Centromeres under pressure: Evolutionary innovation in conflict with conserved function

Centromeres are essential genetic elements that enable spindle microtubule attachment for chromosome segregation during mitosis and meiosis. While this function is preserved across species, centromeres display an array of dynamic features, including: (1) rapidly evolving DNA; (2) wide evolutionary diversity in size, shape and organization; (3) evidence of mutational processes to generate homogenized repetitive arrays that characterize centromeres in several species; (4) tolerance to changes in position, as in the case of neocentromeres; and (5) intrinsic fragility derived by sequence composition and secondary DNA structures. Centromere drive underlies rapid centromere DNA evolution due to the “selfish” pursuit to bias meiotic transmission and promote the propagation of stronger centromeres. Yet, the origins of other dynamic features of centromeres remain unclear. Here, we review our current understanding of centromere evolution and plasticity. We also detail the mutagenic processes proposed to shape the divergent genetic nature of centromeres. Changes to centromeres are not simply evolutionary relics, but ongoing shifts that on one side promote centromere flexibility, but on the other can undermine centromere integrity and function with potential pathological implications such as genome instability

INVESTIGATING INVASION IN DUCTAL CARCINOMA IN SITU WITH TOPOGRAPHICAL SINGLE CELL GENOME SEQUENCING

Synchronous Ductal Carcinoma in situ (DCIS-IDC) is an early stage breast cancer invasion in which it is possible to delineate genomic evolution during invasion because of the presence of both in situ and invasive regions within the same sample. While laser capture microdissection studies of DCIS-IDC examined the relationship between the paired in situ (DCIS) and invasive (IDC) regions, these studies were either confounded by bulk tissue or limited to a small set of genes or markers. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS), which combines laser-catapulting with single cell DNA sequencing to measure genomic copy number profiles from single tumor cells while preserving their spatial context. We applied TSCS to sequence 1,293 single cells from 10 synchronous DCIS patients. We also applied deep-exome sequencing to the in situ, invasive and normal tissues for the DCIS-IDC patients. Previous bulk tissue studies had produced several conflicting models of tumor evolution. Our data support a multiclonal invasion model, in which genome evolution occurs within the ducts and gives rise to multiple subclones that escape the ducts into the adjacent tissues to establish the invasive carcinomas. In summary, we have developed a novel method for single cell DNA sequencing, which preserves spatial context, and applied this method to understand clonal evolution during the transition between carcinoma in situ to invasive ductal carcinoma

Substitution Patterns Are GC-Biased in Divergent Sequences across the Metazoans

The fastest-evolving regions in the human and chimpanzee genomes show a remarkable excess of weak (A,T) to strong (G,C) nucleotide substitutions since divergence from their common ancestor. We investigated the phylogenetic extent and possible causes of this weak to strong (W→S) bias in divergent sequences (BDS) using recently sequenced genomes and recombination maps from eight trios of eukaryotic species. To quantify evidence for BDS, we inferred substitution histories using an efficient maximum likelihood approach with a context-dependent evolutionary model. We then annotated all lineage-specific substitutions in terms of W→S bias and density on the chromosomes. Finally, we used the inferred substitutions to calculate a BDS score—a log odds ratio between substitution type and density—and assessed its statistical significance with Fisher's exact test. Applying this approach, we found significant BDS in the coding and noncoding sequence of human, mouse, dog, stickleback, fruit fly, and worm. We also observed a significant lack of W→S BDS in chicken and yeast. The BDS score varies between species and across the chromosomes within each species. It is most strongly correlated with different genomic features in different species, but a strong correlation with recombination rates is found in several species. Our results demonstrate that a W→S substitution bias in fast-evolving sequences is a widespread phenomenon. The patterns of BDS observed suggest that a recombination-associated process, such as GC-biased gene conversion, is involved in the production of the bias in many species, but the strength of the BDS likely depends on many factors, including genome stability, variability in recombination rate over time and across the genome, the frequency of meiosis, and the amount of outcrossing in each species

EVOLUTION OF THE CIRCADIAN CLOCK IN EXTREME ENVIRONMENT: LESSONS FROM CAVEFISH.

Evolution has been strongly influenced by the daily cycles of temperature and light imposed by the rotation of the Earth. Fascinating demonstrations of this are seen in extreme environments such as caves where some animals have remained completely isolated from the day-night cycle for millions of years. Most of these species show convergent evolution, sharing a range of striking physical properties such as eye loss. One fundamental issue is whether “hypogean” species retain a functional circadian clock. This highly conserved, physiological timing mechanism allows organisms to anticipate daily environmental changes and is synchronized primarily by light. The Somalian cavefish, Phreatichthys andruzzii does possess a circadian clock that is entrained by a daily regular feeding time but strikingly, not by light. Under constant conditions the P. andruzzii clock oscillates with an extremely long period and also lacks normal temperature compensation. We document multiple mutations affecting a light-induced clock gene, Period2 as well as the genes encoding the extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin. Remarkably, we show that ectopic expression of zebrafish homologs of these opsins rescues light induced clock gene expression in P. andruzzii cells. Thus, by studying this natural mutant we provide direct evidence for a peripheral light-sensing function of extra-retinal opsins in vertebrates. Furthermore, the properties of this cavefish illustrate that evolution in constant darkness leads not only to anatomical changes but also to loss of gene function linked with the detection and anticipation of the day-night cycle

