Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Reduced sister chromatid cohesion acts as a tumor penetrance modifier.

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.

Dissecting the regulatory activity and sequence content of loci with exceptional numbers of transcription factor associations.

DNA-associated proteins (DAPs) classically regulate gene expression by binding to regulatory loci such as enhancers or promoters. As expanding catalogs of genome-wide DAP binding maps reveal thousands of loci that, unlike the majority of conventional enhancers and promoters, associate with dozens of different DAPs with apparently little regard for motif preference, an understanding of DAP association and coordination at such regulatory loci is essential to deciphering how these regions contribute to normal development and disease. In this study, we aggregated publicly available ChIP-seq data from 469 human DAPs assayed in three cell lines and integrated these data with an orthogonal data set of 352 nonredundant, in vitro-derived motifs mapped to the genome within DNase I hypersensitivity footprints to characterize regions with high numbers of DAP associations. We establish a generalizable definition for high occupancy target (HOT) loci and identify putative driver DAP motifs in HepG2 cells, including HNF4A, SP1, SP5, and ETV4, that are highly prevalent and show sequence conservation at HOT loci. The number of different DAPs associated with an element is positively associated with evidence of regulatory activity, and by systematically mutating 245 HOT loci with a massively parallel mutagenesis assay, we localized regulatory activity to a central core region that depends on the motif sequences of our previously nominated driver DAPs. In sum, this work leverages the increasingly large number of DAP motif and ChIP-seq data publicly available to explore how DAP associations contribute to genome-wide transcriptional regulation.

Genome-wide DNA methylation analysis implicates enrichment of interferon pathway in African American patients with Systemic Lupus Erythematosus and European Americans with lupus nephritis.

Systemic Lupus Erythematosus (SLE) is a chronic, multisystem, inflammatory autoimmune disease that disproportionately affects women. Trends in SLE prevalence and clinical course differ by ancestry, with those of African American ancestry presenting with more active, severe and rapidly progressive disease than European Americans. Previous research established altered epigenetic signatures in SLE patients compared to controls. However, the contribution of aberrant DNA methylation (DNAm) to the risk of SLE by ancestry and differences among patients with SLE-associated Lupus Nephritis (LN) has not been well described. We evaluated the DNA methylomes of 87 individuals including 41 SLE patients, with and without LN, and 46 controls enrolled in an ancestry diverse, well-characterized cohort study of established SLE (41 SLE patients [20 SLE-LN+, 21 SLE-LN-] and 46 sex-, race- and age-matched controls; 55% African American, 45% European American). Participants were genotyped using the Infinium Global Diversity Array (GDA), and genetic ancestry was estimated using principal components. Genome-wide DNA methylation was initially measured using the Illumina MethylationEPIC 850K Beadchip array followed by methylation-specific qPCR to validate the methylation status at putative loci. Differentially Methylated Positions (DMP) were identified using a case-control approach adjusted for ancestry. We identified a total of 51 DMPs in CpGs among SLE patients compared to controls. Genes proximal to these CpGs were highly enriched for involvement in type I interferon signaling. DMPs among European American SLE patients with LN were similar to African American SLE patients with and without LN. Our findings were validated using an orthogonal, methyl-specific PCR for three SLE-associated DMPs near or proximal to MX1, USP18, and IFITM1. Our study confirms previous reports that DMPs in CpGs associated with SLE are enriched in type I interferon genes. However, we show that European American SLE patients with LN have similar DNAm patterns to African American SLE patients irrespective of LN, suggesting that aberrant DNAm alters activity of type I interferon pathway leading to more severe disease independent of ancestry.

Genomic analysis reveals HDAC1 regulates clinically relevant transcriptional programs in Pancreatic cancer.

Novel strategies are needed to combat multidrug resistance in pancreatic ductal adenocarcinoma (PDAC). We applied genomic approaches to understand mechanisms of resistance in order to better inform treatment and precision medicine. Altered function of chromatin remodeling complexes contribute to chemoresistance. Our study generates and analyzes genomic and biochemical data from PDAC cells overexpressing HDAC1, a histone deacetylase involved in several chromatin remodeling complexes. We characterized the impact of overexpression on drug response, gene expression, HDAC1 binding, and chromatin structure using RNA-sequencing and ChIP-sequencing for HDAC1 and H3K27 acetylation. Integrative genomic analysis shows that HDAC1 overexpression promotes activation of key resistance pathways including epithelial to mesenchymal transition, cell cycle, and apoptosis through global chromatin remodeling. Target genes are similarly altered in patient tissues and show correlation with patient survival. We also demonstrate that direct targets of HDAC1 that also show altered chromatin are enriched near genes associated with altered GTPase activity. HDAC1 target genes identified using in vitro methods and observed in patient tissues were used to develop a clinically relevant nine-transcript signature associated with patient prognosis. Integration of multiple genomic and biochemical data types enables understanding of multidrug resistance and tumorigenesis in PDAC, a disease in desperate need of novel treatment strategies.

Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers based on five-year survival rates. Genes contributing to chemoresistance represent novel therapeutic targets that can improve treatment response. Increased expression of ANGPTL4 in tumors correlates with poor outcomes in pancreatic cancer. METHODS: We used statistical analysis of publicly available gene expression data (TCGA-PAAD) to test whether expression of ANGPTL4 and its downstream targets, ITGB4 and APOL1, were correlated with patient survival. We measured the impact of ANGPTL4 overexpression in a common pancreatic cancer cell line, MIA PaCa-2 cells, using CRISPRa for overexpression and DsiRNA for knockdown. We characterized global gene expression changes associated with high levels of ANGPTL4 and response to gemcitabine treatment using RNA-sequencing. Gemcitabine dose response curves were calculated on modified cell lines by measuring cell viability with CellTiter-Glo (Promega). Impacts on cell migration were measured using a time course scratch assay. RESULTS: We show that ANGPTL4 overexpression leads to in vitro resistance to gemcitabine and reduced survival times in patients. Overexpression of ANGPTL4 induces transcriptional signatures of tumor invasion and metastasis, proliferation and differentiation, and inhibition of apoptosis. Analyses revealed an overlapping signature of genes associated with both ANGPTL4 activation and gemcitabine response. Increased expression of the genes in this signature in patient PDAC tissues was significantly associated with shorter patient survival. We identified 42 genes that were both co-regulated with ANGPTL4 and were responsive to gemcitabine treatment. ITGB4 and APOL1 were among these genes. Knockdown of either of these genes in cell lines overexpressing ANGPTL4 reversed the observed gemcitabine resistance and inhibited cellular migration associated with epithelial to mesenchymal transition (EMT) and ANGPTL4 overexpression. CONCLUSIONS: These data suggest that ANGPTL4 promotes EMT and regulates the genes APOL1 and ITGB4. Importantly, we show that inhibition of both targets reverses chemoresistance and decreases migratory potential. Our findings have revealed a novel pathway regulating tumor response to treatment and suggest relevant therapeutic targets in pancreatic cancer.

Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC. METHODS: To assess cellular resistance mechanisms common to four cytotoxic chemotherapies (gemcitabine, 5-fluorouracil, irinotecan, and oxaliplatin) used to treat PDAC patients, we performed four genome-wide CRISPR activation (CRISPRact) and CRISPR knock-out (CRISPRko) screens in two common PDAC cell lines (Panc-1 and BxPC3). We used pathway analysis to identify gene sets enriched among our hits and conducted RNA-sequencing and chromatin immunoprecipitation-sequencing (ChIP-seq) to characterize top hits from our screen. We used scratch assays to assess changes in cellular migration with HDAC1 overexpression. RESULTS: Our data revealed activation of ABCG2, a well-described efflux pump, as the most consistent mediator of resistance in each of our screens. CRISPR-mediated activation of genes involved in transcriptional co-repressor complexes also conferred resistance to multiple drugs. Expression of many of these genes, including HDAC1, is associated with reduced survival in PDAC patients. Up-regulation of HDAC1 in vitro increased promoter occupancy and expression of several genes involved in the epithelial-to-mesenchymal transition (EMT). These cells also displayed phenotypic changes in cellular migration consistent with activation of the EMT pathway. The expression changes resulting from HDAC1 activation were also observed with activation of several other co-repressor complex members. Finally, we developed a publicly available analysis tool, PancDS, which integrates gene expression profiles with our screen results to predict drug sensitivity in resected PDAC tumors and cell lines. CONCLUSION: Our results provide a comprehensive resource for identifying cellular mechanisms of drug resistance in PDAC, mechanistically implicate HDAC1, and co-repressor complex members broadly, in multi-drug resistance, and provide an analytical tool for predicting treatment response in PDAC tumors and cell lines.

A genome-wide interactome of DNA-associated proteins in the human liver.

Large-scale efforts like the ENCODE Project have made tremendous progress in cataloging the genomic binding patterns of DNA-associated proteins (DAPs), such as transcription factors (TFs). However, most chromatin immunoprecipitation-sequencing (ChIP-seq) analyses have focused on a few immortalized cell lines whose activities and physiology differ in important ways from endogenous cells and tissues. Consequently, binding data from primary human tissue are essential to improving our understanding of in vivo gene regulation. Here, we identify and analyze more than 440,000 binding sites using ChIP-seq data for 20 DAPs in two human liver tissue samples. We integrated binding data with transcriptome and phased WGS data to investigate allelic DAP interactions and the impact of heterozygous sequence variation on the expression of neighboring genes. Our tissue-based data set exhibits binding patterns more consistent with liver biology than cell lines, and we describe uses of these data to better prioritize impactful noncoding variation. Collectively, our rich data set offers novel insights into genome function in human liver tissue and provides a valuable resource for assessing disease-related disruptions.

