Suchergebnisse

All organisms regulate transcription of their genes. To understand this process, a complete understanding of how transcription factors find their targets in cellular nuclei is essential. The DNA sequence and other variables are known to influence this binding, but the distribution of transcription factor binding patterns remains mostly unexplained in metazoan genomes. Here, we investigate the role of chromosome conformation in the trajectories of transcription factors. Using molecular dynamics simulations, we uncover the principles of their diffusion on chromatin. Chromosome contacts play a conflicting role: at low density they enhance transcription factor traffic, but at high density they lower it by volume exclusion. Consistently, we observe that in human cells, highly occupied targets, where protein binding is promiscuous, are found at sites engaged in chromosome loops within uncompacted chromatin. In summary, we provide a framework for understanding the search trajectories of transcription factors, highlighting the key contribution of genome conformation. ; We acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness (Centro de Excelencia Severo Ochoa 2013–2017, SEV-2012–0208, Plan Nacional BFU2012-37168), of the CERCA Programme/Generalitat de Catalunya, of the European Research Council (ERC Synergy Grant 609989), and of the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007–2013) under REA grant agreement 608959.

The increasing throughput of DNA sequencing technologies creates a need for faster algorithms. The fate of most reads is to be mapped to a reference sequence, typically a genome. Modern mappers rely on heuristics to gain speed at a reasonable cost for accuracy. In the seeding heuristic, short matches between the reads and the genome are used to narrow the search to a set of candidate locations. Several seeding variants used in modern mappers show good empirical performance but they are difficult to calibrate or to optimize for lack of theoretical results. Here we develop a theory to estimate the probability that the correct location of a read is filtered out during seeding, resulting in mapping errors. We describe the properties of simple exact seeds, skip seeds and MEM seeds (Maximal Exact Match seeds). The main innovation of this work is to use concepts from analytic combinatorics to represent reads as abstract sequences, and to specify their generative function to estimate the probabilities of interest. We provide several algorithms, which together give a workable solution for the problem of calibrating seeding heuristics for short reads. We also provide a C implementation of these algorithms in a library called Sesame. These results can improve current mapping algorithms and lay the foundation of a general strategy to tackle sequence alignment problems. The Sesame library is open source and available for download at https://github.com/gui11aume/sesame. ; We acknowledge the financial support of the Spanish Ministry of Economy, Industry and Competitiveness (Centro de Excelencia Severo Ochoa 2013–2017, Plan Estatal PGC2018-099807-B-I00), of the CERCA Programme/Generalitat de Catalunya, and of the European Research Council (Synergy Grant 609989). RC was supported by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement 608959. We also acknowledge support of the Spanish Ministry of Economy and Competitiveness (MEIC) to the EMBL partnership.

The main obstacle to curing HIV is the presence of latent proviruses in the bodies of infected patients. The partial success of reactivation therapies suggests that the genomic context of integrated proviruses can interfere with treatment. Here we developed a method called Barcoded HIV ensembles (B-HIVE) to map the chromosomal locations of thousands of individual proviruses while tracking their transcriptional activities in an infected cell population. B-HIVE revealed that, in Jurkat cells, the expression of HIV is strongest close to endogenous enhancers. The insertion site also affects the response to latency-reversing agents, because we found that phytohemagglutinin and vorinostat reactivated proviruses inserted at distinct genomic locations. From these results, we propose that combinations of drugs targeting all areas of the genome will be most effective. Overall, our data suggest that the insertion context of HIV is a critical determinant of the viral response to reactivation therapies. ; This research was supported by the Government of Catalonia and the Spanish Ministry of Economy and Competitiveness (Plan Nacional BFU2012-37168, Centro de Excelencia Severo Ochoa 2013-2017 SEV-2012-0208). J.P.M. and A.M. were supported by a grant from the Spanish Ministry of Economy and Competitiveness and FEDER (SAF2013-46077-R). E.Z. and G.J.F. are supported by the European Research Council (Synergy Grant 609989).

Background: Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. The molecular mechanisms of those biases are still poorly understood because it is difficult to isolate the contributions of DNA repair from those of DNA damage. Results: Here, we develop a genome-wide assay whereby the same DNA lesion is repaired in different genomic contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated if the break is repaired through single-strand annealing. The resolution of the mismatch showed a 60-80% bias in favor of the strand with the longest 3' flap. The location of the lesion in the genome and the type of mismatch had little influence on the bias. Instead, we observe a complete reversal of the bias when the longest 3' flap is moved to the opposite strand by changing the position of the double-strand break in the reporter. Conclusions: These results suggest that the processing of the double-strand break has a major influence on the repair of mismatches during a single-strand annealing. ; We acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2020-06377), the Spanish Ministry of Economy, Industry and Competitiveness ("Centro de Excelencia Severo Ochoa 2013-2017", Plan Estatal PGC2018-099807-B-I00), of the CERCA Programme/Generalitat de Catalunya, and of the European Research Council (Synergy Grant 609989). VOP was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie programme (665385). We also acknowledge the support of the Spanish Ministry of Economy and Competitiveness (MEIC) to the EMBL partnership

Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect. ; This work was supported by the Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa 2013-2017," SEV-2012-0208, Plan Nacional BFU2012-37168, NWO-ALW VICI, ERC Advanced Grant 293662 (B.v.S.), ERC Synergy Grant 609989 (G.J.F.), and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 608959 (R.C.).