Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations.

Mutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDSs). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. We applied long-read transcriptome sequencing (LRTS) to 44 MDS and CLL patients, as well as two pairs of isogenic cell lines with and without SF3B1 mutations, and found >60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3' splice sites. Our data highlighted a constrained window at canonical 3' splice sites in which dynamic splice-site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA-binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3' splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E Our work presents the hitherto most-complete LRTS study of the SF3B1 mutation in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosises result most likely from the different cell types expanded during carcinogenesis rather than different mechanisms of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases.

Oncogenic role and target properties of the lysine-specific demethylase KDM1A in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), epigenetic alterations are considered to centrally shape the transcriptional signatures that drive disease evolution and underlie its biological and clinical subsets. Characterizations of epigenetic regulators, particularly histone-modifying enzymes, are very rudimentary in CLL. In efforts to establish effectors of the CLL-associated oncogene T-cell leukemia 1A (TCL1A), we identified here the lysine-specific histone demethylase KDM1A to interact with the TCL1A protein in B cells in conjunction with an increased catalytic activity of KDM1A. We demonstrate that KDM1A is upregulated in malignant B cells. Elevated KDM1A and associated gene expression signatures correlated with aggressive disease features and adverse clinical outcomes in a large prospective CLL trial cohort. Genetic Kdm1a knockdown in Eµ-TCL1A mice reduced leukemic burden and prolonged animal survival, accompanied by upregulated p53 and proapoptotic pathways. Genetic KDM1A depletion also affected milieu components (T, stromal, and monocytic cells), resulting in significant reductions in their capacity to support CLL-cell survival and proliferation. Integrated analyses of differential global transcriptomes (RNA sequencing) and H3K4me3 marks (chromatin immunoprecipitation sequencing) in Eµ-TCL1A vs iKdm1aKD;Eµ-TCL1A mice (confirmed in human CLL) implicate KDM1A as an oncogenic transcriptional repressor in CLL which alters histone methylation patterns with pronounced effects on defined cell death and motility pathways. Finally, pharmacologic KDM1A inhibition altered H3K4/9 target methylation and revealed marked anti-B-cell leukemic synergisms. Overall, we established the pathogenic role and effector networks of KDM1A in CLL via tumor-cell intrinsic mechanisms and its impacts in cells of the microenvironment. Our data also provide rationales to further investigate therapeutic KDM1A targeting in CLL.

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

LPS-induced modules of co-expressed genes in equine peripheral blood mononuclear cells.

BACKGROUND: Lipopolysaccharide (endotoxin, LPS) is a strong inducer of the innate immune response. It is widespread in our environment, e.g. in house dust and contributes to asthma. Compared to humans, horses are even more sensitive to LPS. However, data on LPS effects on the equine transcriptome are very limited. Using RNA-seq we analysed LPS-induced differences in the gene expression in equine peripheral blood mononuclear cells at the gene and gene-network level in two half-sib families and one group of unrelated horses. RESULTS: 24 h-LPS challenge of equine immune cells resulted in substantial changes in the transcriptomic profile (1,265 differentially expressed genes) showing partial overlap with human data. One of the half-sib families showed a specific response different from the other two groups of horses. We also identified co-expressed gene modules that clearly differentiated 24 h-LPS- from non-stimulated samples. These modules consisted of 934 highly interconnected genes and included genes involved in the immune response (e.g. IL6, CCL22, CXCL6, CXCL2), however, none of the top ten hub genes of the modules have been annotated as responsive to LPS in gene ontology. CONCLUSIONS: Using weighted gene co-expression network analysis we identified ten co-expressed gene modules significantly regulated by in vitro stimulation with LPS. Apart from 47 genes (5%) all other genes highly interconnected within the most up- and down-regulated modules were also significantly differentially expressed (FDR < 0.05). The LPS-regulated module hub genes have not yet been described as having a role in the immune response to LPS (e.g. VAT1 and TTC25).

