Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

Abiotic Stress Phenotypes Are Associated with Conserved Genes Derived from Transposable Elements.

Plant phenomics offers unique opportunities to accelerate our understanding of gene function and plant response to different environments, and may be particularly useful for studying previously uncharacterized genes. One important type of poorly characterized genes is those derived from transposable elements (TEs), which have departed from a mobility-driven lifestyle to attain new adaptive roles for the host (exapted TEs). We used phenomics approaches, coupled with reverse genetics, to analyze T-DNA insertion mutants of both previously reported and novel protein-coding exapted TEs in the model plant Arabidopsis thaliana. We show that mutations in most of these exapted TEs result in phenotypes, particularly when challenged by abiotic stress. We built statistical multi-dimensional phenotypic profiles and compared them to wild-type and known stress responsive mutant lines for each particular stress condition. We found that these exapted TEs may play roles in responses to phosphate limitation, tolerance to high salt concentration, freezing temperatures, and arsenic toxicity. These results not only experimentally validate a large set of putative functional exapted TEs recently discovered through computational analysis, but also uncover additional novel phenotypes for previously well-characterized exapted TEs in A. thaliana.

Phylogenetic and Genomic Analyses Resolve the Origin of Important Plant Genes Derived from Transposable Elements.

Once perceived as merely selfish, transposable elements (TEs) are now recognized as potent agents of adaptation. One way TEs contribute to evolution is through TE exaptation, a process whereby TEs, which persist by replicating in the genome, transform into novel host genes, which persist by conferring phenotypic benefits. Known exapted TEs (ETEs) contribute diverse and vital functions, and may facilitate punctuated equilibrium, yet little is known about this process. To better understand TE exaptation, we designed an approach to resolve the phylogenetic context and timing of exaptation events and subsequent patterns of ETE diversification. Starting with known ETEs, we search in diverse genomes for basal ETEs and closely related TEs, carefully curate the numerous candidate sequences, and infer detailed phylogenies. To distinguish TEs from ETEs, we also weigh several key genomic characteristics including repetitiveness, terminal repeats, pseudogenic features, and conserved domains. Applying this approach to the well-characterized plant ETEs MUG and FHY3, we show that each group is paraphyletic and we argue that this pattern demonstrates that each originated in not one but multiple exaptation events. These exaptations and subsequent ETE diversification occurred throughout angiosperm evolution including the crown group expansion, the angiosperm radiation, and the primitive evolution of angiosperms. In addition, we detect evidence of several putative novel ETE families. Our findings support the hypothesis that TE exaptation generates novel genes more frequently than is currently thought, often coinciding with key periods of evolution.

A call for benchmarking transposable element annotation methods.

DNA derived from transposable elements (TEs) constitutes large parts of the genomes of complex eukaryotes, with major impacts not only on genomic research but also on how organisms evolve and function. Although a variety of methods and tools have been developed to detect and annotate TEs, there are as yet no standard benchmarks-that is, no standard way to measure or compare their accuracy. This lack of accuracy assessment calls into question conclusions from a wide range of research that depends explicitly or implicitly on TE annotation. In the absence of standard benchmarks, toolmakers are impeded in improving their tools, annotators cannot properly assess which tools might best suit their needs, and downstream researchers cannot judge how accuracy limitations might impact their studies. We therefore propose that the TE research community create and adopt standard TE annotation benchmarks, and we call for other researchers to join the authors in making this long-overdue effort a success.

Discovery of novel genes derived from transposable elements using integrative genomic analysis.

Complex eukaryotes contain millions of transposable elements (TEs), comprising large fractions of their nuclear genomes. TEs consist of structural, regulatory, and coding sequences that are ordinarily associated with transposition, but that occasionally confer on the organism a selective advantage and may thereby become exapted. Exapted transposable element genes (ETEs) are known to play critical roles in diverse systems, from vertebrate adaptive immunity to plant development. Yet despite their evident importance, most ETEs have been identified fortuitously and few systematic searches have been conducted, suggesting that additional ETEs may await discovery. To explore this possibility, we develop a comprehensive systematic approach to searching for ETEs. We use TE-specific conserved domains to identify with high precision genes derived from TEs and screen them for signatures of exaptation based on their similarities to reference sets of known ETEs, conventional (non-TE) genes, and TE genes across diverse genetic attributes including repetitiveness, conservation of genomic location and sequence, and levels of expression and repressive small RNAs. Applying this approach in the model plant Arabidopsis thaliana, we discover a surprisingly large number of novel high confidence ETEs. Intriguingly, unlike known plant ETEs, several of the novel ETE families form tandemly arrayed gene clusters, whereas others are relatively young. Our results not only identify novel TE-derived genes that may have practical applications but also challenge the notion that TE exaptation is merely a relic of ancient life, instead suggesting that it may continue to fundamentally drive evolution.

An atlas of over 90,000 conserved noncoding sequences provides insight into crucifer regulatory regions.

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.

A gene family derived from transposable elements during early angiosperm evolution has reproductive fitness benefits in Arabidopsis thaliana.