Evolution of mammalian genome architecture through retrotransposition

Retrotransposons, mobile DNA elements that replicate via a copy and paste mechanism, are a major component of mammalian genome architecture. They account for at least one-third of the human genome and are major drivers of lineage-specific gain and loss of DNA. While there are many examples of how specific retrotransposons have impacted evolution, their interaction with large-scale genome architecture remains poorly characterised. Throughout my thesis I investigated two fundamental questions regarding genome evolution and retrotransposons. Firstly, how does genome architecture shape retrotransposon accumulation? Secondly, how does retrotransposon accumulation in turn impact on genome architecture? The current model of retrotransposon accumulation largely relies on local sequence composition. However, this model fails to account for genome-wide chromatin structure, an important factor that regulates DNA accessibility to insertion machinery. By analysing retrotransposon accumulation at open chromatin sites I showed that genome structure strongly associates with retrotransposon accumulation patterns. In addition, by mapping retrotransposon accumulation patterns of non-human mammals back to human, I was able to observe large-scale positional conservation of lineage-specific retrotransposons. These findings suggest that through conservation of synteny, gene regulation and nuclear organisation, retrotransposon accumulation in mammalian genomes follows similar evolutionary trajectories. Beneath the conserved structural framework of mammalian genomes there exists a high degree of lineage-specific turnover of DNA. Outside of whole genome duplication, retrotransposons are the largest contributing factor to genome growth. In contrast to this, accumulation of retrotransposons can also increase the probability of unequal crossing over causing DNA loss through large deletion events. Using multiple pairwise alignments I calculated regional levels of lineage-specific DNA gain and loss in the human and mouse genomes. I found that while lineage-specific DNA loss overlapped with open chromatin regions in both genomes, different sources for lineage-specific DNA gain drove divergence in genome architecture. These findings reveal the turbulent nature of lineage-specific evolution of large-scale genome architecture, ultimately questioning the evolutionary stability of structural chromosomal domains. In addition to analysing large-scale genome architecture I performed two separate analyses on retrotransposons in the bovine genome. Due to the presence of BovB retrotransposons, the bovine retrotransposon landscape is clearly distinct from other placental mammals. For the first analysis, I identified bovine-specific retrotransposon associated gene coexpression networks. Following the genomic distribution of bovine retrotransposons, my results show that gene expression strongly associates with genome architecture. For the second analysis, I characterised retrotransposons surrounding tandem duplicate copies of the bovine NK-lysin gene. My results were consistent with retrotransposon accumulation causing genomic rearrangements via non-allelic homologous recombination. Altogether, my thesis reveals hidden interactions between retrotransposon accumulation, and mammalian genome structure and function. By re-purposing publicly available datasets I have characterised various aspects of the complex co-evolutionary relationships between retrotransposons and the genomes in which they reside in.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

Development and application of mammalian molecular cytogenetic tools for genome reconstruction, evolution and reproductive screening

Chromosomal analysis enables a genome-wide overview of an organism, it can provide information when used to study cellular function, the taxonomic relationship between divergent species and disease phenotypes. Consequently, chromosomal analysis is used to identify chromosomal rearrangements in an individual, which can be associated with disease and/or reproductive complications, or within a population, which is associated with speciation and reproductive isolation. The techniques used to examine the chromosomes of an organism have improved considerably over the past four decades. Observations were traditionally achieved through the production of Giemsa stained chromosomes which permitted banding analysis, therefore enabling the detection of differences in chromosome morphology and number, to more specific, molecular cytogenetic approaches (fluorescence in situ hybridisation - FISH) which can be used to identify sub-microscopic differences. Today, genome sequencing facilitates genome-wide analysis at a higher resolution than previously possible; sequence information can be used in a multitude of ways, including identification of specific mutations which result in disease, investigating homologous segments between divergent species and for ascertaining potential drug targets. However, without a physical genetic map it is now apparent that by themselves genome sequence assemblies fail to provide sufficient information regarding certain biological questions, in particular genome organisation throughout times of mammalian evolution. However, it is now apparent that map-based chromosome-level assemblies are required for deeper analysis of the genome. With this in mind, the purpose of this work was to extend upon, and develop efficient cytogenetic tools to screen for chromosomal rearrangements in mammalian species, in the context evolutionarily events and to examine chromosomal rearrangements that manifest as fertility problems in a range of agricultural and zoological animals. Using traditional karyotyping techniques, Ducos et al (2007) demonstrated that the translocation incidence rate was 0.47% in unproven boars. In this work, a large number of boars (>1000) were analysed using a FISH-based screening device, whereby 13 unique chromosomal translocations were detected, resulting in an incidence rate of 1%. Therefore, the results in this work demonstrate that the incidence rate is under reported in the current literature. Before this work, karyotype analysis was the only technique used to identify chromosomal rearrangements in cattle. As a consequence of the success observed in pigs, a FISH-based device was developed to screen for chromosomal translocations in cattle. Using this technology, heterozygous and homozygous 1;29 translocations were identified, and an unreported 12;23 reciprocal translocation

