An endothelial regulatory module links blood pressure regulation with elite athletic performance.

The control of transcription is crucial for homeostasis in mammals. A previous selective sweep analysis of horse racing performance revealed a 19.6 kb candidate regulatory region 50 kb downstream of the Endothelin3 (EDN3) gene. Here, the region was narrowed to a 5.5 kb span of 14 SNVs, with elite and sub-elite haplotypes analyzed for association to racing performance, blood pressure and plasma levels of EDN3 in Coldblooded trotters and Standardbreds. Comparative analysis of human HiCap data identified the span as an enhancer cluster active in endothelial cells, interacting with genes relevant to blood pressure regulation. Coldblooded trotters with the sub-elite haplotype had significantly higher blood pressure compared to horses with the elite performing haplotype during exercise. Alleles within the elite haplotype were part of the standing variation in pre-domestication horses, and have risen in frequency during the era of breed development and selection. These results advance our understanding of the molecular genetics of athletic performance and vascular traits in both horses and humans.

Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis- and psoriasis-associated genes.

BACKGROUND: Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear. OBJECTIVE: We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate. METHODS: We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies. RESULTS: We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis. CONCLUSIONS: Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.

Promoter anchored interaction landscape of THP-1 macrophages captures early immune response processes.

Macrophages are highly plastic immune cells with temporally distinct transcriptome changes upon lipopolysaccride (LPS) activation. However, to what extent transcriptome reprogramming is mediated via spatial chromatin looping is not well studied. We generated high resolution chromatin interaction maps for LPS-stimulated THP-1 macrophages (0 and 2 h) using capture Hi-C. Success of LPS stimulation was validated with transcriptome sequencing. Circa 2900 genes changed their interaction profile upon LPS stimulation and those gaining interactions were enriched for LPS response relevant processes, suggesting a substantial role for distal regulation. Immune and cardiovascular risk variants were enriched within the interacting regions, thereby providing insights into macrophage biology.

HiCapTools: a software suite for probe design and proximity detection for targeted chromosome conformation capture applications.

Summary: Folding of eukaryotic genomes within nuclear space enables physical and functional contacts between regions that are otherwise kilobases away in sequence space. Targeted chromosome conformation capture methods (T2C, chi-C and HiCap) are capable of informing genomic contacts for a subset of regions targeted by probes. We here present HiCapTools, a software package that can design sequence capture probes for targeted chromosome capture applications and analyse sequencing output to detect proximities involving targeted fragments. Two probes are designed for each feature while avoiding repeat elements and non-unique regions. The data analysis suite processes alignment files to report genomic proximities for each feature at restriction fragment level and is isoform-aware for gene features. Statistical significance of contact frequencies is evaluated using an empirically derived background distribution. Targeted chromosome conformation capture applications are invaluable for locating target genes of disease-associated variants found by genome-wide association studies. Hence, we believe our software suite will prove to be useful for a wider user base within clinical and functional applications. Availability: https://github.com/sahlenlab/HiCapTools. Contact: pelinak@kth.se. Supplementary information: Supplementary data are available at Bioinformatics online.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation.

Optical nanopore sensing offers great potential in single-molecule detection, genotyping, or DNA sequencing for high-throughput applications. However, one of the bottle-necks for fluorophore-based biomolecule sensing is the lack of an optically optimized membrane with a large array of nanopores, which has large pore-to-pore distance, small variation in pore size and low background photoluminescence (PL). Here, we demonstrate parallel detection of single-fluorophore-labeled DNA strands (450 bps) translocating through an array of silicon nanopores that fulfills the above-mentioned requirements for optical sensing. The nanopore array was fabricated using electron beam lithography and anisotropic etching followed by electrochemical etching resulting in pore diameters down to ∼7 nm. The DNA translocation measurements were performed in a conventional wide-field microscope tailored for effective background PL control. The individual nanopore diameter was found to have a substantial effect on the translocation velocity, where smaller openings slow the translocation enough for the event to be clearly detectable in the fluorescence. Our results demonstrate that a uniform silicon nanopore array combined with wide-field optical detection is a promising alternative with which to realize massively-parallel single-molecule detection.

Enhancer mutations modulate the severity of chemotherapy-induced myelosuppression.

Non-small cell lung cancer is often diagnosed at advanced stages, and many patients are still treated with classical chemotherapy. The unselective nature of chemotherapy often results in severe myelosuppression. Previous studies showed that protein-coding mutations could not fully explain the predisposition to myelosuppression. Here, we investigate the possible role of enhancer mutations in myelosuppression susceptibility. We produced transcriptome and promoter-interaction maps (using HiCap) of three blood stem-like cell lines treated with carboplatin or gemcitabine. Taking advantage of publicly available enhancer datasets, we validated HiCap results in silico and in living cells using epigenetic CRISPR technology. We also developed a network approach for interactome analysis and detection of differentially interacting genes. Differential interaction analysis provided additional information on relevant genes and pathways for myelosuppression compared with differential gene expression analysis at the bulk level. Moreover, we showed that enhancers of differentially interacting genes are highly enriched for variants associated with differing levels of myelosuppression. Altogether, our work represents a prominent example of integrative transcriptome and gene regulatory datasets analysis for the functional annotation of noncoding mutations.

