ResearchPad - genomic-signal-processing https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Association test using Copy Number Profile Curves (CONCUR) enhances power in rare copy number variant analysis]]> https://www.researchpad.co/article/elastic_article_14642 Copy number variants comprise a large proportion of variation in human genomes. Large rare CNVs, especially those disrupting genes or changing the dosages of genes, can carry relatively strong risks for neurodevelopmental and neuropsychiatric disorders. Kernel-based association methods have been developed for the analysis of rare CNVs and shown to be a valuable tool. Kernel methods model the collective effect of rare CNVs using flexible kernel functions that capture the characteristics of CNVs and measure CNV similarity of individual pairs. Typically kernels are created by summarizing similarity within an artificially defined “CNV locus” and then collapsing across all loci. In this work, we propose a new kernel-based test, CONCUR, that is based on the CNV location information contained in standard processing of the variants and which obviates the need for arbitrarily defined CNV loci. CONCUR quantifies similarity between individual pairs as the common area under their copy number profile curves and is designed to detect CNV dosage, length and dosage-length interaction effects. In simulation studies and real data analysis, we demonstrate the ability of the CONCUR test to detect CNV effects under diverse CNV architectures with power and robustness over existing methods.

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<![CDATA[Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway]]> https://www.researchpad.co/article/5c784fbdd5eed0c484007497

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

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<![CDATA[A genome-wide scan for diversifying selection signatures in selected horse breeds]]> https://www.researchpad.co/article/5c5b5289d5eed0c4842bcb02

The genetic differentiation of the current horse population was evolutionarily created by natural or artificial selection which shaped the genomes of individual breeds in several unique ways. The availability of high throughput genotyping methods created the opportunity to study this genetic variation on a genome-wide level allowing detection of genome regions divergently selected between separate breeds as well as among different horse types sharing similar phenotypic features. In this study, we used the population differentiation index (FST) that is generally used for measuring locus-specific allele frequencies variation between populations, to detect selection signatures among six horse breeds maintained in Poland. These breeds can be classified into three major categories, including light, draft and primitive horses, selected mainly in terms of type (utility), exterior, performance, size, coat color and appearance. The analysis of the most pronounced selection signals found in this study allowed us to detect several genomic regions and genes connected with processes potentially important for breed phenotypic differentiation and associated with energy homeostasis during physical effort, heart functioning, fertility, disease resistance and motor coordination. Our results also confirmed previously described association of loci on ECA3 (spanning LCORL and NCAPG genes) and ECA11 (spanning LASP1 gene) with the regulation of body size in our draft and primitive (small size) horses. The efficiency of the applied FST-based approach was also confirmed by the identification of a robust selection signal in the blue dun colored Polish Konik horses at the locus of TBX3 gene, which was previously shown to be responsible for dun coat color dilution in other horse breeds. FST-based method showed to be efficient in detection of diversifying selection signatures in the analyzed horse breeds. Especially pronounced signals were observed at the loci responsible for fixed breed-specific features. Several candidate genes under selection were proposed in this study for traits selected in separate breeds and horse types, however, further functional and comparative studies are needed to confirm and explain their effect on the observed genetic diversity of the horse breeds.

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<![CDATA[Viral loads correlate with upregulation of PD-L1 and worse patient prognosis in Epstein–Barr Virus-associated gastric carcinoma]]> https://www.researchpad.co/article/5c59fec6d5eed0c484135434

Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC), one of four major gastric cancer types, consists of clonal growth of EBV-infected epithelial cells. However, the significance of viral loads in each tumor cell has not been evaluated. EBV-DNA is stably maintained in episomal form in the nucleus of each cancer cell. To estimate EBV copy number per genome (EBV-CN), qPCR of viral EBNA1 and host GAPDH, standardized by Namalwa DNA (one copy/genome), was applied to the formalin-fixed paraffin embedded (FFPE) surgically resected EBVaGC specimens (n = 43) and EBVaGC cell lines (SNU-719 and NCC-24). In surgical specimens, the cancer cell ratio (CCR) was determined with image analysis, and EBV-CN was obtained by adjusting qPCR value with CCR. Fluorescent in situ hybridization (FISH) was also applied to the FFPE sections using the whole EBV-genome as a probe. In surgical specimens, EBV-CN obtained by qPCR/CCR was between 1.2 and 185 copies with a median of 9.9. EBV-CN of SNU-719 and NCC-24 was 42.0 and 1.1, respectively. A linear correlation was observed with qPCR/CCR data up to 20 copies/genome (40 signals/nucleus), the limit of FISH analysis. In addition, substantial variation in the number of EBV foci was observed. Based on qPCR/CCR, high EBV-CN (>10 copies) correlated with PD-L1 expression in cancer cells (P = 0.015), but not with other pathological indicators. Furthermore, EBVaGC with high EBV-CN showed worse disease-specific survival (P = 0.041). Our findings suggest that cancer cell viral loads may contribute to expression of the immune checkpoint molecule and promotion of cancer progression in EBVaGC.

