported compartment dynamics requires more careful positioning relative to established models of genome organization. Specific concerns are outlined below:

(1) One of the major conclusions of the study is that compartment maturation does not require ongoing DNA replication. However, the interpretation would benefit from more precise wording. Thymidine arrest still permits licensing, replisome assembly, and other S-phase-associated chromatin changes upstream of bulk DNA synthesis. Therefore, their data, as presented, demonstrate independence from DNA synthesis per se, but not necessarily from the broader replication program. Please clarify this distinction in the text and interpretations throughout the manuscript.

(2) A major conceptual issue that is not addressed at all is the well-established anti-correlation between cohesin-mediated loop extrusion and A/B compartmentalization. Numerous studies have shown that loss of cohesin or reduced loop extrusion leads to stronger compartment signals, whereas increased cohesin residence or enhanced extrusion weakens compartmentalization. Given this framework, an obvious alternative explanation for the authors' observations is that the abrupt increase in compartment strength at G1/S, and its decline toward G2, could reflect cell-cycle-dependent modulation of cohesin activity rather than a compartment-intrinsic "maturation" program.

The manuscript does not explicitly consider this possibility, nor does it examine loop extrusion-related features (such as loop strength, insulation, or stripe patterns) across the same cell-cycle stages. Without discussing or analyzing this widely accepted model, it is difficult to distinguish whether the reported compartment dynamics represent a novel architectural mechanism or an indirect consequence of known changes in extrusion behavior during the cell cycle. I strongly encourage the authors to analyze their data to determine if they observe anti-correlated loop changes at the same time they observe compartment changes. Ideally, the authors would remove loop extrusion during interphase using well-established cohesin degrons available in mESCs and determine if the relative differences in compartment dynamics persist.

(3) The proposed "peninsula-like" A-domain structures are inferred from ensemble Hi-C data and polymer modeling, rather than directly observed physical conformations. That is, single-cell imaging data clearly have shown that Hi-C (especially ensemble Hi-C) cannot uniquely specify physical conformations and that different underlying structures can produce similar contact patterns. The "peninsula" language, as written, risks being interpreted as a literal structural model rather than a conceptual visualization. Instead of risking this as just another nuanced Hi-C feature in the field, the authors could strengthen the manuscript by either (i) explicitly framing the peninsula model as a heuristic description of contact redistribution rather than a definitive physical architecture, or (ii) discussing alternative structural scenarios that could give rise to similar Hi-C patterns. Clarifying this distinction would improve the rigor and help readers better understand what aspects of A-compartment consolidation are directly supported by the data versus model-based extrapolations. For example, it would be useful to clarify whether the observed increase in long-range A-A contacts reflects spatial extension of internal A regions, changes in loop extrusion dynamics, increased compartment mixing within the A state, or population-averaged heterogeneity across alleles.

(4) The extension of the analysis to additional cell types using HiRES single-cell data is a valuable addition and supports the idea that compartment maturation is not unique to mESCs. However, the limitations of these data, in particular, the limited phase resolution, in addition to the pseudo-bulk aggregation and variable coverage, should be emphasized more clearly in the main text. Framing these results as evidence for conservation in principle, rather than definitive proof of identical dynamics across tissues, would be a more appropriate framing.