Validating the concept of mutational signatures with isogenic cell models.

The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems-the exposures to different mutational processes in a patient's lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures

Fast ancestral gene order reconstruction of genomes with unequal gene content

Feijão P, Soares de Araujo FE. Fast ancestral gene order reconstruction of genomes with unequal gene content. BMC Bioinformatics. 2016;17(S14): 413.Background During evolution, genomes are modified by large scale structural events, such as rearrangements, deletions or insertions of large blocks of DNA. Of particular interest, in order to better understand how this type of genomic evolution happens, is the reconstruction of ancestral genomes, given a phylogenetic tree with extant genomes at its leaves. One way of solving this problem is to assume a rearrangement model, such as Double Cut and Join (DCJ), and find a set of ancestral genomes that minimizes the number of events on the input tree. Since this problem is NP-hard for most rearrangement models, exact solutions are practical only for small instances, and heuristics have to be used for larger datasets. This type of approach can be called event-based. Another common approach is based on finding conserved structures between the input genomes, such as adjacencies between genes, possibly also assigning weights that indicate a measure of confidence or probability that this particular structure is present on each ancestral genome, and then finding a set of non conflicting adjacencies that optimize some given function, usually trying to maximize total weight and minimizing character changes in the tree. We call this type of methods homology-based. Results In previous work, we proposed an ancestral reconstruction method that combines homology- and event-based ideas, using the concept of intermediate genomes, that arise in DCJ rearrangement scenarios. This method showed better rate of correctly reconstructed adjacencies than other methods, while also being faster, since the use of intermediate genomes greatly reduces the search space. Here, we generalize the intermediate genome concept to genomes with unequal gene content, extending our method to account for gene insertions and deletions of any length. In many of the simulated datasets, our proposed method had better results than MLGO and MGRA, two state-of-the-art algorithms for ancestral reconstruction with unequal gene content, while running much faster, making it more scalable to larger datasets. Conclusion Studing ancestral reconstruction problems under a new light, using the concept of intermediate genomes, allows the design of very fast algorithms by greatly reducing the solution search space, while also giving very good results. The algorithms introduced in this paper were implemented in an open-source software called RINGO (ancestral Reconstruction with INtermediate GenOmes), available at https://github.com/pedrofeijao/RINGO

A High-Resolution Map of Synteny Disruptions in Gibbon and Human Genomes

Gibbons are part of the same superfamily (Hominoidea) as humans and great apes, but their karyotype has diverged faster from the common hominoid ancestor. At least 24 major chromosome rearrangements are required to convert the presumed ancestral karyotype of gibbons into that of the hominoid ancestor. Up to 28 additional rearrangements distinguish the various living species from the common gibbon ancestor. Using the northern white-cheeked gibbon (2n = 52) (Nomascus leucogenys leucogenys) as a model, we created a high-resolution map of the homologous regions between the gibbon and human. The positions of 100 synteny breakpoints relative to the assembled human genome were determined at a resolution of about 200 kb. Interestingly, 46% of the gibbon–human synteny breakpoints occur in regions that correspond to segmental duplications in the human lineage, indicating a common source of plasticity leading to a different outcome in the two species. Additionally, the full sequences of 11 gibbon BACs spanning evolutionary breakpoints reveal either segmental duplications or interspersed repeats at the exact breakpoint locations. No specific sequence element appears to be common among independent rearrangements. We speculate that the extraordinarily high level of rearrangements seen in gibbons may be due to factors that increase the incidence of chromosome breakage or fixation of the derivative chromosomes in a homozygous state

Design, synthesis and characterization of the synthetic yeast genome

With the rapid development of DNA synthesis technologies, synthetic biology has made tremendous progress in the past 15 years, in particular for synthetic genomics. Synthetic genomics is a nascent field of synthetic biology, which aims to design new biological systems/organisms to satisfy human needs. Conventional synthetic biology focuses on the redesign, construction and modeling of biological parts, pathways or genomes that do not exist in nature, while synthetic genomics encompasses technologies that allow the generation of chemically synthesized larger parts of genomes or whole genomes, with simultaneous redesign of an organism’s genetic material. Synthetic genomics is painting a blueprint for a new era of biology and holds great potential for a multitude of applications, such as pharmaceuticals, biofuels and rapid generation of vaccines against emerging diseases. Chapter One gives an introduction of the current state of the art and challenges of synthetic genomics and the objectives of this study. Chapter Two demonstrates the design and construction strategy of two megabase-long synthetic yeast chromosomes, SynII and SynVII. Chapter Three describes the full characterization of SynII and SynVII. Chapter Four introduces the SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution) system and its application in SynII and SynVII. Taken together, this work demonstrates the utility of synthetic yeast for understanding biological systems and its potential for industrial applications

