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Polo, S. E.

Publications and source records attributed to Polo, S. E..

9 recordsLinked to original sources

New histone deposition recruits the DNA methylation maintenance machinery at sites of DNA damage repair

Faithful inheritance of DNA methylation contributes to the memory of epigenetic states and protects against disease. While the mechanisms underlying DNA methylation maintenance at replication forks are well characterized, whether and how DNA methylation is altered or maintained at sites of DNA damage repair is still poorly understood. Here, by exploiting sequencing, imaging and proteomic approaches in mammalian cells exposed to UV radiation, we show that the majority of DNA methylation marks are maintained during UV damage repair and we dissect the molecular machinery involved in DNA methylation control. We detect the recruitment to sites of repair synthesis of the DNMT1 and DNMT3A DNA methylating enzymes, driven by the DNMT1 cofactor UHRF1 and by UV damage repair endonucleases. We also uncover a crosstalk with histone dynamics, whereby newly deposited H3.3 histones at UV damage sites promote the recruitment of DNMT1. Functionally, we reveal the importance of the DNA methylation maintenance machinery for the transcriptional response to UV damage and sustained cell proliferation. This work provides a comprehensive picture of DNA methylation control mechanisms following DNA damage, with important implications for our understanding of human diseases with an altered methylome.

molecular biology↗

Non-canonical ATR signalling via NBS1 phosphorylation propagates fork slowing from stressed to unperturbed nuclear regions

DNA replication forks frequently encounter obstacles and remodel into four-way junctions to actively slow DNA fork progression. Fork slowing can also spread to undamaged forks via an ATR-dependent mechanism that remained elusive. Here, using mild genotoxic stress, we show that fork slowing and reversal require full ATR activity, but no canonical ATR activators and signalling partners, defining a non-canonical ATR pathway distinct from origin firing control. Phospho-proteomics in S-phase cells under checkpoint-blind replication stress identified a subset of ATR-dependent phospho-sites, such as S343 on NBS1, the regulatory subunit of the MRN complex. This residue is essential to stimulate MRN exonuclease activity in vitro and required in cells for global fork slowing upon mild DNA damage. Ultimately, local UV-C micro-irradiation reveals that ATR-dependent MRN-stimulated resection dampens DNA synthesis at lesions and propagates fork slowing to undamaged chromatin, supporting MRN-mediated ssDNA exposure as a mean to coordinate replication slowdown across the nucleus.

cell biology↗

Proteomic profiling of UV damage repair patches uncovers histone chaperones with central functions in chromatin repair

DNA damage compromises not only genome stability but also the integrity of the chromatin template, which plays a central role in controlling cell identity. Our understanding of chromatin repair mechanisms is very incomplete. To bridge this knowledge gap, here we devise a novel proteomic strategy to characterize dynamic changes in the chromatin landscape during the repair of UV-induced DNA lesions in human cells, in a quantitative, unbiased and time-resolved manner. Thus, we identify the histone chaperones DNAJC9 and MCM2 as central players in chromatin repair. We demonstrate that DNAJC9 and MCM2 are independently recruited to sites of UV damage repair. DNAJC9 provides new H3-H4 histones to CAF-1 and HIRA chaperones for deposition into chromatin and also stimulates old H3-H4 histone recovery. DNAJC9 cooperates with MCM2 to coordinate old and new histone dynamics during UV damage repair. Together, our proteomic dataset provides a molecular framework for further dissecting epigenome maintenance mechanisms.

molecular biology↗

Mitotic chromatin marking governs asymmetric segregation of DNA damage

The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However, most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here, we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation, and the incorporation of newly synthesized histones at UV damage sites, that both prevent local mitotic phosphorylations on histone H3 serine residues. Functionally, this chromatin-marking pathway drives the asymmetric segregation of UV damage in the cell progeny with consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions.

molecular biology↗

The histone chaperone activity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

molecular biology↗

Aberrant DNA repair is a vulnerability in histone H3.3-mutant brain tumors

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent oncogenic mechanism, which fosters genome instability and tumor cell growth in H3.3 mutant pHGG, thus opening new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme Polynucleotide Kinase 3-Phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

cancer biology↗

Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA

Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilizing the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark.

molecular biology↗

Histone chaperones and modifiers cooperate to maintain heterochromatin integrity following DNA damage.

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil 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. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

molecular biology↗

The histone chaperones FACT and ANP32E reshape the chromatin landscape during DNA damage repair through H2A.Z/H2A.X histone variant exchange

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, by investigating the mechanisms governing the distribution of H2A.X in mammalian chromatin, we demonstrate that this histone variant is deposited de novo at sites of DNA damage in a repair synthesis-coupled manner. Our mechanistic studies further identify the histone chaperone FACT (Facilitates Chromatin Transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, FACT potentiates H2A.X-dependent signaling of DNA damage and, together with ANP32E (Acidic Nuclear Phosphoprotein 32 Family Member E), orchestrates a H2A.Z/H2A.X exchange reaction that reshapes the chromatin landscape at repair sites. We propose that this mechanism promotes chromatin accessibility and helps tailoring DNA damage signaling to repair progression.\n\nHIGHLIGHTSO_LIH2A.X, but not H2A.Z, is deposited de novo at sites of DNA damage repair\nC_LIO_LIFACT promotes new H2A.X deposition coupled to repair synthesis\nC_LIO_LIFACT and ANP32E chaperones orchestrate H2A.Z/H2A.X exchange in damaged chromatin\nC_LIO_LIFACT stimulates H2A.X-dependent signaling of DNA damage\nC_LI

molecular biology↗