Novel equine tissue miRNAs and breed-related miRNA expressed in serum.

BACKGROUND: MiRNAs regulate multiple genes at the post-transcriptional level and therefore play an important role in many biological processes. It has been suggested that miRNA exported outside the cells contribute to inter-cellular communication. Consequently, circulating miRNAs are of particular interest and are promising biomarkers for many diseases. The number of miRNAs annotated in the horse genome is much lower compared to model organisms like human and mouse. We therefore aimed to identify novel equine miRNAs for tissue types and breed in serum. RESULTS: We analysed 71 small RNA-seq libraries derived from nine tissues (gluteus medius, platysma, masseter muscle, heart, liver, cartilage, bone, total blood and serum) using miRDeep2 and miRdentify tools. Known miRNAs represented between 2.3 and 62.9 % of the reads in 71 libraries. A total of 683 novel miRNAs were identified. Breed and tissue type affected the number of miRNAs detected and interestingly, affected its average intensity. A total of 50 miRNAs in serum proved to be potential biomarkers to differentiate specific breed types, of which miR-122, miR-200, miR-483 were over-expressed and miR-328 was under-expressed in ponies compared to Warmbloods. The different miRNAs profiles, as well as the differences in their expression levels provide a foundation for more hypotheses based on the novel miRNAs discovered. CONCLUSIONS: We identified 683 novel equine miRNAs expressed in seven solid tissues, blood and serum. Additionally, our approach evidenced that such data supported identification of specific miRNAs as markers of functions related to breeds or disease tissues.

SparkSeq: fast, scalable and cloud-ready tool for the interactive genomic data analysis with nucleotide precision.

UNLABELLED: Many time-consuming analyses of next -: generation sequencing data can be addressed with modern cloud computing. The Apache Hadoop-based solutions have become popular in genomics BECAUSE OF: their scalability in a cloud infrastructure. So far, most of these tools have been used for batch data processing rather than interactive data querying. The SparkSeq software has been created to take advantage of a new MapReduce framework, Apache Spark, for next-generation sequencing data. SparkSeq is a general-purpose, flexible and easily extendable library for genomic cloud computing. It can be used to build genomic analysis pipelines in Scala and run them in an interactive way. SparkSeq opens up the possibility of customized ad hoc secondary analyses and iterative machine learning algorithms. This article demonstrates its scalability and overall fast performance by running the analyses of sequencing datasets. Tests of SparkSeq also prove that the use of cache and HDFS block size can be tuned for the optimal performance on multiple worker nodes. AVAILABILITY AND IMPLEMENTATION: Available under open source Apache 2.0 license: https://bitbucket.org/mwiewiorka/sparkseq/.

Hedgehog dysregulation contributes to tissue-specific inflammaging of resident macrophages.

Age-associated low-grade sterile inflammation, commonly referred to as inflammaging, is a recognized hallmark of aging, which contributes to many age-related diseases. While tissue-resident macrophages are innate immune cells that secrete many types of inflammatory cytokines in response to various stimuli, it is not clear whether they have a role in driving inflammaging. Here we characterized the transcriptional changes associated with physiological aging in mouse resident macrophage populations across different tissues and sexes. Although the age-related transcriptomic signatures of resident macrophages were strikingly tissue-specific, the differentially expressed genes were collectively enriched for those with important innate immune functions such as antigen presentation, cytokine production, and cell adhesion. The brain-resident microglia had the most wide-ranging age-related alterations, with compromised expression of tissue-specific genes and relatively exaggerated responses to endotoxin stimulation. Despite the tissue-specific patterns of aging transcriptomes, components of the hedgehog (Hh) signaling pathway were decreased in aged macrophages across multiple tissues. In vivo suppression of Hh signaling in young animals increased the expression of pro-inflammatory cytokines, while in vitro activation of Hh signaling in old macrophages, in turn, suppressed the expression of these inflammatory cytokines. This suggests that hedgehog signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Overall, our data represent a resourceful catalog of tissue-specific and sex-specific transcriptomic changes in resident macrophages of peritoneum, liver, and brain, during physiological aging.