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<![CDATA[Genetic architecture of human thinness compared to severe obesity]]> https://www.researchpad.co/article/5c536c4ed5eed0c484a49d48

The variation in weight within a shared environment is largely attributable to genetic factors. Whilst many genes/loci confer susceptibility to obesity, little is known about the genetic architecture of healthy thinness. Here, we characterise the heritability of thinness which we found was comparable to that of severe obesity (h2 = 28.07 vs 32.33% respectively), although with incomplete genetic overlap (r = -0.49, 95% CI [-0.17, -0.82], p = 0.003). In a genome-wide association analysis of thinness (n = 1,471) vs severe obesity (n = 1,456), we identified 10 loci previously associated with obesity, and demonstrate enrichment for established BMI-associated loci (pbinomial = 3.05x10-5). Simulation analyses showed that different association results between the extremes were likely in agreement with additive effects across the BMI distribution, suggesting different effects on thinness and obesity could be due to their different degrees of extremeness. In further analyses, we detected a novel obesity and BMI-associated locus at PKHD1 (rs2784243, obese vs. thin p = 5.99x10-6, obese vs. controls p = 2.13x10-6 pBMI = 2.3x10-13), associations at loci recently discovered with much larger sample sizes (e.g. FAM150B and PRDM6-CEP120), and novel variants driving associations at previously established signals (e.g. rs205262 at the SNRPC/C6orf106 locus and rs112446794 at the PRDM6-CEP120 locus). Our ability to replicate loci found with much larger sample sizes demonstrates the value of clinical extremes and suggest that characterisation of the genetics of thinness may provide a more nuanced understanding of the genetic architecture of body weight regulation and may inform the identification of potential anti-obesity targets.

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<![CDATA[An improved DNA array-based classification method for the identification of Salmonella serotypes shows high concordance between traditional and genotypic testing]]> https://www.researchpad.co/article/5c1028fed5eed0c484248bb4

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10−1 ng/μl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination.

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<![CDATA[A Simple Model for the Influence of Meiotic Conversion Tracts on GC Content]]> https://www.researchpad.co/article/5989dad1ab0ee8fa60bb64f0

A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3) of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias.

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<![CDATA[Genome-Wide Association Study Reveals Multiple Loci Influencing Normal Human Facial Morphology]]> https://www.researchpad.co/article/5989db3fab0ee8fa60bd62dd

Numerous lines of evidence point to a genetic basis for facial morphology in humans, yet little is known about how specific genetic variants relate to the phenotypic expression of many common facial features. We conducted genome-wide association meta-analyses of 20 quantitative facial measurements derived from the 3D surface images of 3118 healthy individuals of European ancestry belonging to two US cohorts. Analyses were performed on just under one million genotyped SNPs (Illumina OmniExpress+Exome v1.2 array) imputed to the 1000 Genomes reference panel (Phase 3). We observed genome-wide significant associations (p < 5 x 10−8) for cranial base width at 14q21.1 and 20q12, intercanthal width at 1p13.3 and Xq13.2, nasal width at 20p11.22, nasal ala length at 14q11.2, and upper facial depth at 11q22.1. Several genes in the associated regions are known to play roles in craniofacial development or in syndromes affecting the face: MAFB, PAX9, MIPOL1, ALX3, HDAC8, and PAX1. We also tested genotype-phenotype associations reported in two previous genome-wide studies and found evidence of replication for nasal ala length and SNPs in CACNA2D3 and PRDM16. These results provide further evidence that common variants in regions harboring genes of known craniofacial function contribute to normal variation in human facial features. Improved understanding of the genes associated with facial morphology in healthy individuals can provide insights into the pathways and mechanisms controlling normal and abnormal facial morphogenesis.

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<![CDATA[Expanding the Understanding of Biases in Development of Clinical-Grade Molecular Signatures: A Case Study in Acute Respiratory Viral Infections]]> https://www.researchpad.co/article/5989dabeab0ee8fa60bafcee

Background

The promise of modern personalized medicine is to use molecular and clinical information to better diagnose, manage, and treat disease, on an individual patient basis. These functions are predominantly enabled by molecular signatures, which are computational models for predicting phenotypes and other responses of interest from high-throughput assay data. Data-analytics is a central component of molecular signature development and can jeopardize the entire process if conducted incorrectly. While exploratory data analysis may tolerate suboptimal protocols, clinical-grade molecular signatures are subject to vastly stricter requirements. Closing the gap between standards for exploratory versus clinically successful molecular signatures entails a thorough understanding of possible biases in the data analysis phase and developing strategies to avoid them.

