A. Gordon Robertson

Genome-wide relationship between histone H3 lysine 4 mono- and tri-methylation and transcription factor binding

We characterized the relationship of H3K4me1 and H3K4me3 at distal and proximal regulatory elements by comparing ChIP-seq profiles for these histone modifications and for two functionally different transcription factors: STAT1 in the immortalized HeLa S3 cell line, with and without interferon-gamma (IFNG) stimulation; and FOXA2 in mouse adult liver tissue. In unstimulated and stimulated HeLa cells, respectively, we determined approximately 270,000 and approximately 301,000 H3K4me1-enriched regions, and approximately 54,500 and approximately 76,100 H3K4me3-enriched regions. In mouse adult liver, we determined approximately 227,000 and approximately 34,800 H3K4me1 and H3K4me3 regions. Seventy-five percent of the approximately 70,300 STAT1 binding sites in stimulated HeLa cells and 87% of the approximately 11,000 FOXA2 sites in mouse liver were distal to known gene TSS; in both cell types, approximately 83% of these distal sites were associated with at least one of the two histone modifications, and H3K4me1 was associated with over 96% of marked distal sites. After filtering against predicted transcription start sites, 50% of approximately 26,800 marked distal IFNG-stimulated STAT1 binding sites, but 95% of approximately 5800 marked distal FOXA2 sites, were associated with H3K4me1 only. Results for HeLa cells generated additional insights into transcriptional regulation involving STAT1. STAT1 binding was associated with 25% of all H3K4me1 regions in stimulated HeLa cells, suggesting that a single transcription factor can interact with an unexpectedly large fraction of regulatory regions. Strikingly, for a large majority of the locations of stimulated STAT1 binding, the dominant H3K4me1/me3 combinations were established before activation, suggesting mechanisms independent of IFNG stimulation and high-affinity STAT1 binding.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

BioBloom tools: fast, accurate and memory-efficient host species sequence screening using bloom filters

Large datasets can be screened for sequences from a specific organism, quickly and with low memory requirements, by a data structure that supports time- and memory-efficient set membership queries. Bloom filters offer such queries but require that false positives be controlled. We present BioBloom Tools, a Bloom filter-based sequence-screening tool that is faster than BWA, Bowtie 2 (popular alignment algorithms) and FACS (a membership query algorithm). It delivers accuracies comparable with these tools, controls false positives and has low memory requirements. Availability and implementaion: www.bcgsc.ca/platform/bioinfo/software/biobloomtools Contact: cjustin@bcgsc.ca or ibirol@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Stiffness of pancreatic cancer cells is associated with increased invasive potential

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Youngs modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Youngs modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

DNA defects, epigenetics, and gene expression in cancer-adjacent breast: a study from The Cancer Genome Atlas

Recurrence rates after breast-conserving therapy may depend on genomic characteristics of cancer-adjacent, benign-appearing tissue. Studies have not evaluated recurrence in association with multiple genomic characteristics of cancer-adjacent breast tissue. To estimate the prevalence of DNA defects and RNA expression subtypes in cancer-adjacent, benign-appearing breast tissue at least 2 cm from the tumor margin, cancer-adjacent, pathologically well-characterized, benign-appearing breast tissue specimens from The Cancer Genome Atlas project were analyzed for DNA sequence, copy-number variation, DNA methylation, messenger RNA (mRNA) sequence, and mRNA/microRNA expression. Additional samples were also analyzed by at least one of these genomic data types and associations between genomic characteristics of normal tissue and overall survival were assessed. Approximately 40% of cancer-adjacent, benign-appearing tissues harbored genomic defects in DNA copy number, sequence, methylation, or in RNA sequence, although these defects did not significantly predict 10-year overall survival. Two mRNA/microRNA expression phenotypes were observed, including an active mRNA subtype that was identified in 40% of samples. Controlling for tumor characteristics and the presence of genomic defects, this active subtype was associated with significantly worse 10-year survival among estrogen receptor (ER)-positive cases. This multi-platform analysis of breast cancer-adjacent samples produced genomic findings consistent with current surgical margin guidelines, and provides evidence that extratumoral RNA expression patterns in cancer-adjacent tissue predict overall survival among patients with ER-positive disease.

