An ATM D-compartmentalization in DNA damage response

Abstract

How chromatin configuration impacts DNA repair is an emerging question. A recent study by Arnould et al. shows that ATM orchestrates a new chromatin compartment (D compartment) following DNA double-strand breaks and establishes that this compartment enhances cellular response to such breaks but also introduces a risk to genome integrity.

Chromatin conformation and spatial organization within the cell nucleus stand as a pivotal element in essential genome functions [1]. Studies utilizing chromosome-conformation-capture techniques, such as Hi-C, have now made it clear that chromatin is organized into topologically associating domains (TADs) [2]. The Hi-C maps have also unveiled the segregation of these TADs into two distinct, spatially segregated, chromatin compartments: the A compartment, consisting of actively transcribed chromatin (euchromatin), and the B compartment, encompassing silenced chromatin (heterochromatin) [3]. Moreover, studies have identified multiple subcompartments that correspond to unique combinations of epigenetic marks, including A1 and A2 within euchromatin, and B1-B4 within heterochromatin [4], further refining our understanding of hierarchical chromatin organization. While these discoveries represent significant progress in the field, numerous questions remain unclear, including the impact of chromatin configurations and spatial chromatin clustering on DNA repair. Arnould et al.’s study in the October issue of Nature delves into this specific area [5].

In this study, the authors used a specialized cell line called DIvA (short for DNA double-strand break (DSB) inducible via AsiSI) that they previously developed [6]. The DIvA cells allowed them to induce multiple well-annotated DSBs across the genome by activating the AsiSI restriction enzyme with 4-hydroxytamoxifen (OHT) [6]. By analyzing the differential Hi-C contact matrix in OHT-treated DIvA cells, Arnould et al. found a substantial increase in intra-TAD contact frequencies within TADs that had experienced DSB compared to undamaged TADs [6]. Simultaneously, contacts with neighboring adjacent domains showed a notable decrease, providing them the initial indication that TADs with a DSB are effectively isolated from their surrounding environment.

To explore the underlying mechanism, the authors then examined the role of two PI3K-like protein kinases, ATM and DNA-PK, which are pivotal regulators of DSB repair [7]. Utilizing Hi-C analysis on DIvA cells exposed to ATM and DNA-PK inhibitors, Arnould et al. found that ATM inhibition led to a decrease in intra-TAD contacts following DSBs. Intriguingly, consistent with prior research suggesting that cohesin supports TAD formation by facilitating loop extrusion [8], the authors found that depleting the cohesin subunit SCC1 resulted in reduced intra-TAD contacts post-DSBs. These findings thus lend support to a model in which ATM triggers cohesin-mediated loop extrusion after DSBs [9], effectively sequestering the damaged TADs from the surrounding chromatin.

To extend this finding, the authors analyzed the Hi-C data with particular focus on long-distance interactions occurring within the nuclear environment. Their investigation unveiled a tendency for DSBs to form clusters. This clustering phenomenon is not limited to DSBs occurring on the same chromosome; it also encompasses DSBs located on different chromosomes and within entire TADs. This observation prompted Arnould et al. to delve into the underlying mechanisms governing DSB clustering. They found that the clustering of DSBs relies on the activity of ATM but does not necessitate the involvement of DNA-PK. Notably, this propensity for clustering is particularly pronounced during the G1 phase of the cell cycle, implying that the regulation of DSB clustering is intertwined with ATM and the cell cycle, with a heightened influence during G1. In addition, by employing the half-fluorescence recovery after photobleaching (half-FRAP) technique [10], the authors demonstrated that DSB clustering primarily arises from polymer-polymer phase separation, as opposed to liquid-liquid phase separation.

Another remarkable discovery in this study involved the identification of a novel chromatin subcompartment that forms following DSBs. The determination of chromatin compartments often involves the use of principal component analysis on Hi-C contact matrix [3]. Utilizing this methodology, Arnould et al. found that DSBs induce a distinct subcompartment within the A compartment. They introduced the term “D compartment” to describe this specific region, as it primarily arises due to the presence of DSBs. Additional investigation, however, showed that the D compartment is not exclusively comprised of damaged chromatin; it also encompasses a subset of DNA damage response (DDR)-upregulated genes, suggesting that DSBs triggers the formation of a chromatin compartment encompassing both damaged TADs and a subset of DDR genes that undergo upregulation post-DSBs. Subsequent experiments revealed that the D compartment plays a role in activating DDR genes and that the D compartment formation is fostered after R-loop accrual, consistent with earlier research linking R-loops to phase separation and chromatin compartmentalization [11].

Finally, the authors illustrated a drawback of DSB clustering, which contrasts with its potential benefits. This drawback primarily stems from the possibility of DSB clustering bringing two DSBs into close proximity, leading to translocations. In this context, promoting D compartment formation led to an increase in translocations, while disrupting D compartment formation reduced the occurrence of translocation events. Additionally, by analyzing interchromosomal translocations in different cancer types, they identified that genes located in the D compartment and upregulated following DNA damage have more translocation breakpoints compared to upregulated genes not associated with the D compartment, suggesting that the physical proximity of DDR upregulated genes with DSBs in the D compartment could contribute to some of the observed translocations in cancer genomes.

In closing, Arnould et al. provide compelling evidence for a link between chromatin architecture and DDR, which has both advantageous and detrimental consequences. Prior studies identified multiple mechanisms behind DSB clustering, implicating the MRN complex, the formin-family actin filament nucleator FMN2, the linker of the nuclear and cytoplasmic skeleton complex, and the mechanical forces mediated by the cytoskeleton [12]. This new work by Arnould et al. sheds light on how alterations in chromatin configuration induced by DSBs also translate into global chromosome reorganization within the nucleus, further regulating the DDR. Additional research is imperative to comprehend: 1) the frequency and kinetics of DDR genes directed toward the D compartment, 2) the extent of variation in compartmentalization across different cell and tissue types, and 3) the specific targets of ATM that strengthen damaged TAD contacts, promoting their clustering and compartmentalization. This report by Arnould et al. is being published at a moment in time where both different folding and spatial organization of chromatin as modulators of essential genome function are the focus of great research interest. Unraveling these intricate relationships necessitates meticulous investigations.

Figure 1. Schematic of D compartment formation following DNA double-strand break (DSB) in the nucleus.

Chromatin is structured into topologically associating domains (TADs) or contact domains that are further subdivided into distinct A and B compartments, along with various subcompartments (such as A1, A2 and B1-B4), associated with active and repressed chromatin, respectively (A). Upon the induction of DSBs, the damaged TADs become spatially isolated from the surrounding environment and give rise to a specialized compartment referred to as the “D compartment” (B). The formation of the D compartment is orchestrated by factors such as ATM activity, the accumulation of R-loops, and polymer-polymer phase separation. Notably, the D compartment comprises not just DSBs but also genes upregulated in response to DNA damage and this spatial arrangement contributes to the activation of those DNA damage response (DDR)-upregulated genes (C). However, there is a trade-off involved, as the close proximity of DSBs and DDR genes within the D compartment can potentially lead to translocation events that pose a risk to genome integrity.