Evolution of Gene Regulatory Networks Controlling Body Plan Development

Evolutionary change in animal morphology results from alteration of the functional organization of the gene regulatory networks (GRNs) that control development of the body plan. A major mechanism of evolutionary change in GRN structure is alteration of cis-regulatory modules that determine regulatory gene expression. Here we consider the causes and consequences of GRN evolution. Although some GRN subcircuits are of great antiquity, other aspects are highly flexible and thus in any given genome more recent. This mosaic view of the evolution of GRN structure explains major aspects of evolutionary process, such as hierarchical phylogeny and discontinuities of paleontological change

Mouse Obox and Crxos modulate preimplantation transcriptional profiles revealing similarity between paralogous mouse and human homeobox genes

Background: ETCHbox genes are eutherian-specific homeobox genes expressed during preimplantation develop-ment at a time when the first cell lineage decisions are being made. The mouse has an unusual repertoire of ETCH-box genes with several gene families lost in evolution and the remaining two, Crxos and Obox, greatly divergent in sequence and number. Each has undergone duplication to give a double homeodomain Crxos locus and a large cluster of over 60 Obox loci. The gene content differences between species raise important questions about how evolution can tolerate loss of genes implicated in key developmental events. Results: We find that Crxos internal duplication occurred in the mouse lineage, while Obox duplication was stepwise, generating subgroups with distinct sequence and expression. Ectopic expression of three Obox genes and a Crxos transcript in primary mouse embryonic cells followed by transcriptome sequencing allowed investigation into their functional roles. We find distinct transcriptomic influences for different Obox subgroups and Crxos, including modula-tion of genes related to zygotic genome activation and preparation for blastocyst formation. Comparison with similar experiments performed using human homeobox genes reveals striking overlap between genes downstream of mouse Crxos and genes downstream of human ARGFX. Conclusions: Mouse Crxos and human ARGFX homeobox genes are paralogous rather than orthologous, yet they have evolved to regulate a common set of genes. This suggests there was compensation of function alongside gene loss through co-option of a different locus. Functional compensation by non-orthologous genes with dissimilar sequences is unusual but may indicate underlying distributed robustness. Compensation may be driven by the strong evolutionary pressure for successful early embryo development

CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2

<p>Abstract</p> <p>Background</p> <p>Elucidating the pattern of evolutionary changes in drug-metabolizing genes is an important subject not only for evolutionary but for biomedical research. We investigated the pattern of divergence and polymorphisms of macaque <it>CYP1A1 </it>and <it>CYP1A2 </it>genes, which are major drug-metabolizing genes in humans. In humans, <it>CYP1A2 </it>is specifically expressed in livers while <it>CYP1A1 </it>has a wider gene expression pattern in extrahepatic tissues. In contrast, macaque <it>CYP1A2 </it>is expressed at a much lower level than <it>CYP1A1 </it>in livers. Interestingly, a previous study has shown that <it>Macaca fascicularis CYP1A2 </it>harbored unusually high genetic diversity within species. Genomic regions showing high genetic diversity within species is occasionally interpreted as a result of balancing selection, where natural selection maintains highly diverged alleles with different functions. Nevertheless many other forces could create such signatures.</p> <p>Results</p> <p>We found that the <it>CYP1A1/2 </it>gene copy number and orientation has been highly conserved among mammalian genomes. The signature of gene conversion between <it>CYP1A1 </it>and <it>CYP1A2 </it>was detected, but the last gene conversion event in the simian primate lineage occurred before the <it>Catarrhini-Platyrrhini </it>divergence. The high genetic diversity of macaque <it>CYP1A2 </it>therefore cannot be explained by gene conversion between <it>CYP1A1 </it>and <it>CYP1A2</it>. By surveying <it>CYP1A2 </it>polymorphisms in total 91 <it>M. fascicularis </it>and <it>M. mulatta</it>, we found several null alleles segregating in these species, indicating functional constraint on <it>CYP1A2 </it>in macaques may have weakened after the divergence between humans and macaques. We propose that the high genetic diversity in macaque <it>CYP1A2 </it>is partly due to the degeneration of CpG sites, which had been maintained at a high level by purifying selection, and the rapid degeneration process was initiated by the loss of functional constraint on macaque <it>CYP1A2</it>.</p> <p>Conclusions</p> <p>Our findings show that the highly polymorphic <it>CYP1A2 </it>gene in macaques has not been created by balancing selection but by the burst of CpG site degeneration after loss of functional constraint. Because the functional importance of <it>CYP1A1/2 </it>genes is different between humans and macaques, we have to be cautious in extrapolating a drug-testing data using substrates metabolized by <it>CYP1A </it>genes from macaques to humans, despite of their somewhat overlapping substrate specificity.</p