Chromodomain Helicase DNA-Binding Protein 7 Is Suppressed in the Perinecrotic/Ischemic Microenvironment and Is a Novel Regulator of Glioblastoma Angiogenesis.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453-462.

Pex35 is a regulator of peroxisome abundance.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1.

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

The expression of MHC class II molecules on murine breast tumors delays T-cell exhaustion, expands the T-cell repertoire, and slows tumor growth.

The expression of MHC class II molecules (MHCII) on tumor cells correlates with survival and responsiveness to immunotherapy. However, the mechanisms underlying these observations are poorly defined. Using a murine breast tumor line, we showed that MHCII-expressing tumors grew more slowly than controls and recruited more functional CD4+ and CD8+ T cells. In addition, MHCII-expressing tumors contained more TCR clonotypes expanded to a larger degree than control tumors. Functional CD8+ T cells in tumors depended on CD4+ T cells. However, both CD4+ and CD8+ T cells eventually became exhausted, even in MHCII-expressing tumors. Treatment with anti-CTLA4, but not anti-PD-1 or anti-TIM-3, promoted complete eradication of MHCII-expressing tumors. These results suggest tumor cell expression of MHCII facilitates the local activation of CD4+ T cells, indirectly helps the activation and expansion of CD8+ T cells, and, in combination with the appropriate checkpoint inhibitor, promotes tumor regression.

R2DGC: threshold-free peak alignment and identification for 2D gas chromatography-mass spectrometry in R.

Summary: Comprehensive 2D gas chromatography-mass spectrometry is a powerful method for analyzing complex mixtures of volatile compounds, but produces a large amount of raw data that requires downstream processing to align signals of interest (peaks) across multiple samples and match peak characteristics to reference standard libraries prior to downstream statistical analysis. Very few existing tools address this aspect of analysis and those that do have shortfalls in usability or performance. We have developed an R package that implements retention time and mass spectra similarity threshold-free alignments, seamlessly integrates retention time standards for universally reproducible alignments, performs common ion filtering and provides compatibility with multiple peak quantification methods. We demonstrate that our package's performance compares favorably to existing tools on a controlled mix of metabolite standards separated under variable chromatography conditions and data generated from cell lines. Availability and implementation: R2DGC can be downloaded at https://github.com/rramaker/R2DGC or installed via the Comprehensive R Archive Network (CRAN). Contact: sjcooper@hudsonalpha.org. Supplementary information: Supplementary data are available at Bioinformatics online.

powerTCR: A model-based approach to comparative analysis of the clone size distribution of the T cell receptor repertoire.

Sequencing of the T cell receptor (TCR) repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR.

aRNApipe: a balanced, efficient and distributed pipeline for processing RNA-seq data in high-performance computing environments.

SUMMARY: The wide range of RNA-seq applications and their high-computational needs require the development of pipelines orchestrating the entire workflow and optimizing usage of available computational resources. We present aRNApipe, a project-oriented pipeline for processing of RNA-seq data in high-performance cluster environments. aRNApipe is highly modular and can be easily migrated to any high-performance computing (HPC) environment. The current applications included in aRNApipe combine the essential RNA-seq primary analyses, including quality control metrics, transcript alignment, count generation, transcript fusion identification, alternative splicing and sequence variant calling. aRNApipe is project-oriented and dynamic so users can easily update analyses to include or exclude samples or enable additional processing modules. Workflow parameters are easily set using a single configuration file that provides centralized tracking of all analytical processes. Finally, aRNApipe incorporates interactive web reports for sample tracking and a tool for managing the genome assemblies available to perform an analysis. AVAILABILITY AND DOCUMENTATION: https://github.com/HudsonAlpha/aRNAPipe ; DOI: 10.5281/zenodo.202950. CONTACT: rmyers@hudsonalpha.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns.

BACKGROUND: Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer. METHODS: We measured genome-wide DNA methylation patterns in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array. We overlaid the most significantly differentially methylated sites in the genome with transcription factor binding sites measured by the Encyclopedia of DNA Elements consortium. We used logistic regression and receiver operating characteristic curves to assess the performance of candidate diagnostic models. RESULTS: We identified methylation patterns that have a high predictive power for distinguishing malignant prostate tissue from benign-adjacent prostate tissue, and these methylation signatures were validated using data from The Cancer Genome Atlas Project. Furthermore, by overlaying ENCODE transcription factor binding data, we observed an enrichment of enhancer of zeste homolog 2 binding in gene regulatory regions with higher DNA methylation in malignant prostate tissues. CONCLUSIONS: DNA methylation patterns are greatly altered in prostate cancer tissue in comparison to benign-adjacent tissue. We have discovered patterns of DNA methylation marks that can distinguish prostate cancers with high specificity and sensitivity in multiple patient tissue cohorts, and we have identified transcription factors binding in these differentially methylated regions that may play important roles in prostate cancer development.

