Biology subjects

Vale-Silva, L. A.

Publications and source records attributed to Vale-Silva, L. A..

3 recordsLinked to original sources

bioRxiv2018

SNP-ChIP: A versatile and tag-free method to quantify changes in protein binding across the genome

Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) is the method of choice for mapping genome-wide binding of chromatin-associated factors. However, broadly applicable methods for between-sample comparisons are lacking. Here, we introduce SNP-ChIP, a method that leverages small-scale intra-species polymorphisms, mainly SNPs, for quantitative spike-in normalization of ChIP-seq results. Sourcing spike-in material from the same species ensures antibody cross-reactivity and physiological coherence, thereby eliminating two central limitations of traditional spike-in approaches. We show that SNP-ChIP is robust to changes in sequencing depth and spike-in proportions, and reliably identifies changes in overall protein levels, irrespective of changes in binding distribution. Application of SNP-ChIP to test cases from budding yeast meiosis allowed discovery of novel regulators of the chromosomal protein Red1 and quantitative analysis of the DNA-damage associated histone modification{gamma} -H2AX. SNP-ChIP is fully compatible with the intra-species diversity of humans and most model organisms and thus offers a general method for normalizing ChIP-seq results.

genomics↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2017

Comparative Genomics Of Two Sequential Candida glabrata Clinical Isolates

Candida glabrata is an important fungal pathogen which develops rapidly antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. We were interested in this study to compare genomes of two specific C. glabrata related isolates, one of which was azole-susceptible (DSY562) while the other was azole-resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time lapse of 50 days of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacbBio technology used here yielded 14 major contigs (sizes 0.18 Mb-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 contained each a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (66) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 non-synonymous SNPs (among which the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes were containing a DNA mismatch repair allele of MSH2 known to be involved in the so-called hypermutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points were exhibiting limited variations, even if submitted to the host pressure.

genomics↗