Methodology and Principal Findings

Using a recently introduced data-analytic protocol as a case study, we provide an in-depth examination of the poorly studied biases of the data-analytic protocols related to signature multiplicity, biomarker redundancy, data preprocessing, and validation of signature reproducibility. The methodology and results presented in this work are aimed at expanding the understanding of these data-analytic biases that affect development of clinically robust molecular signatures.

Conclusions and Significance

Several recommendations follow from the current study. First, all molecular signatures of a phenotype should be extracted to the extent possible, in order to provide comprehensive and accurate grounds for understanding disease pathogenesis. Second, redundant genes should generally be removed from final signatures to facilitate reproducibility and decrease manufacturing costs. Third, data preprocessing procedures should be designed so as not to bias biomarker selection. Finally, molecular signatures developed and applied on different phenotypes and populations of patients should be treated with great caution.

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<![CDATA[A Single Cell Level Based Method for Copy Number Variation Analysis by Low Coverage Massively Parallel Sequencing]]> https://www.researchpad.co/article/5989dafbab0ee8fa60bc49c1

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.

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<![CDATA[Genome-wide association analysis identifies resistance loci for bacterial blight in a diverse collection of indica rice germplasm]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc88c

Bacterial blight, which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating rice diseases worldwide. The development and use of disease-resistant cultivars have been the most effective strategy to control bacterial blight. Identifying the genes mediating bacterial blight resistance is a prerequisite for breeding cultivars with broad-spectrum and durable resistance. We herein describe a genome-wide association study involving 172 diverse Oryza sativa ssp. indica accessions to identify loci influencing the resistance to representative strains of six Xoo races. Twelve resistance loci containing 121 significantly associated signals were identified using 317,894 single nucleotide polymorphisms, which explained 13.3–59.9% of the variability in lesion length caused by Xoo races P1, P6, and P9a. Two hotspot regions (L11 and L12) were located within or nearby two cloned R genes (xa25 and Xa26) and one fine-mapped R gene (Xa4). Our results confirmed the relatively high resolution of genome-wide association studies. Moreover, we detected novel significant associations on chromosomes 2, 3, and 6–10. Haplotype analyses of xa25, the Xa26 paralog (MRKc; LOC_Os11g47290), and a Xa4 candidate gene (LOC_11g46870) revealed differences in bacterial blight resistance among indica subgroups. These differences were responsible for the observed variations in lesion lengths resulting from infections by Xoo races P1 and P9a. Our findings may be relevant for future studies involving bacterial blight resistance gene cloning, and provide insights into the genetic basis for bacterial blight resistance in indica rice, which may be useful for knowledge-based crop improvement.

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<![CDATA[GWAS for serum galactose-deficient IgA1 implicates critical genes of the O-glycosylation pathway]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd092

Aberrant O-glycosylation of serum immunoglobulin A1 (IgA1) represents a heritable pathogenic defect in IgA nephropathy, the most common form of glomerulonephritis worldwide, but specific genetic factors involved in its determination are not known. We performed a quantitative GWAS for serum levels of galactose-deficient IgA1 (Gd-IgA1) in 2,633 subjects of European and East Asian ancestry and discovered two genome-wide significant loci, in C1GALT1 (rs13226913, P = 3.2 x 10−11) and C1GALT1C1 (rs5910940, P = 2.7 x 10−8). These genes encode molecular partners essential for enzymatic O-glycosylation of IgA1. We demonstrated that these two loci explain approximately 7% of variability in circulating Gd-IgA1 in Europeans, but only 2% in East Asians. Notably, the Gd-IgA1-increasing allele of rs13226913 is common in Europeans, but rare in East Asians. Moreover, rs13226913 represents a strong cis-eQTL for C1GALT1 that encodes the key enzyme responsible for the transfer of galactose to O-linked glycans on IgA1. By in vitro siRNA knock-down studies, we confirmed that mRNA levels of both C1GALT1 and C1GALT1C1 determine the rate of secretion of Gd-IgA1 in IgA1-producing cells. Our findings provide novel insights into the genetic regulation of O-glycosylation and are relevant not only to IgA nephropathy, but also to other complex traits associated with O-glycosylation defects, including inflammatory bowel disease, hematologic disease, and cancer.

