Two distinct modes for propagation of histone PTMs across the cell cycle

Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how histone marks propagate and demonstrate that chromatin states oscillate within the cell cycle

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.</p

Protección frente a la metilación y origen evolutivo de las islas CpG en el genoma de mamíferos

Esta tesis doctoral ha sido financiada por una beca predoctoral del Programa de Formación del Profesorado Universitario (2006/2010) del Ministerio de Educación del Gobierno de España, proyectos del Plan Nacional de I+D y por el proyecto CONSOLIDER Ingenio 2010.[ES] El objetivo principal de esta tesis doctoral es contribuir a esclarecer cómo se han generado las islas CpG en el genoma de mamíferos y qué mecanismos permiten a estas regiones permanecer desprovistas de metilación. Para ello decidimos llevar a cabo los siguientes análisis: 1. Papel de la replicación del DNA en la protección frente a la metilación de las islas CpG. 2. Contribución de la transcripción al mantenimiento de las islas CpG en estado no metilado. 3. Dinámica de generación de las islas CpG en el genoma de mamíferos.[EN] The main objective of this thesis is to help clarify how CpG islands have been generated in the mammalian genome and what mechanisms allow these regions remain devoid of methylation. To do this we decided to conduct the following tests: 1. Role of DNA replication in the protection against methylation of CpG islands. 2. Contribution of the transcript to the maintenance of CpG islands in unmethylated state. 3. Dynamic generation of CpG islands in the mammalian genome

Genome-wide and sister chromatid-resolved profiling of protein occupancy in replicated chromatin with ChOR-seq and SCAR-seq

Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.Research in the Groth laboratory was supported by the Independent Research Fund Denmark (7016-00042B and 4092-00404), the European Research Council (CoG no. 724436) and the Lundbeck Foundation (R198-2015-269 and R165-2013-15306). Research at CPR is supported by the Novo Nordisk Foundation (NNF14CC0001). N.P. is supported by LabEx ‘Who Am I?’ #ANR-11-LABX-0071 and the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its ‘Investments for the Future’ program and by an INTEGER grant from Agence Nationale de la Recherche (ANR-19-CE12-0030-01). C.G.-A. is the recipient of a Ramón y Cajal Contract RYC2018-025485-I, and her laboratory is supported by grants PID2019-105742GA-100 from the Spanish Government and VIPPIT-2019-IV.2 from the University of Sevilla. M.D. and R.A. were supported by the Independent Research Fund Denmark (6108-00038B) and the European Research Council (StG no. 638173) awarded to R.A

Two distinct modes for propagation of histone PTMs across the cell cycle

The South East Europe Electricitiy Roadmap (SEERMAP) project develops electricity sector scenarios until 2050. The project focuses on 9 countries in South East Europe: Albania, Bosnia and Herzegovina, Bulgaria, Greece, Kosovo*, former YugoslavRepublic of Macedonia, Montenegro, Romania and Serbia. The implications of different investment strategies in the electricity sector are assessed for affordability, energy security, sustainability and security of supply. In addition to analytical work, the project focuses on trainings, capacity building and enhancing dialogue and cooperation within the SEE region. *This designation is without prejudice to positions on status, and it is in line with UNSCR 1244 and the ICI Opinion on the Kosovo declaration of independence