Targeting the Wnt/ß-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974.

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/ß-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/ß-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 µM WNT974±100 µM carboplatin. Viability was evaluated with the ATPlite assay. The IC50 was calculated using a dose-response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P=0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/ß-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast.

To better understand the quantitative characteristics and structure of phenotypic diversity, we measured over 14,000 transcript, protein, metabolite, and morphological traits in 22 genetically diverse strains of Saccharomyces cerevisiae. More than 50% of all measured traits varied significantly across strains [false discovery rate (FDR) = 5%]. The structure of phenotypic correlations is complex, with 85% of all traits significantly correlated with at least one other phenotype (median = 6, maximum = 328). We show how high-dimensional molecular phenomics data sets can be leveraged to accurately predict phenotypic variation between strains, often with greater precision than afforded by DNA sequence information alone. These results provide new insights into the spectrum and structure of phenotypic diversity and the characteristics influencing the ability to accurately predict phenotypes.

High-throughput profiling of amino acids in strains of the Saccharomyces cerevisiae deletion collection.

The measurement of small molecule metabolites on a large scale offers the opportunity for a more complete understanding of cellular metabolism. We developed a high-throughput method to quantify primary amine-containing metabolites in the yeast Saccharomyces cerevisiae by the use of capillary electrophoresis in combination with fluorescent derivatization of cell extracts. We measured amino acid levels in the yeast deletion collection, a set of approximately 5000 strains each lacking a single gene, and developed a computational pipeline for data analysis. Amino acid peak assignments were validated by mass spectrometry, and the overall approach was validated by the result that expected pathway intermediates accumulate in mutants of the arginine biosynthetic pathway. Global analysis of the deletion collection was carried out using clustering methods. We grouped strains based on their metabolite profiles, revealing clusters of mutants enriched for genes encoding mitochondrial proteins, urea cycle enzymes, and vacuolar ATPase functions. One of the most striking profiles, common among several strains lacking ribosomal protein genes, accumulated lysine and a lysine-related metabolite. Mutations in the homologous ribosomal protein genes in the human result in Diamond-Blackfan anemia, demonstrating that metabolite data may have potential value in understanding disease pathology. This approach establishes metabolite profiling as capable of characterizing genes in a large collection of genetic variants.

Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance.

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers based on five-year survival rates. Genes contributing to chemoresistance represent novel therapeutic targets that can improve treatment response. Increased expression of ANGPTL4 in tumors correlates with poor outcomes in pancreatic cancer. Methods We used statistical analysis of publicly available gene expression data (TCGA-PAAD) to test whether expression of ANGPTL4 and its downstream targets, ITGB 4 and APOL1 , were correlated with patient survival. We measured the impact of ANGPTL4 overexpression in a common pancreatic cancer cell line, MIA PaCa-2 cells, using CRISPRa for overexpression and DsiRNA for knockdown. We characterized global gene expression changes associated with high levels of ANGPTL4 and response to gemcitabine treatment using RNA-sequencing. Gemcitabine dose response curves were calculated on modified cell lines by measuring cell viability with CellTiter-Glo (Promega). Impacts on cell migration were measured using a time course scratch assay. Results We show that ANGPTL4 overexpression leads to in vitro resistance to gemcitabine and reduced survival times in patients. Overexpression of ANGPTL4 induces transcriptional signatures of tumor invasion and metastasis, proliferation and differentiation, and inhibition of apoptosis. Analyses revealed an overlapping signature of genes associated with both ANGPTL4 activation and gemcitabine response. Increased expression of the genes in this signature in patient PDAC tissues was significantly associated with shorter patient survival. We identified 42 genes that were both co-regulated with ANGPTL4 and were responsive to gemcitabine treatment. ITGB4 and APOL1 were among these genes. Knockdown of either of these genes in cell lines overexpressing ANGPTL4 reversed the observed gemcitabine resistance and inhibited cellular migration associated with epithelial to mesenchymal transition (EMT) and ANGPTL4 overexpression. Conclusions These data suggest that ANGPTL4 promotes EMT and regulates the genes APOL1 and ITGB4 . Importantly, we show that inhibition of both targets reverses chemoresistance and decreases migratory potential. Our findings have revealed a novel pathway regulating tumor response to treatment and suggest relevant therapeutic targets in pancreatic cancer.