A pharmaceutical model for the molecular evolution of microbial natural products

Abstract Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.Peer reviewe

Gene duplications during experimental evolution of Caenorhabditis elegans : duplication rates and evolutionary responses

Copy-number variants (CNVs) are a ubiquitous form of genetic variation. How often this form of variation arises and its adaptive significance are active areas of contemporary research. This work presents evidence regarding both of these subjects. First, it demonstrates that gene duplications occur at a frequency two orders of magnitude greater than point mutations. Specifically, the gene duplication rate is estimated to be 1.2 x 10-7/gene/generation, compared to a point mutation rate on the order of ~10-9/site/ generation. Second, it was found that populations in a low state of fitness due to mutation accumulation could recover some or all of their fitness over short spans of generations concurrent with an increase in frequency of duplications and deletions that arose during the recovery process. The pattern of frequency increase among CNVs over generations during recovery was consistent with the signature of positive selection. The median size of duplications that were identified after selection for ~200 generations were significantly larger (191.5 kb) than both duplications that occurred spontaneously (2 kb) in the absence of selection and deletions identified after selection for ~200 generations (12.5 kb). The median number of genes contained in the duplications during recovery was 38, evincing the ability of these events to increase the genetic information available for selection to act on. These results clearly demonstrate that gene duplication and deletion processes contribute significantly to the adaptability of populations

An investigation of genomic instability and its impact on cancer development and heterogeneity

Genomic instability (GIN), a genomic state facilitating large scale chromosomal rearrangements, is a hallmark of cancer. GIN can contribute to oncogenesis by disrupting genes, and leading to copy number aberrations (CNAs), the gain or loss of genomic segments. In this thesis I describe two projects linked by the overarching theme of GIN, outlined below: Project 1: Copy-number aberrations (CNAs) contribute to clonal diversity within cancer, with clinical implications. Breast cancer is one such example, but the effect of CNAs on gene expression in intra-tumour subclonal populations has not been properly characterised. Due to sequencing technology limits and lack of computational methods, it is difficult to assess CNAs at a subclonal level. Here, I have benchmarked the ‘InferCNV’ computational method and used it to infer single cell CNA profiles from 14 primary breast cancer single cell RNA-sequencing (scRNA-seq) datasets. I reveal diverse intratumoural heterogeneity involving at least four subclonal populations per tumour. Finally, I identify subclones with expression/CNA profiles indicative of metastatic potential, involving differential regulation of metastasis associated genes such as MUCL1, BST2 and IGFBP5. Project 2: High-grade serous ovarian cancer (HGSOC) is characterised by widespread GIN. Drivers of GIN include deficient DNA repair and amplification of Cyclin E1, however no major cause is known for one third of tumours. Deregulation of repetitive elements may contribute to GIN in HGSOC. It is difficult to investigate repetitive elements from sequencing data as they map to multiple places within the genome. I have quantified repetitive RNA in 99 high-grade serous ovarian cancer (HGSOC) and matched control RNA-seq datasets to determine their potential contribution to GIN. I identified retrotransposons which are deregulated in HGSOC, which may have been active during cancer development. Some of these retrotransposons were enriched at structural variant breakpoints, indicating potential causality. Finally, I identified retrotransposon-associated structural variants in proximity to deregulated oncogenes implicated in homologous DNA repair, which may have modulated their expression and contributed to cancer development. In summary, I have explored both a cause (retrotransposons) and consequence (CNA-based heterogeneity) of GIN in cancer, and shown how GIN can contribute to the modulation of cancer-associated genes which influence cancer development and outcomes

Promoter propagation in prokaryotes

Transcriptional activation or 'rewiring' of silent genes is an important, yet poorly understood, phenomenon in prokaryotic genomes. Anecdotal evidence coming from experimental evolution studies in bacterial systems has shown the promptness of adaptation upon appropriate selective pressure. In many cases, a partial or complete promoter is mobilized to silent genes from elsewhere in the genome. We term hereafter such recruited regulatory sequences as Putative Mobile Promoters (PMPs) and we hypothesize they have a large impact on rapid adaptation of novel or cryptic functions. Querying all publicly available prokaryotic genomes (1362) uncovered >4000 families of highly conserved PMPs (50 to 100 long with =80% nt identity) in 1043 genomes from 424 different genera. The genomes with the largest number of PMP families are Anabaena variabilis (28 families), Geobacter uraniireducens (27 families) and Cyanothece PCC7424 (25 families). Family size varied from 2 to 93 homologous promoters (in Desulfurivibrio alkaliphilus). Some PMPs are present in particular species, but some are conserved across distant genera. The identified PMPs represent a conservative dataset of very recent or conserved events of mobilization of non-coding DNA and thus they constitute evidence of an extensive reservoir of recyclable regulatory sequences for rapid transcriptional rewirin