The benefits of ever-growing numbers of sequenced eukaryotic genomes will not be fully realized until we learn to decipher vast stretches of noncoding DNA, largely composed of transposable elements. Transposable elements persist through self-replication, but some genes once encoded by transposable elements have, through a process called molecular domestication, evolved new functions that increase fitness. Although they have conferred numerous adaptations, the number of such domesticated transposable element genes remains unknown, so their evolutionary and functional impact cannot be fully assessed. Systematic searches that exploit genomic signatures of natural selection have been employed to identify potential domesticated genes, but their predictions have yet to be experimentally verified. To this end, we investigated a family of domesticated genes called MUSTANG (MUG), identified in a previous bioinformatic search of plant genomes. We show that MUG genes are functional. Mutants of Arabidopsis thaliana MUG genes yield phenotypes with severely reduced plant fitness through decreased plant size, delayed flowering, abnormal development of floral organs, and markedly reduced fertility. MUG genes are present in all flowering plants, but not in any non-flowering plant lineages, such as gymnosperms, suggesting that the molecular domestication of MUG may have been an integral part of early angiosperm evolution. This study shows that systematic searches can be successful at identifying functional genetic elements in noncoding regions and demonstrates how to combine systematic searches with reverse genetics in a fruitful way to decipher eukaryotic genomes.

Genomewide SNP variation reveals relationships among landraces and modern varieties of rice.

Rice, the primary source of dietary calories for half of humanity, is the first crop plant for which a high-quality reference genome sequence from a single variety was produced. We used resequencing microarrays to interrogate 100 Mb of the unique fraction of the reference genome for 20 diverse varieties and landraces that capture the impressive genotypic and phenotypic diversity of domesticated rice. Here, we report the distribution of 160,000 nonredundant SNPs. Introgression patterns of shared SNPs revealed the breeding history and relationships among the 20 varieties; some introgressed regions are associated with agronomic traits that mark major milestones in rice improvement. These comprehensive SNP data provide a foundation for deep exploration of rice diversity and gene-trait relationships and their use for future rice improvement.

Transposon-mediated expansion and diversification of a family of ULP-like genes.

Transposons comprise a major component of eukaryotic genomes, yet it remains controversial whether they are merely genetic parasites or instead significant contributors to organismal function and evolution. In plants, thousands of DNA transposons were recently shown to contain duplicated cellular gene fragments, a process termed transduplication. Although transduplication is a potentially rich source of novel coding sequences, virtually all appear to be pseudogenes in rice. Here we report the results of a genome-wide survey of transduplication in Mutator-like elements (MULEs) in Arabidopsis thaliana, which shows that the phenomenon is generally similar to rice transduplication, with one important exception: KAONASHI (KI). A family of more than 97 potentially functional genes and apparent pseudogenes, evidently derived at least 15 MYA from a cellular small ubiquitin-like modifier-specific protease gene, KI is predominantly located in potentially autonomous non-terminal inverted repeat MULEs and has evolved under purifying selection to maintain a conserved peptidase domain. Similar to the associated transposase gene but unlike cellular genes, KI is targeted by small RNAs and silenced in most tissues but has elevated expression in pollen. In an Arabidopsis double mutant deficient in histone and DNA methylation with elevated KI expression compared to wild type, at least one KI-MULE is mobile. The existence of KI demonstrates that transduplicated genes can retain protein-coding capacity and evolve novel functions. However, in this case, our evidence suggests that the function of KI may be selfish rather than cellular.

The evolutionary fate of MULE-mediated duplications of host gene fragments in rice.

DNA transposons are known to frequently capture duplicated fragments of host genes. The evolutionary impact of this phenomenon depends on how frequently the fragments retain protein-coding function as opposed to becoming pseudogenes. Gene fragment duplication by Mutator-like elements (MULEs) has previously been documented in maize, Arabidopsis, and rice. Here we present a rigorous genome-wide analysis of MULEs in the model plant Oryza sativa (domesticated rice). We identify 8274 MULEs with intact termini and target-site duplications (TSDs) and show that 1337 of them contain duplicated host gene fragments. Through a detailed examination of the 5% of duplicated gene fragments that are transcribed, we demonstrate that virtually all cases contain pseudogenic features such as fragmented conserved protein domains, frameshifts, and premature stop codons. In addition, we show that the distribution of the ratio of nonsynonymous to synonymous amino acid substitution rates for the duplications agrees with the expected distribution for pseudogenes. We conclude that MULE-mediated host gene duplication results in the formation of pseudogenes, not novel functional protein-coding genes; however, the transcribed duplications possess characteristics consistent with a potential role in the regulation of host gene expression.

MUSTANG is a novel family of domesticated transposase genes found in diverse angiosperms.

While transposons have traditionally been viewed as genomic parasites or "junk DNA," the discovery of transposon-derived host genes has fueled an ongoing debate over the evolutionary role of transposons. In particular, while mobility-related open reading frames have been known to acquire host functions, the contribution of these types of events to the evolution of genes is not well understood. Here we report that genome-wide searches for Mutator transposase-derived host genes in Arabidopsis thaliana (Columbia-0) and Oryza sativa ssp. japonica (cv. Nipponbare) (domesticated rice) identified 121 sequences, including the taxonomically conserved MUSTANG1. Syntenic MUSTANG1 orthologs in such varied plant species as rice, poplar, Arabidopsis, and Medicago truncatula appear to be under purifying selection. However, despite the evidence of this pathway of gene evolution, MUSTANG1 belongs to one of only two Mutator-like gene families with members in both monocotyledonous and dicotyledonous plants, suggesting that Mutator-like elements seldom evolve into taxonomically widespread host genes.