Prenylcysteine Oxidase 1 Is a Key Regulator of Adipogenesis.

The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1-/-/Apoe-/- mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe-/- mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing completely prevented the differentiation of 3T3-L1 preadipocytes, by acting as an upstream regulator of several key players, such as FABP4, PPARγ, C/EBPα. Proteomic analysis, performed by quantitative label-free mass spectrometry, further strengthened the role of PCYOX1 in adipogenesis by expanding the list of its downstream targets. Finally, the absence of Pcyox1 reduces the inflammatory markers in adipose tissue. These findings render PCYOX1 a novel adipogenic factor with possible pathophysiological or therapeutic potential.

SPP1/osteopontin: a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?

Degenerative ascending aortic aneurysm (AscAA) is a silent and potentially fatal disease characterized by excessive vascular inflammation and fibrosis. We aimed to characterize the cellular and molecular signature for the fibrotic type of endothelial mesenchymal transition (EndMT) that has previously been described in degenerative AscAA. Patients undergoing elective open-heart surgery for AscAA and/or aortic valve repair were recruited. Gene expression in the intima-media of the ascending aorta was measured in 22 patients with non-dilated and 24 with dilated aortas, and candidate genes were identified. Protein expression was assessed using immunohistochemistry. Interacting distal gene enhancer regions were identified using targeted chromosome conformation capture (HiCap) in untreated and LPS-treated THP1 cells, and the associated transcription factors were analyzed. Differential expression analysis identified SPP1 (osteopontin) as a key gene in the signature of fibrotic EndMT in patients with degenerative AscAA. The aortic intima-media expression of SPP1 correlated with the expression of inflammatory markers, the level of macrophage infiltration, and the aortic diameter. HiCap analysis, followed by transcription factor binding analysis, identified ETS1 as a potential regulator of SPP1 expression under inflammatory conditions. In conclusion, the present findings suggest that SPP1 may be involved in the development of the degenerative type of AscAA. KEY MESSAGES: In the original manuscript titled "SPP1/osteopontin, a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?" by David Freiholtz, Otto Bergman, Saliendra Pradhananga, Karin Lång, Flore-Anne Poujade, Carl Granath, Christian Olsson, Anders Franco-Cereceda, Pelin Sahlén, Per Eriksson, and Hanna M Björck, we present novel findings on regulatory factors on osteopontin (SPP1) expression in immune cells involved in degenerative ascending aortic aneurysms (AscAA). The central findings convey: SPP1 is a potential driver of the fibrotic endothelial-to-mesenchymal transition in AscAA. SPP1/osteopontin expression in AscAA is predominately by immune cells. ETS1 is a regulatory transcription factor of SPP1 expression in AscAA immune cells.

Targeted Chromosome Conformation Capture (HiCap).

Targeted chromosome conformation capture (HiCap) is an experimental method for detecting spatial interactions of genomic features such as promoters and/or enhancers. The protocol first describes the design of sequence capture probes. After that, it provides details on the chromosome conformation capture adapted for next-generation sequencing (Hi-C). Finally, the methodology for coupling Hi-C with sequence capture technology is described.

Current challenges in understanding the role of enhancers in disease.

Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies.

Variants That Differentiate Wolf and Dog Populations Are Enriched in Regulatory Elements.

Research on the genetics of domestication most often focuses on the protein-coding exons. However, exons cover only a minor part (1-2%) of the canine genome, whereas functional mutations may be located also in regions beyond the exome, in regulatory regions. Therefore, a large proportion of phenotypical differences between dogs and wolves may remain genetically unexplained. In this study, we identified variants that have high allelic frequency differences (i.e., highly differentiated variants) between wolves and dogs across the canine genome and investigated the potential functionality. We found that the enrichment of highly differentiated variants was substantially higher in promoters than in exons and that such variants were enriched also in enhancers. Several enriched pathways were identified including oxytocin signaling, carbohydrate digestion and absorption, cancer risk, and facial and body features, many of which reflect phenotypes of potential importance during domestication, including phenotypes of the domestication syndrome. The results highlight the importance of regulatory mutations during dog domestication and motivate the functional annotation of the noncoding part of the canine genome.

A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and HiCap Bulk Data in Human Liver.

The liver is the largest solid organ and a primary metabolic hub. In recent years, intact cell nuclei were used to perform single-nuclei RNA-seq (snRNA-seq) for tissues difficult to dissociate and for flash-frozen archived tissue samples to discover unknown and rare cell subpopulations. In this study, we performed snRNA-seq of a liver sample to identify subpopulations of cells based on nuclear transcriptomics. In 4282 single nuclei, we detected, on average, 1377 active genes and we identified seven major cell types. We integrated data from 94,286 distal interactions (p < 0.05) for 7682 promoters from a targeted chromosome conformation capture technique (HiCap) and mass spectrometry proteomics for the same liver sample. We observed a reasonable correlation between proteomics and in silico bulk snRNA-seq (r = 0.47) using tissue-independent gene-specific protein abundancy estimation factors. We specifically looked at genes of medical importance. The DPYD gene is involved in the pharmacogenetics of fluoropyrimidine toxicity and some of its variants are analyzed for clinical purposes. We identified a new putative polymorphic regulatory element, which may contribute to variation in toxicity. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and we investigated all known risk genes. We identified a complex regulatory landscape for the SLC2A2 gene with 16 candidate enhancers. Three of them harbor somatic motif breaking and other mutations in HCC in the Pan Cancer Analysis of Whole Genomes dataset and are candidates to contribute to malignancy. Our results highlight the potential of a multi-omics approach in the study of human diseases.

