Pervasive lesion segregation shapes cancer genome evolution.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains.

BACKGROUND: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. RESULTS: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. CONCLUSIONS: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.

CTCF maintains regulatory homeostasis of cancer pathways.

BACKGROUND: CTCF binding to DNA helps partition the mammalian genome into discrete structural and regulatory domains. Complete removal of CTCF from mammalian cells causes catastrophic genome dysregulation, likely due to widespread collapse of 3D chromatin looping and alterations to inter- and intra-TAD interactions within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction of CTCF expression are viable, albeit with increased cancer incidence. Here, we exploit chronic Ctcf hemizygosity to reveal its homeostatic roles in maintaining genome function and integrity. RESULTS: We find that Ctcf hemizygous cells show modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these are enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, we observe dysregulation of the expression of several hundred genes, which are concentrated in cancer-related pathways, and are caused by changes in transcriptional regulation. Chromatin structure is preserved but some loop interactions are destabilized; these are often found around differentially expressed genes and their enhancers. Importantly, the transcriptional alterations identified in vitro are recapitulated in mouse tumors and also in human cancers. CONCLUSIONS: This multi-dimensional genomic and epigenomic profiling of a Ctcf hemizygous mouse model system shows that chronic depletion of CTCF dysregulates steady-state gene expression by subtly altering transcriptional regulation, changes which can also be observed in primary tumors.

Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5' end and a more flexible 3' end with the possibility of 3' tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage).

Starvation and re-feeding affect Hsp expression, MAPK activation and antioxidant enzymes activity of European sea bass (Dicentrarchus labrax).

In the context of food deprivation in fish (wild and farmed), understanding of cellular responses is necessary in order to develop strategies to minimize stress caused by starvation in the aquaculture section. The present study evaluates the effects of long term starvation (1F-3S: one-month feeding-three-month starvation) and starvation/re-feeding (2S-2F: two-month starvation-two-month re-feeding) compared to the control group (4F-0S: four-month feeding-zero month starvation) on cellular stress response and antioxidant defense in organs, like the intestine, the liver, the red and white muscle of European sea bass Dicentrarchus labrax. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK) and the extracellular signal-regulated kinases (ERK-1/2). For the determination of the effect of the oxidative stress caused by food deprivation and/or re-feeding, the (maximum) activities of antioxidant enzymes such as glutathione peroxidise (GPx), catalase (CAT) and superoxide dismutase (SOD) as well as the determination of thiobarbituric acid reactive substances (TBARS) were studied. The experimental feeding trials caused a tissue distinct and differential response on the cellular and antioxidant capacity of sea bass not only during the stressful process of starvation but also in re-feeding. Specifically, the intestine phosphorylation of ERKs and antioxidant enzymatic activities increased in the 2S-2F fish group, while in the 1F-3S group an increase was detected in the levels of the same proteins except for GPx. In the liver and the red muscle of 2S-2F fish, decreased Hsp70 and phosphorylated p38 MAPK levels and increased Hsp90 levels were observed. Additionally, SOD activity decreased in the red muscle of 2S-2F and 1F-3S groups. In the liver and red muscle of 1F-3S group Hsp70 levels increased, while the activation of p38 MAPK in the liver decreased. In the white muscle, Hsp90 levels decreased and the phosphorylation of p38 MAPK increased in both feeding regimes compared to control. In the same tissue, GPx and catalase levels were decreased in 2S-2F regime, while SOD levels were decreased in 1F-3S regime.