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Caption: In this artistic work by S. Colmenares, the heterochromatin compartment is depicted as the Sun. Heterochromatic ‘flares’ are released during repair to allow the recruitment of repair components (yellow and blue dots).
Our Question:
Our Question:
While homologous recombination (HR) is the most accurate pathway to repair double-strand breaks (DSBs), misregulation of HR results in chromosomal rearrangements and aneuploidy. This "aberrant recombination" occurs when ectopic (non-homologous) sequences, rather than sister chromatids or homologous chromosomes, are used as templates for repair. In heterochromatin, which occupies 10-30% of the genome, the risk of ectopic exchanges is extremely high. This is because up to millions of similar repeated sequences are present in heterochromatin of non-homologous chromosomes, which can be used as templates for repair. How do cells repair heterochromatic DSBs without generating massive genome instability?
Answering this question will resolve one of the most interesting mysteries in biology: how do human cells maintain a stable genome despite being mostly composed of repeated sequences?
Why is it important?
Aberrant recombination and chromosome rearrangements are responsible for cancer formation, infertility, developmental disorders, neurological and neuromuscular diseases, and aging. Understanding the molecular mechanisms involved in heterochromatin repair is expected to provide more effective preventive, diagnostic and therapeutic strategies for these human diseases and aging-related disorders.
Our approach:
Our approach:
We work with mouse, human, and Drosophila cell models, and we integrate multidisciplinary genetic, biochemical, -omics, computational, and high-resolution imaging approaches. The Drosophila model system (fruit fly) is currently the best model for heterochromatin repair studies because in this system heterochromatin is organized in a distinct nuclear domain (Figure 1), facilitating the use of cytological approaches to study repair. To characterize repair progression, we express fluorescent-tagged repair components and follow their recruitment to foci at DSBs relative to the heterochromatin domain (Movie 1).
What we discovered:
What we discovered:
Heterochromatin undergoes a global reorganization in response to damage: the entire domain expands and forms dynamic protrusions, while heterochromatic repair foci relocalize to the euchromatic space (Movie 1).
DSBs are repaired by HR, but with significant differences from euchromatin. Repair starts within the domain (Movie 2), but can be completed only after a striking relocalization of repair sites to the nuclear periphery [2]. This separation of repair steps in space and time requires SUMOylation and the Smc5/6 complex [1,2], and is essential to prevent aberrant recombination and genome instability [1]. This movement likely prevents aberrant recombination while enabling "safe" repair by isolating the broken DNA and its homologous templates from the bulk of heterochromatic repeats during repair.
Figure 1. Drosophila melanogaster.
Figure 2. Heterochromatin and euchromatin form distinct domains in the nucleus of Drosophila cells.
Movie 1: A Drosophila nucleus shows early repair foci that form within the heterochromatin domain and relocalize to outside the domain during repair.
Ryu et al., Nature Cell Biology, 2015
We also discovered that the movement of repair sites occurs in two steps:
1 - Inside the heterochromatin domain, repair sites move largely with diffusive motions. Relocalization to the surface of the heterochromatin domain relies on capillary forces generated by immiscible condensates: the heterochromatin domain and repair foci. Biomolecular condensates are membraneless structures with unique properties that separate them from the surrounding environment, like oil in water. The heterochromatin domain forms condensates through its heterochromatin protein 1 (HP1), while at repair sites condensate formation is driven by Nup98.
2 - Once at the heterochromatin domain periphery, repair sites are "pulled" to the nuclear periphery by a striking network of nuclear actin filaments and their associated myosins. Myosins interact with the heterochromatin repair component Smc5/6 that bridges the motors with chromatin and generate directed motions by "walking" along the filaments.
Our studies revealed that relocalization occurs through actin filaments assembled at repair sites (Movie 2). Myosins activated by Smc5/6 ride along these ‘highways’ for repair, enabling the directed motion of heterochromatic repair sites to the nuclear periphery.
Caridi et al., Nature, 2018
Current projects:
Current projects:
Our studies revealed the importance of nuclear dynamics in heterochromatic DSB repair, and raised new and exciting questions:
What mechanisms control these dynamics?
We employ genomic and proteomic approaches to identify new molecular components involved in the spatial and temporal regulation of heterochromatin repair, and we characterize their functions in the pathway with a combination of genetic, imaging, genomic mapping, and biochemical approaches.
Our current projects are addressing the following questions:
What is the composition and regulation of Nup98 condensates in heterochromatin?
How does the "silent" epigenetic state of heterochromatin contributes to repair?
How are nuclear actin filaments regulated to coordinate repair responses in heterochromatin?
How is HR repair halted inside the heterochromatin domain and resumed at the nuclear periphery?
How is Smc5/6 regulated in heterochromatin?
How does deregulation of these processes affect genome stability in cancer cells and during aging?
Deregulation of heterochromatin repair is potentially one of the most powerful driving forces for cancer and other genome-instability disorders, and a major cause for genome instability and cell lethality in old organisms. We are interested in establishing the consequences of inactivating heterochromatin repair pathways to genome stability and aging. We expect these studies to allow the development of better therapeutic approaches for cancer and other aging-related disorders.
Our current projects include establishing how dysregulation of Nup98 condensates contribute to acute myeloid leukemia (AML), a very agressive form of blood cancer with limited therapeutic options.
References:
[1] Chiolo I. et al., Chiolo I. (2011) Double-strand breaks in heterochromatin move outside a dynamic HP1a domain to complete recombinational repair. Cell 144:732-44.
[2] Ryu T. et al., Chiolo I. (2015) Heterochromatic breaks move to the nuclear periphery to continue recombinational repair Nature Cell Biology 17:1401-11.
[3] Caridi et al., ..Chiolo I. (2018). Nuclear F-actin and myosins drive relocalization of heterochromatin breaks. Nature, 559:35-7.
[4] Merigliano et al., Chiolo I. (2025). Off-pore nucleoporins relocalize heterochromatic breaks through phase separation. Molecular Cell. 85(12):2355-2373.e11.
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