Developing molecular tools for probing and modulating genomic spatial adjacency

In addition to the vast information encoded in DNA sequence, the genome has physical features that are also essential for its function, including its organization in threedimensional space. The development of high-throughput technology has greatly advanced our understanding of the spatial organization of the genome but has also raised more questions. In this thesis, we developed molecular tools to address the remaining challenges regarding the interplay between genomic organization and function. By breaking down the subject from the global architecture of the genome into an ensemble of spatially adjacent chromatin segments, we came up with different methods covering various aspects. We demonstrated in Paper I that global spatial information can be transferred in the format of DNA sequence encoding pairwise spatial proximity between two distinct molecular objects. We have shown that by growing network from pairwise relationship encoded in DNA sequence, spatial features at a global scale can be recovered. The results from this work highlighted the potential of using pairwise adjacency as a fundamental unit for recording the spatial organization of complex molecular systems. The high programmability and versatility of nucleic acids make them an ideal medium for encoding this information. With the aim of studying the pairwise relationship between genomic DNA in cells, we devised a CRISPR-dCas9 system for different purposes by leveraging its high programmability for genome targeting. In Paper III, we have shown that the re-designed guide RNA can direct dCas9 to a pair of genomic loci, inducing DNA contacts. This system can be applied as a modulation tool to introduce pairwise contacts for decoding functional implications in cells. In Paper IV, we developed a method for the direct detection of pairwise interactions between genomic loci at the single-cell level in situ. This method is achieved by conjugating oligonucleotide tags to Cas9 and using the tags for probing the spatial adjacency between a pair of genomic loci targeted by Cas9 Meanwhile, we developed an efficient method to fabricate and purify DNA origami with modifications in Paper II. This method makes the production of functionalized nanostructures more time and material-efficient compared to established techniques. The ease of production allows broader applications of functionalized nanostructures, including characterizing the effect of nanoscale distance on biochemical assays, as shown in Paper IV

Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus

International audienceReconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements , and protein divergence into a single evolutionary framework

Male Germline Chromatin

Spermatogenesis requires radical restructuring of germline chromatin at multiple stages, involving coordinated waves of DNA methylation/demethylation, histone modification, and the replacement and removal that occurs before, during, and after meiosis. This Special Issue will draw together papers that address all aspects of chromatin organization and dynamics in the male germ line, in humans, and in model organisms. In particular, we will invite authors to discuss novel methods for studying germline chromatin structure, the interplay between chromatin structure and susceptibility to DNA damage and mutation, chromatin modifications associated with epigenetic inheritance in the early embryo, and the impact this work has for understanding natural fertility and improving assisted reproduction techniques

Chromosome rearrangements and population genomics

Chromosome rearrangements result in changes to the physical linkage and order of sequences in the genome. Although we have known about these mutations for more than a century, we still lack a detailed understanding of how they become fixed and what their effect is on other evolutionary processes. Analysing genome sequences provides a way to address this knowledge gap. In this thesis I compare genome assemblies and use population genomic inference to gain a better understanding of the role that chromosome rearrangements play in evolution. I focus on butterflies in the genus Brenthis, where chromosome numbers are known to vary between species. In chapter 2, I present a genome assembly of Brenthis ino and show that its genome has been shaped by many chromosome rearrangements, including a Z-autosome fusion that is still segregating. In chapter 3, I investigate how synteny information in genome sequences can be used to infer ancestral linkage groups and inter-chromosomal rearrangements, implementing the methods in a command-line tool. In chapter 4, I test whether chromosome fissions and fusions have acted as barriers to gene flow between B. ino and its sister species B. daphne. I find that chromosomes involved in rearrangements have experienced less post-divergence gene flow than the rest of the genome, suggesting that rearrangements have promoted speciation. Finally, in chapter 5, I investigate how chromosome rearrangements have become fixed in B. ino, B. daphne, and a third species, B. hecate. I show that genetic drift is unlikely to be a strong enough force to have fixed very underdominant rearrangements, and that there is only weak evidence that chromosome fusions have become fixed through positive natural selection. In summary, this work provides methods for researching chromosome evolution as well as new results about how rearrangements evolve and impact the speciation process

Bioinformatics

This book is divided into different research areas relevant in Bioinformatics such as biological networks, next generation sequencing, high performance computing, molecular modeling, structural bioinformatics, molecular modeling and intelligent data analysis. Each book section introduces the basic concepts and then explains its application to problems of great relevance, so both novice and expert readers can benefit from the information and research works presented here