AmpliconReconstructor integrates NGS and optical mapping to resolve the complex structures of focal amplifications.

Oncogene amplification, a major driver of cancer pathogenicity, is often mediated through focal amplification of genomic segments. Recent results implicate extrachromosomal DNA (ecDNA) as the primary driver of focal copy number amplification (fCNA) - enabling gene amplification, rapid tumor evolution, and the rewiring of regulatory circuitry. Resolving an fCNA's structure is a first step in deciphering the mechanisms of its genesis and the fCNA's subsequent biological consequences. We introduce a computational method, AmpliconReconstructor (AR), for integrating optical mapping (OM) of long DNA fragments (>150 kb) with next-generation sequencing (NGS) to resolve fCNAs at single-nucleotide resolution. AR uses an NGS-derived breakpoint graph alongside OM scaffolds to produce high-fidelity reconstructions. After validating its performance through multiple simulation strategies, AR reconstructed fCNAs in seven cancer cell lines to reveal the complex architecture of ecDNA, a breakage-fusion-bridge and other complex rearrangements. By reconstructing the rearrangement signatures associated with an fCNA's generative mechanism, AR enables a more thorough understanding of the origins of fCNAs.

Loss of Heterozygosity Drives Adaptation in Hybrid Yeast.

Hybridization is often considered maladaptive, but sometimes hybrids can invade new ecological niches and adapt to novel or stressful environments better than their parents. The genomic changes that occur following hybridization that facilitate genome resolution and/or adaptation are not well understood. Here, we examine hybrid genome evolution using experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae × Saccharomyces uvarum and their parentals. We evolved these strains in nutrient-limited conditions for hundreds of generations and sequenced the resulting cultures identifying numerous point mutations, copy number changes, and loss of heterozygosity (LOH) events, including species-biased amplification of nutrient transporters. We focused on a particularly interesting example, in which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecific hybrids. Using allele replacement methods, we tested the fitness of different alleles in hybrid and S. cerevisiae strain backgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S. cerevisiae and the hybrids. This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosity and demonstrates that even infrequent outcrossing may have lasting impacts on adaptation.

Differential paralog divergence modulates genome evolution across yeast species.

Evolutionary outcomes depend not only on the selective forces acting upon a species, but also on the genetic background. However, large timescales and uncertain historical selection pressures can make it difficult to discern such important background differences between species. Experimental evolution is one tool to compare evolutionary potential of known genotypes in a controlled environment. Here we utilized a highly reproducible evolutionary adaptation in Saccharomyces cerevisiae to investigate whether experimental evolution of other yeast species would select for similar adaptive mutations. We evolved populations of S. cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. uvarum and S. cerevisiae for ~200-500 generations in sulfate-limited continuous culture. Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter gene, SUL1. However, while amplification of the SUL1 locus was detected in S. paradoxus and S. mikatae populations, S. uvarum cultures instead selected for amplification of the paralog, SUL2. We measured the relative fitness of strains bearing deletions and amplifications of both SUL genes from different species, confirming that, converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric promoter-ORF constructs, we were able to delineate the cause of this differential fitness effect primarily to the promoter of S. uvarum SUL1. Our data show evidence of differential sub-functionalization among the sulfate transporters across Saccharomyces species through recent changes in noncoding sequence. Furthermore, these results show a clear example of how such background differences due to paralog divergence can drive changes in genome evolution.

DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis.

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing.

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function.

Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed.

The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?

The nucleolus is a prominent nuclear structure that is the site of ribosomal RNA (rRNA) transcription, and hence ribosome biogenesis. Cellular demand for ribosomes, and hence rRNA, is tightly linked to cell growth and the rRNA makes up the majority of all the RNA within a cell. To fulfill the cellular demand for rRNA, the ribosomal RNA (rDNA) genes are amplified to high copy number and transcribed at very high rates. As such, understanding the rDNA has profound consequences for our comprehension of genome and transcriptional organization in cells. In this review, we address the question of whether the nucleolus is a raft adrift the sea of nuclear DNA, or actively contributes to genome organization. We present evidence supporting the idea that the nucleolus, and the rDNA contained therein, play more roles in the biology of the cell than simply ribosome biogenesis. We propose that the nucleolus and the rDNA are central factors in the spatial organization of the genome, and that rapid alterations in nucleolar structure in response to changing conditions manifest themselves in altered genomic structures that have functional consequences. Finally, we discuss some predictions that result from the nucleolus having a central role in nuclear organization.

tRNA gene identity affects nuclear positioning.

