Comprehensive detection of structural variation and transposable element differences between wild type laboratory lineages of C. elegans.

Genomic structural variations (SVs) and transposable elements (TEs) can be significant contributors to genome evolution, altered gene expression, and risk of genetic diseases. Recent advancements in long-read sequencing have greatly improved the quality of de novo genome assemblies and enhanced the detection of sequence variants at the scale of hundreds or thousands of bases. Comparisons between two diverged wild isolates of Caenorhabditis elegans, the Bristol and Hawaiian strains, have been widely utilized in the analysis of small genetic variations. Genetic drift, including SVs and rearrangements of repeated sequences such as TEs, can occur over time from long-term maintenance of wild type isolates within the laboratory. To comprehensively detect both large and small structural variations as well as TEs due to genetic drift, we generated de novo genome assemblies and annotations for each strain from our lab collection using both long- and short-read sequencing and compared our assemblies and annotations with that of other lab wild type strains. Within our lab assemblies, we annotate over 3.1Mb of sequence divergence between the Bristol and Hawaiian isolates: 337,584 SNPs, 94,503 small insertion-deletions (<50bp), and 4,334 structural variations (>50bp). Further, we define the location and movement of specific DNA TEs between N2 Bristol and CB4856 Hawaiian wild type isolates. Specifically, we find the N2 Bristol genome has 20.6% more TEs from the Tc1/mariner family than the CB4856 Hawaiian genome. Moreover, we identified Zator elements as the most abundant and mobile TE family in the genome. Using specific TE sequences with unique SNPs, we also identify 38 TEs that moved intrachromosomally and 9 TEs that moved interchromosomally between the N2 Bristol and CB4856 Hawaiian genomes. By comparing the de novo genome assembly of our lab collection Bristol isolate to the VC2010 Bristol assembly, we also reveal that lab lineages display over 2 Mb of total variation: 1,162 SNPs, 1,528 indels, and 897 SVs with 95% of the variation due to SVs. Overall, our work demonstrates the unique contribution of SVs and TEs to variation and genetic drift between wild type laboratory strains assumed to be isogenic despite growing evidence of genetic drift and phenotypic variation.

Ultrasensitive Circulating Tumor DNA Pilot Study Distinguishes Complete Response and Partial Response With Immunotherapy in Patients With Metastatic Renal Cell Carcinoma.

PURPOSE: Circulating tumor DNA (ctDNA) has been validated across multiple indications in the adjuvant and surveillance settings. We evaluated whether targeted digital sequencing (TARDIS) may distinguish a partial response (PR) from a complete response (CR) among patients with metastatic renal cell carcinoma (mRCC) receiving immune checkpoint inhibitor (ICI) therapy. MATERIALS AND METHODS: Eligible patients had mRCC that yielded a PR or CR to ICI therapy. Peripheral blood was obtained at a single time point for ctDNA analysis. TARDIS was used for quantification of average variant allele fractions (VAFs). Our primary objective was to determine the association between VAFs and depth of response (PR v CR). A secondary objective was to determine whether VAFs were associated with disease progression. RESULTS: Twelve patients were analyzed, nine of whom achieved a PR (75%). Patients received either nivolumab monotherapy (50%) or nivolumab plus ipilimumab (50%). ctDNA analysis incorporated an average of 30 patient-specific mutations (range, 19-35); average coverage depth was 103,342 reads per target. TARDIS quantified a significant difference in VAFs between PR and CR (median, 0.181% [IQR, 0.077%-0.420%] v 0.007% [IQR, 0.0%-0.028%], respectively [P = .014]). Of the 12 patients in the series, six patients demonstrated radiographic progression subsequent to ctDNA assessment. Patients who progressed on subsequent scans had significantly higher ctDNA than those who maintained their response (median, 0.362% [IQR, 0.181%-2.71%] v 0.033% [IQR, 0.007%-0.077%], respectively [P = .026]). CONCLUSION: In this pilot study, TARDIS accurately differentiated PR from CR among patients with mRCC receiving immunotherapy, and also prospectively identified patients at risk for subsequent progression. Given these findings, we envision subsequent studies that validate these results and investigate the utility of this assay to discern appropriate candidates for discontinuation of immunotherapy.

Basis of specificity for a conserved and promiscuous chromatin remodeling protein.

Eukaryotic genomes are organized dynamically through the repositioning of nucleosomes. Isw2 is an enzyme that has been previously defined as a genome-wide, nonspecific nucleosome spacing factor. Here, we show that Isw2 instead acts as an obligately targeted nucleosome remodeler in vivo through physical interactions with sequence-specific factors. We demonstrate that Isw2-recruiting factors use small and previously uncharacterized epitopes, which direct Isw2 activity through highly conserved acidic residues in the Isw2 accessory protein Itc1. This interaction orients Isw2 on target nucleosomes, allowing for precise nucleosome positioning at targeted loci. Finally, we show that these critical acidic residues have been lost in the Drosophila lineage, potentially explaining the inconsistently characterized function of Isw2-like proteins. Altogether, these data suggest an 'interacting barrier model,' where Isw2 interacts with a sequence-specific factor to accurately and reproducibly position a single, targeted nucleosome to define the precise border of phased chromatin arrays.

