Tracking Cancer Evolution Reveals Constrained Routes to Metastases: TRACERx Renal.

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.

Genetic perturbation of the maize methylome.

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.

Post-conversion targeted capture of modified cytosines in mammalian and plant genomes.

We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.

Broad chromosomal domains of histone modification patterns in C. elegans.

Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development.

Euchromatic subdomains in rice centromeres are associated with genes and transcription.

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.

Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content.

Following the domestication of maize over the past approximately 10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Representative Sequencing: Unbiased Sampling of Solid Tumor Tissue.

Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mm biopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB but low clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Targeted Bisulfite Sequencing Using the SeqCap Epi Enrichment System.

Cytosine methylation has been shown to have a role in a host of biological processes. In mammalian biology these include stem cell differentiation, embryonic development, genomic imprinting, inflammation, and silencing of transposable elements. Given the central importance of these processes, it is not surprising to find aberrant cytosine methylation patterns associated with many disorders in humans, including cancer, cardiovascular disease, and neurological disease. While whole genome shotgun bisulfite sequencing (WGBS) has recently become feasible, generating high sequence coverage data for the entire genome is expensive, both in terms of money and analysis time, when generally only a small subset of the genome is of interest to most researchers. This report details a procedure for the targeted enrichment of bisulfite treated DNA via SeqCap Epi, allowing high resolution focus of next generation sequencing onto a subset of the genome for high resolution cytosine methylation analysis. Regions ranging in size from only a few kb up to over 200 Mb may be targeted, including the use of the SeqCap Epi CpGiant design which is designed to target 5.5 million CpGs in the human genome. Finally, multiple samples may be multiplexed and sequenced together to provide an inexpensive method of generating methylation data for a large number of samples in a high throughput fashion.

Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development.

Lens fiber cell differentiation involves extensive reconstruction of the cell's architecture, including the degradation and elimination of all membrane-bound organelles via a process that has been likened to apoptosis. Using caspase reporter assays under conditions in which nonspecific cleavage of the reporter peptides by the proteasome has been inhibited, we investigated whether any specific caspase activities are temporally correlated with this process of organelle loss. Extracts from neonatal mouse lenses contained strong VEID-7-amino-4-trifluoromethylcoumarin (AFC) and minor IETD-AFC and LEVD-AFC cleavage activities, but no DEVD-AFC cleavage activity. Further testing suggested that the VEID-AFC and IETD-AFC cleavage activities were likely due to the same enzyme. In lens extracts from rat embryos, VEID-AFC cleavage activity increased during the period when organelles are eliminated, between embryonic days 15.5 and 18.5, whereas procaspase-6 protein levels decreased, suggesting that this enzyme is responsible for VEID-AFC cleavage. By contrast, in extracts from alpha AE7 transgenic mouse lenses in which apoptosis was induced, strong DEVD-AFC cleavage activity and activated caspase-3 protein were detected. Thus, within the same tissue, different caspase activities can predominate depending on the context, normal differentiation versus apoptosis. These results highlight the difference between normal fiber cell differentiation and apoptosis and the capacity of the lens to differentially regulate these two processes.

Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest.

Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650-12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergent-resistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nm) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B' regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B'-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B' subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B' and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.