The three-dimensional organization of genomes is dynamic and plays a critical role in the regulation of cellular development and phenotypes. Here we use proximity-based ligation methods (i.e. chromosome conformation capture [3C] and circularized chromosome confrmation capture [4C]) to explore the spatial organization of tRNA genes and their locus-specific interactions with the ribosomal DNA. Directed replacement of one lysine and two leucine tRNA loci shows that tRNA spatial organization depends on both tRNA coding sequence identity and the surrounding chromosomal loci. These observations support a model whereby the three-dimensional, spatial organization of tRNA loci within the nucleus utilizes tRNA gene-specific signals to affect local interactions, though broader organization of chromosomal regions are determined by factors outside the tRNA genes themselves.

Cellular dynamics of tRNAs and their genes.

This discussion focuses on the cellular dynamics of tRNA transcription, processing, and turnover. Early tRNA biosynthesis steps are shared among most tRNAs, while later ones are often individualized for specific tRNAs. In yeast, tRNA transcription and early processing occur coordinately in the nucleolus, requiring topological arrangement of approximately 300 tRNA genes and early processing enzymes to this site; later processing events occur in the nucleoplasm or cytoplasm. tRNA nuclear export requires multiple exporters which function in parallel and the export process is coupled with other cellular events. Nuclear-cytoplasmic tRNA subcellular movement is not unidirectional as a retrograde pathway delivers mature cytoplasmic tRNAs to the nucleus. Despite the long half-lives, there are multiple pathways to turnover damaged tRNAs or normal tRNAs upon cellular stress.

Spatial organization of genes as a component of regulated expression.

The DNA of living cells is highly compacted. Inherent in this spatial constraint is the need for cells to organize individual genetic loci so as to facilitate orderly retrieval of information. Complex genetic regulatory mechanisms are crucial to all organisms, and it is becoming increasingly evident that spatial organization of genes is one very important mode of regulation for many groups of genes. In eukaryotic nuclei, it appears not only that DNA is organized in three-dimensional space but also that this organization is dynamic and interactive with the transcriptional state of the genes. Spatial organization occurs throughout evolution and with genes transcribed by all classes of RNA polymerases in all eukaryotic nuclei, from yeast to human. There is an increasing body of work examining the ways in which this organization and consequent regulation are accomplished. In this review, we discuss the diverse strategies that cells use to preferentially localize various classes of genes.

The repertoire and dynamics of evolutionary adaptations to controlled nutrient-limited environments in yeast.

The experimental evolution of laboratory populations of microbes provides an opportunity to observe the evolutionary dynamics of adaptation in real time. Until very recently, however, such studies have been limited by our inability to systematically find mutations in evolved organisms. We overcome this limitation by using a variety of DNA microarray-based techniques to characterize genetic changes -- including point mutations, structural changes, and insertion variation -- that resulted from the experimental adaptation of 24 haploid and diploid cultures of Saccharomyces cerevisiae to growth in either glucose, sulfate, or phosphate-limited chemostats for approximately 200 generations. We identified frequent genomic amplifications and rearrangements as well as novel retrotransposition events associated with adaptation. Global nucleotide variation detection in ten clonal isolates identified 32 point mutations. On the basis of mutation frequencies, we infer that these mutations and the subsequent dynamics of adaptation are determined by the batch phase of growth prior to initiation of the continuous phase in the chemostat. We relate these genotypic changes to phenotypic outcomes, namely global patterns of gene expression, and to increases in fitness by 5-50%. We found that the spectrum of available mutations in glucose- or phosphate-limited environments combined with the batch phase population dynamics early in our experiments allowed several distinct genotypic and phenotypic evolutionary pathways in response to these nutrient limitations. By contrast, sulfate-limited populations were much more constrained in both genotypic and phenotypic outcomes. Thus, the reproducibility of evolution varies with specific selective pressures, reflecting the constraints inherent in the system-level organization of metabolic processes in the cell. We were able to relate some of the observed adaptive mutations (e.g., transporter gene amplifications) to known features of the relevant metabolic pathways, but many of the mutations pointed to genes not previously associated with the relevant physiology. Thus, in addition to answering basic mechanistic questions about evolutionary mechanisms, our work suggests that experimental evolution can also shed light on the function and regulation of individual metabolic pathways.

Mapping novel traits by array-assisted bulk segregant analysis in Saccharomyces cerevisiae.

We demonstrate a new method, microarray-assisted bulk segregant analysis, for mapping traits in yeast by genotyping pooled segregants. We apply a probabilistic model to the progeny of a single cross and as little as two microarray hybridizations to reliably map an auxotrophic marker, a Mendelian trait, and a major-effect quantitative trait locus.