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Chromatin conformation regulates the coordination between DNA replication and transcription [array]

ABSTRACT: Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure we analysed the replication initiation landscape, elongation kinetics, and timing of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or triple-KO in three histone H1 genes. We found that loss of chromatin compaction in H1-depleted cells alters dramatically the initiation patterns triggering the accumulation of stalled forks and DNA damage as a consequence of transcription-replication conflicts, whereas reductions in nucleosome content due to the lack of HMGB1 causes faster fork progression without perturbing the initiation landscape or fork stability. Thus, perturbations in the integrity of the chromatin template elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure, such as those occurring during cellular aging or in some developmental disorders or cancer, contribute to genomic instability.

ORGANISM(S): Mus musculus

PROVIDER: GSE99739 | GEO | 2018/02/14

REPOSITORIES: GEO

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Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

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Chromatin conformation regulates the coordination between DNA replication and transcription [array]

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