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<![CDATA[Admixture mapping in two Mexican samples identifies significant associations of locus ancestry with triglyceride levels in the BUD13/ZNF259/APOA5 region and fine mapping points to rs964184 as the main driver of the association signal]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcb9e

We carried out an admixture mapping study of lipid traits in two samples from Mexico City. Native American locus ancestry was significantly associated with triglyceride levels in a broad region of chromosome 11 overlapping the BUD13, ZNF259 and APOA5 genes. In our fine-mapping analysis of this region using dense genome-wide data, rs964184 is the only marker included in the 99% credible set of SNPs, providing strong support for rs964184 as the causal variant within this region. The frequency of the allele associated with increased triglyceride concentrations (rs964184-G) is between 30–40% higher in Native American populations from Mexico than in European populations. The evidence currently available for this variant indicates that it may be exerting its effect through three potential mechanisms: 1) modification of enhancer activity, 2) regulation of the expression of several genes in cis and/or trans, or 3) modification of the methylation patterns of the promoter of the APOA5 gene.

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<![CDATA[Integrative Tissue-Specific Functional Annotations in the Human Genome Provide Novel Insights on Many Complex Traits and Improve Signal Prioritization in Genome Wide Association Studies]]> https://www.researchpad.co/article/5989db27ab0ee8fa60bd0875

Extensive efforts have been made to understand genomic function through both experimental and computational approaches, yet proper annotation still remains challenging, especially in non-coding regions. In this manuscript, we introduce GenoSkyline, an unsupervised learning framework to predict tissue-specific functional regions through integrating high-throughput epigenetic annotations. GenoSkyline successfully identified a variety of non-coding regulatory machinery including enhancers, regulatory miRNA, and hypomethylated transposable elements in extensive case studies. Integrative analysis of GenoSkyline annotations and results from genome-wide association studies (GWAS) led to novel biological insights on the etiologies of a number of human complex traits. We also explored using tissue-specific functional annotations to prioritize GWAS signals and predict relevant tissue types for each risk locus. Brain and blood-specific annotations led to better prioritization performance for schizophrenia than standard GWAS p-values and non-tissue-specific annotations. As for coronary artery disease, heart-specific functional regions was highly enriched of GWAS signals, but previously identified risk loci were found to be most functional in other tissues, suggesting a substantial proportion of still undetected heart-related loci. In summary, GenoSkyline annotations can guide genetic studies at multiple resolutions and provide valuable insights in understanding complex diseases. GenoSkyline is available at http://genocanyon.med.yale.edu/GenoSkyline.

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<![CDATA[The Drosophila speciation factor HMR localizes to genomic insulator sites]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc2ca

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR’s function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species.

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<![CDATA[High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion]]> https://www.researchpad.co/article/5989dad1ab0ee8fa60bb658d

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50–80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

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<![CDATA[Influence of Rotational Nucleosome Positioning on Transcription Start Site Selection in Animal Promoters]]> https://www.researchpad.co/article/5989da68ab0ee8fa60b92667

The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters’ sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters’ primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence–induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals.

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<![CDATA[Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens]]> https://www.researchpad.co/article/5989d9f5ab0ee8fa60b6fc7a

Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables—TCR and absolute copy numbers of HER2 and CEP17—by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status.

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<![CDATA[4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments]]> https://www.researchpad.co/article/5989db1aab0ee8fa60bcdeb9

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or “bait”) that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

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<![CDATA[A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells]]> https://www.researchpad.co/article/5989db05ab0ee8fa60bc83d6

The inactivation of p53 creates a major challenge for inducing apoptosis in cancer cells. An attractive strategy is to identify and subsequently target the survival signals in p53 defective cancer cells. Here we uncover a RUNX2-mediated survival signal in p53 defective cancer cells. The inhibition of this signal induces apoptosis in cancer cells but not non-transformed cells. Using the CRISPR technology, we demonstrate that p53 loss enhances the apoptosis caused by RUNX2 knockdown. Mechanistically, RUNX2 provides the survival signal partially through inducing MYC transcription. Cancer cells have high levels of activating histone marks on the MYC locus and concomitant high MYC expression. RUNX2 knockdown decreases the levels of these histone modifications and the recruitment of the Menin/MLL1 (mixed lineage leukemia 1) complex to the MYC locus. Two inhibitors of the Menin/MLL1 complex induce apoptosis in p53 defective cancer cells. Together, we identify a RUNX2-mediated epigenetic mechanism of the survival of p53 defective cancer cells and provide a proof-of-principle that the inhibition of this epigenetic axis is a promising strategy to kill p53 defective cancer cells.

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