Heterochromatin maintenance in response to DNA damage – HEROD

Building on the unique and broad expertise of our research consortium, we will use a combination of powerful novel approaches to explore the existence of dedicated mechanisms for the maintenance of heterochromatin structure and function in response to DNA damage in mammalian cells.
Specifically, we intend to elucidate the response to UV lesions and DNA double-strand breaks (DSBs) in two types of heterochromatin compartments, characterized by distinct sets of epigenetic marks: constitutive pericentric heterochromatin domains, and the inactive X chromosome in female cells, a typical example of facultative heterochromatin. We recently developed unique cellular models for tracking the response to DNA damage in heterochromatin domains and innovative methods for the local induction of DNA lesions in these chromatin compartments.
We will employ these new tools to examine the influence of higher-order chromatin organization on DNA damage repair by analyzing how the heterochromatic state affects DNA repair efficiency and DSB repair pathway choice. Reciprocally, through a wide range of structural and functional assays, we will determine how genotoxic stress impacts heterochromatin integrity, in terms of histone deposition, histone modifications, chromatin compaction and transcriptional status. This will ultimately lead us to dissect the pathways to heterochromatin integrity in response to genotoxic stress, with the identification of key molecular players among candidate histone chaperones and histone modifiers.

We have established powerful cellular models for examining the response to UV damage and DNA double-strand breaks (DSBs) in constitutive and facultative heterochromatin domains.
In particular, we have set up an innovative approach based on UVC laser micro-irradiation in mouse fibroblasts for targeting UV damage to pericentric heterochromatin and for tracking the heterochromatin response to UV in real time. Thus, we have uncovered profound alterations in heterochromatin compaction during repair, orchestrated by the UV damage sensor DDB2. Mechanistically, DDB2 promotes linker histone displacement from damaged chromatin and DDB2-mediated chromatin decompaction facilitates the access of downstream repair factors to the core of heterochromatin domains. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We have unveiled a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV, SETDB1 coordinating histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. These findings reveal core principles of constitutive heterochromatin maintenance in response to UV damage.
Following up on this work, we have initiated studies to examine the DSB response in facultative heterochromatin in female mammals. We have established imaging methods for analysing various facultative heterochromatin features and examined how those are affected by the DSB-inducing agent neocarzinostatin. In parallel, we are setting up methods to induce sequence-specific DSBs in facultative heterochromatin domains on the inactive X chromosome using a degradable Cas9 nuclease.

Altogether, this work should shed new light on the fundamental mechanisms involved in heterochromatin maintenance following DNA damage. Beyond the DNA damage response, our findings may also provide a molecular framework for understanding heterochromatin maintenance during other disruptive events in both normal and pathological conditions, like DNA replication, cell differentiation, aging and cancer diseases.

Fortuny A, Chansard A, Caron P, Chevallier O, Leroy O, Renaud O, Polo SE. Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance. Biorxiv doi.org/10.1101/818914

DNA damage challenges both genome integrity and its organization with histone proteins into chromatin, which governs cell identity. How chromatin structure is altered after DNA damage while preserving its functions and the epigenetic information that it conveys is thus a fundamental and yet still unanswered question. The proposed project aims to address this topical issue by focusing on higher-order heterochromatin domains. Building on the unique and broad expertise of our research consortium, we will use a combination of powerful novel approaches to explore the existence of dedicated mechanisms for the maintenance of heterochromatin structure and function in response to DNA damage in mammalian cells.
Specifically, we intend to elucidate the response to UV lesions and DNA double-strand breaks (DSBs) in two types of heterochromatin compartments, characterized by distinct sets of epigenetic marks: constitutive pericentric heterochromatin domains, and the inactive X chromosome in female cells, a typical example of facultative heterochromatin. We recently developed unique cellular models for tracking the response to DNA damage in heterochromatin domains and innovative methods for the local induction of DNA lesions in these chromatin compartments.
We will employ these new tools to examine the influence of higher-order chromatin organization on DNA damage repair by analyzing how the heterochromatic state affects DNA repair efficiency and DSB repair pathway choice. Reciprocally, through a wide range of structural and functional assays, we will determine how genotoxic stress impacts heterochromatin integrity, in terms of histone deposition, histone modifications, chromatin compaction and transcriptional status. This will ultimately lead us to dissect the pathways to heterochromatin integrity in response to genotoxic stress, with the identification of key molecular players among candidate histone chaperones and histone modifiers. Altogether, this work should shed new light on the fundamental mechanisms involved in heterochromatin maintenance following DNA damage.