THOR: targeted high-throughput ortholog reconstructor

Low-coverage genomes (LCGs) are becoming an increasingly important source of data for phylogenetic studies. However, assembly of these genomes is time consuming, difficult and lags behind sequence generation. THOR is a fast, stringent application for targeted reconstruction of sequence orthologs in unassembled LCGs. Using a 4x coverage set of mouse whole-genome sequence reads, THOR could partially or completely reconstruct 416/1000 human promoter ortholog regions in approximately 7.3 min/promoter. THORs reconstruction rate improves markedly with both higher-coverage, and less divergent target species.

Tumour-suppressor microRNAs regulate ovarian cancer cell physical properties and invasive behaviour

The activities of pathways that regulate malignant transformation can be influenced by microRNAs (miRs). Recently, we showed that increased expression of five tumour-suppressor miRs, miR-508-3p, miR-508-5p, miR-509-3p, miR-509-5p and miR-130b-3p, correlate with improved clinical outcomes in human ovarian cancer patients, and that miR-509-3p attenuates invasion of ovarian cancer cell lines. Here, we investigate the mechanism underlying this reduced invasive potential by assessing the impact of these five miRs on the physical properties of cells. Human ovarian cancer cells (HEYA8, OVCAR8) that are transfected with miR mimics representing these five miRs exhibit decreased invasion through collagen matrices, increased cell size and reduced deformability as measured by microfiltration and microfluidic assays. To understand the molecular basis of altered invasion and deformability induced by these miRs, we use predicted and validated mRNA targets that encode structural and signalling proteins that regulate cell mechanical properties. Combined with analysis of gene transcripts by real-time PCR and image analysis of F-actin in single cells, our results suggest that these tumour-suppressor miRs may alter cell physical properties by regulating the actin cytoskeleton. Our findings provide biophysical insights into how tumour-suppressor miRs can regulate the invasive behaviour of ovarian cancer cells, and identify potential therapeutic targets that may be implicated in ovarian cancer progression.

A-to-I miR-378a-3p editing can prevent melanoma progression via regulation of PARVA expression

Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) editing. The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined. Here we report that miR-378a–3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells. The function of the edited form is different from its wild-type counterpart. The edited form of miR-378a-3p preferentially binds to the 3′-UTR of the PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype. Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo. These results further emphasize the role of RNA editing in melanoma progression.In melanoma, reduced ADAR1 impairs A-to-I microRNA editing. Here, the authors show that miR-378a-3p undergoes this editing in non-metastatic cells and the edited form of miR-378a-3p binds to the PARVA oncogene, inhibiting its expression and preventing melanoma progression and metastasis.