Whole-genome sequencing and gene network modules predict gemcitabine/carboplatin-induced myelosuppression in non-small cell lung cancer patients.

Gemcitabine/carboplatin chemotherapy commonly induces myelosuppression, including neutropenia, leukopenia, and thrombocytopenia. Predicting patients at risk of these adverse drug reactions (ADRs) and adjusting treatments accordingly is a long-term goal of personalized medicine. This study used whole-genome sequencing (WGS) of blood samples from 96 gemcitabine/carboplatin-treated non-small cell lung cancer (NSCLC) patients and gene network modules for predicting myelosuppression. Association of genetic variants in PLINK found 4594, 5019, and 5066 autosomal SNVs/INDELs with p ≤ 1 × 10-3 for neutropenia, leukopenia, and thrombocytopenia, respectively. Based on the SNVs/INDELs we identified the toxicity module, consisting of 215 unique overlapping genes inferred from MCODE-generated gene network modules of 350, 345, and 313 genes, respectively. These module genes showed enrichment for differentially expressed genes in rat bone marrow, human bone marrow, and human cell lines exposed to carboplatin and gemcitabine (p < 0.05). Then using 80% of the patients as training data, random LASSO reduced the number of SNVs/INDELs in the toxicity module into a feasible prediction model consisting of 62 SNVs/INDELs that accurately predict both the training and the test (remaining 20%) data with high (CTCAE 3-4) and low (CTCAE 0-1) maximal myelosuppressive toxicity completely, with the receiver-operating characteristic (ROC) area under the curve (AUC) of 100%. The present study shows how WGS, gene network modules, and random LASSO can be used to develop a feasible and tested model for predicting myelosuppressive toxicity. Although the proposed model predicts myelosuppression in this study, further evaluation in other studies is required to determine its reproducibility, usability, and clinical effect.

High-Resolution Regulatory Maps Connect Vascular Risk Variants to Disease-Related Pathways.

BACKGROUND: Genetic variant landscape of coronary artery disease is dominated by noncoding variants among which many occur within putative enhancers regulating the expression levels of relevant genes. It is crucial to assign the genetic variants to their correct genes both to gain insights into perturbed functions and better assess the risk of disease. METHODS: In this study, we generated high-resolution genomic interaction maps (≈750 bases) in aortic endothelial, smooth muscle cells and THP-1 (human leukemia monocytic cell line) macrophages stimulated with lipopolysaccharide using Hi-C coupled with sequence capture targeting 25 429 features, including variants associated with coronary artery disease. We also sequenced their transcriptomes and mapped putative enhancers using chromatin immunoprecipitation with an antibody against H3K27Ac. RESULTS: The regions interacting with promoters showed strong enrichment for enhancer elements and validated several previously known interactions and enhancers. We detected interactions for 727 risk variants obtained by genome-wide association studies and identified novel, as well as established genes and functions associated with cardiovascular diseases. We were able to assign potential target genes for additional 398 genome-wide association studies variants using haplotype information, thereby identifying additional relevant genes and functions. Importantly, we discovered that a subset of risk variants interact with multiple promoters and their expression levels were strongly correlated. CONCLUSIONS: In summary, we present a catalog of candidate genes regulated by coronary artery disease-related variants and think that it will be an invaluable resource to further the investigation of cardiovascular pathologies and disease.

Allele specific chromatin signals, 3D interactions, and motif predictions for immune and B cell related diseases.

Several Genome Wide Association Studies (GWAS) have reported variants associated to immune diseases. However, the identified variants are rarely the drivers of the associations and the molecular mechanisms behind the genetic contributions remain poorly understood. ChIP-seq data for TFs and histone modifications provide snapshots of protein-DNA interactions allowing the identification of heterozygous SNPs showing significant allele specific signals (AS-SNPs). AS-SNPs can change a TF binding site resulting in altered gene regulation and are primary candidates to explain associations observed in GWAS and expression studies. We identified 17,293 unique AS-SNPs across 7 lymphoblastoid cell lines. In this set of cell lines we interrogated 85% of common genetic variants in the population for potential regulatory effect and we identified 237 AS-SNPs associated to immune GWAS traits and 714 to gene expression in B cells. To elucidate possible regulatory mechanisms we integrated long-range 3D interactions data to identify putative target genes and motif predictions to identify TFs whose binding may be affected by AS-SNPs yielding a collection of 173 AS-SNPs associated to gene expression and 60 to B cell related traits. We present a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles and novel strategies to detect the regulated genes.

The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation.

BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics.

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution.

Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp--close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.