DNA encodes the genetic instructions for life in a long, flexible molecular chain that is packaged up neatly to fit inside cells. Short sections of DNA are wound around proteins to form bundles called nucleosomes, and then spun into chromatin fibres, a more compact form of DNA. While nucleosomes are a fundamental part of this space-saving packaging process, they also play a key regulatory role in gene expression, which is where genes are decoded into working proteins. Placing nucleosomes at regular intervals along DNA invariably controls which parts of the DNA ­ and which genes ­ the cell's machinery can access and 'read' to make proteins. But the nucleosomes' positions are not fixed, and gene expression is a dynamic process. The cell often uncoils and repackages its DNA while molecular motors called chromatin remodelling proteins move nucleosomes up and down the DNA, exposing some genes and obstructing others. One group of chromatin remodelling proteins are called Imitation Switch (ISWI) complexes. It has long been thought that these complexes position nucleosomes with little regard to the underlying DNA sequence or the genes encoded, that is to say in a non-specific way. However, this theory has not been thoroughly tested. It is possible that ISWI complexes actually place nucleosomes on certain parts of DNA at particular times in an organism's development, or in response to other environmental factors. Except how such precision is achieved remains unknown. To test this alternative theory of nucleosome positioning, Donovan et al. studied ISWI proteins and nucleosomes in common baker's yeast. This involved systematically removing sections of ISWI proteins to see whether the complexes could still position nucleosomes, and which parts of the proteins where essential for the job. By doing so, Donovan et al. identified multiple 'targeting' proteins that bind to ISWI proteins and deliver the complexes to specific target sequences of DNA. From there, the complex remodels the nucleosome, positioning it at a specific distance from its landing site on DNA, as further experiments showed. This research provides a new model for explaining how nucleosomes are positioned to package DNA and control gene expression. Donovan et al. have identified a new mechanism of interaction between nucleosomes and chromatin remodelling proteins of the ISWI variety. It is possible that more interactions of this kind will be discovered with further research.

Elevated Temperatures Cause Transposon-Associated DNA Damage in C. elegans Spermatocytes.

Sexually reproducing organisms use meiosis to generate haploid gametes and faithfully transmit their genome to the next generation. In comparison to oogenesis in many organisms, spermatogenesis is particularly sensitive to small temperature fluctuations, and spermatocytes must develop within a very narrow isotherm [1-4]. Although failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility in several organisms, the mechanisms of temperature-induced male infertility have not been fully elucidated [5]. Here, we show that upon exposure to a brief 2°C temperature increase, Caenorhabditis elegans spermatocytes exhibit up to a 25-fold increase in double-strand DNA breaks (DSBs) throughout meiotic prophase I and a concurrent reduction in male fertility. We demonstrate that these heat-induced DSBs in spermatocytes are independent of the endonuclease SPO-11. Further, we find that the production of these heat-induced DSBs in spermatocytes correlate with heat-induced mobilization of Tc1/mariner transposable elements, which are known to cause DSBs and alter genome integrity [6, 7]. Moreover, we define the specific sequences and regions of the male genome that preferentially experience these heat-induced de novo Tc1 insertions. In contrast, oocytes do not exhibit changes in DSB formation or Tc1 transposon mobility upon temperature increases. Taken together, our data suggest spermatocytes are less tolerant of higher temperatures because of an inability to effectively repress the movement of specific mobile DNA elements that cause excessive DNA damage and genome alterations, which can impair fertility.

Microbial Communities in Bioswale Soils and Their Relationships to Soil Properties, Plant Species, and Plant Physiology.

Bioswales and other forms of green infrastructure can be effective means to reduce environmental stresses in urban ecosystems; however, few studies have evaluated the ecology of these systems, or the role that plant selection and microbial assembly play in their function. For the current study, we examined the relationship between plant transpiration rates for five commonly planted herbaceous species in three bioswales in New York City, as well as bioswale soil microbial composition and soil chemistry. Soils were sampled near individual plants, with distinction made between upper (bioswale inlet) and lower slopes (bioswale outlet). We found high variation in transpiration rates across species, and that Nepeta × faassenii was the highest conductor (13.65 mmol H2O m-2s-1), while Panicum virgatum was the lowest conductor (2.67 mmol H2O m-2s-1) (p < 0.001). There was significant variation in percent N of leaves and soil, which did not relate to the higher water conductance in bioswales. Significantly higher C, N, and water content on the high end of bioswale slopes suggest storm water run-off is mostly absorbed on the inlet side. Bacterial and fungal communities were significantly clustered by bioswale and by plant species within each bioswale implying there are micro-environmental controls on the soil microbial composition, and that plant composition matters for microbial assemblages within bioswales. Plants with higher transpiration rates were associated with greater fungal and bacterial diversity at the level of the bioswale and at scale of the individual plant, suggesting a possible link between plant physiological traits and soil microbial communities. These data suggest that the specific plant palette selected for planting bioswales can have deterministic effects on the surrounding microbial communities which may further influence functions such as transpiration and nutrient cycling. These results may have implications for bioswale management to improve urban water quality and reduce stress on sewage systems after storm events by revising plant species palette selection based on the functional consequences of plant-microbial associations in engineered green infrastructure.

Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning.

Regulation of chromatin structure is essential for controlling access of DNA to factors that require association with specific DNA sequences. Here we describe the development and validation of engineered chromatin remodeling proteins (E-ChRPs) for inducing programmable changes in nucleosome positioning by design. We demonstrate that E-ChRPs function both in vitro and in vivo to specifically reposition target nucleosomes and entire nucleosomal arrays. We show that induced, systematic positioning of nucleosomes over yeast Ume6 binding sites leads to Ume6 exclusion, hyperacetylation, and transcriptional induction at target genes. We also show that programmed global loss of nucleosome-free regions at Reb1 targets is generally inhibitory with mildly repressive transcriptional effects. E-ChRPs are compatible with multiple targeting modalities, including the SpyCatcher and dCas9 moieties, resulting in high versatility and enabling diverse future applications. Thus, engineered chromatin remodeling proteins represent a simple and robust means to probe and disrupt DNA-dependent processes in different chromatin contexts.