The chromatin accessibility landscape of primary human cancers

Cancer chromatin accessibility landscape The Cancer Genome Atlas (TCGA) provides a high-quality resource of molecular data on a large variety of human cancers. Corces et al. used a recently modified assay to profile chromatin accessibility to determine the accessible chromatin landscape in 410 TCGA samples from 23 cancer types (see the Perspective by Taipale). When the data were integrated with other omics data available for the same tumor samples, inherited risk loci for cancer predisposition were revealed, transcription factors and enhancers driving molecular subtypes of cancer with patient survival differences were identified, and noncoding mutations associated with clinical prognosis were discovered. Science, this issue p. eaav1898; see also p. 401 Chromatin accessibility profiling identifies principles of epigenetic regulation in 23 primary human cancers. INTRODUCTION Cancer is one of the leading causes of death worldwide. Although the 2% of the human genome that encodes proteins has been extensively studied, much remains to be learned about the noncoding genome and gene regulation in cancer. Genes are turned on and off in the proper cell types and cell states by transcription factor (TF) proteins acting on DNA regulatory elements that are scattered over the vast noncoding genome and exert long-range influences. The Cancer Genome Atlas (TCGA) is a global consortium that aims to accelerate the understanding of the molecular basis of cancer. TCGA has systematically collected DNA mutation, methylation, RNA expression, and other comprehensive datasets from primary human cancer tissue. TCGA has served as an invaluable resource for the identification of genomic aberrations, altered transcriptional networks, and cancer subtypes. Nonetheless, the gene regulatory landscapes of these tumors have largely been inferred through indirect means. RATIONALE A hallmark of active DNA regulatory elements is chromatin accessibility. Eukaryotic genomes are compacted in chromatin, a complex of DNA and proteins, and only the active regulatory elements are accessible by the cell’s machinery such as TFs. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) quantifies DNA accessibility through the use of transposase enzymes that insert sequencing adapters at these accessible chromatin sites. ATAC-seq enables the genome-wide profiling of TF binding events that orchestrate gene expression programs and give a cell its identity. RESULTS We generated high-quality ATAC-seq data in 410 tumor samples from TCGA, identifying diverse regulatory landscapes across 23 cancer types. These chromatin accessibility profiles identify cancer- and tissue-specific DNA regulatory elements that enable classification of tumor subtypes with newly recognized prognostic importance. We identify distinct TF activities in cancer based on differences in the inferred patterns of TF-DNA interaction and gene expression. Genome-wide correlation of gene expression and chromatin accessibility predicts tens of thousands of putative interactions between distal regulatory elements and gene promoters, including key oncogenes and targets in cancer immunotherapy, such as MYC, SRC, BCL2, and PDL1. Moreover, these regulatory interactions inform known genetic risk loci linked to cancer predisposition, nominating biochemical mechanisms and target genes for many cancer-linked genetic variants. Lastly, integration with mutation profiling by whole-genome sequencing identifies cancer-relevant noncoding mutations that are associated with altered gene expression. A single-base mutation located 12 kilobases upstream of the FGD4 gene, a regulator of the actin cytoskeleton, generates a putative de novo binding site for an NKX TF and is associated with an increase in chromatin accessibility and a concomitant increase in FGD4 gene expression. CONCLUSION The accessible genome of primary human cancers provides a wealth of information on the susceptibility, mechanisms, prognosis, and potential therapeutic strategies of diverse cancer types. Prediction of interactions between DNA regulatory elements and gene promoters sets the stage for future integrative gene regulatory network analyses. The discovery of hundreds of noncoding somatic mutations that exhibit allele-specific regulatory effects suggests a pervasive mechanism for cancer cells to manipulate gene expression and increase cellular fitness. These data may serve as a foundational resource for the cancer research community. Cancer gene regulatory landscape. Chromatin accessibility profiling of 23 human cancer types (left) in 410 tumor samples from TCGA revealed 562,709 DNA regulatory elements. The activity of these DNA elements organized cancer subtypes, identified TF proteins and regulatory elements controlling cancer gene expression, and suggested molecular mechanisms for cancer-associated inherited variants and somatic mutations in the noncoding genome. See main article for abbreviations of cancer types. Ref., reference; Var., variant. We present the genome-wide chromatin accessibility profiles of 410 tumor samples spanning 23 cancer types from The Cancer Genome Atlas (TCGA). We identify 562,709 transposase-accessible DNA elements that substantially extend the compendium of known cis-regulatory elements. Integration of ATAC-seq (the assay for transposase-accessible chromatin using sequencing) with TCGA multi-omic data identifies a large number of putative distal enhancers that distinguish molecular subtypes of cancers, uncovers specific driving transcription factors via protein-DNA footprints, and nominates long-range gene-regulatory interactions in cancer. These data reveal genetic risk loci of cancer predisposition as active DNA regulatory elements in cancer, identify gene-regulatory interactions underlying cancer immune evasion, and pinpoint noncoding mutations that drive enhancer activation and may affect patient survival. These results suggest a systematic approach to understanding the noncoding genome in cancer to advance diagnosis and therapy.