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

An Integrative miRNA-mRNA Expression Analysis Reveals Striking Transcriptomic Similarities between Severe Equine Asthma and Specific Asthma Endotypes in Humans.

Severe equine asthma is an incurable obstructive respiratory condition affecting 10-15% of horses in temperate climates. Upon exposure to airborne antigens from hay feeding, affected horses show neutrophilic airway inflammation and bronchoconstriction, leading to increased respiratory effort. The resulting implications range from welfare concerns to economic impacts on equestrian sports and horse breeding. Immunological and pathophysiological characteristics of severe equine asthma show important parallels with allergic and severe neutrophilic human asthma. Our study aimed at investigating regulatory networks underlying the pathophysiology of the disease by profiling miRNA and mRNA expression in lung tissue samples from asthmatic horses compared with healthy controls. We sequenced small RNAs and mRNAs from lungs of seven asthmatic horses in exacerbation, five affected horses in remission, and eight healthy control horses. Our comprehensive differential expression analyses, combined with the miRNA-mRNA negative correlation approach, revealed a strong similarity on the transcriptomic level between severe equine asthma and severe neutrophilic asthma in humans, potentially through affecting Th17 cell differentiation. This study also showed that several dysregulated miRNAs and mRNAs are involved in airway remodeling. These results present a starting point for a better transcriptomic understanding of severe equine asthma and its similarities to asthma in humans.

lncRNA expression predicts mRNA abundance.

Aim: Long noncoding RNAs (lncRNAs) have been reported to influence multiple gene regulatory processes. Technological advances in RNA-seq platforms allow detection of low-abundance RNA species such as lncRNAs. This study examined the relationship between expression of lncRNAs and their putative partner mRNAs. Methods: We analyzed total RNA-seq data from mouse macrophages under various inflammatory and intervention conditions. Results: The macrophage expression of lncRNAs is strongly regulated by an inflammatory stimulus. Moreover, the expression of a majority of lncRNAs was correlated or anti-correlated with the partner mRNA(s), across the different treatment conditions. This relationship was maintained even in cells from a distinct genotype. Conclusion: These results suggest a previously unappreciated tight coupling of lncRNA and mRNA expression during macrophage responses to various microenvironmental perturbations.

Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression.

BACKGROUND: Currently no methods are available to predict the clinical outcome of individual horses with equine sarcoid (ES) disease. OBJECTIVE: To investigate if whole blood microRNA (miRNA) profiles can predict the long-term development of ES tumors. ANIMALS: Five horses with regression and 5 with progression of ES lesions monitored over 5-7 years and 5 control horses free of ES for at least 5 years. METHODS: For this cohort study, RNA extracted from whole blood samples from the regression, progression, and control groups was used for high throughput sequencing. Known and novel miRNAs were identified using miRDeep2 and differential expression analysis was carried out by the DESeq2 algorithm. Target gene and pathway prediction as well as enrichment and network analyses were conducted using TarBase, mirPath, and metaCore from GeneGo. RESULTS: Fourteen miRNAs were differentially expressed between regression and progression groups after accounting for the control condition: 4 miRNAs (28.6%) were upregulated and 10 miRNAs (71.4%) were downregulated with >2-fold change. Seven of the 10 downregulated miRNAs are encoded in an miRNA cluster on equine chromosome 24, homologous to the well-known 14q32 cluster in humans. Their target genes show enrichment for pathways involved in viral carcinogenesis. CONCLUSIONS AND CLINICAL IMPORTANCE: Whole blood miRNA expression profiles are associated with long-term ES growth in horses and warrant further validation as prognostic biomarkers in a larger study cohort. Deregulation of miRNAs on equine chromosome 24 might represent a trigger for ES development.

MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers.

Serum and whole blood microRNA (miRNA) fingerprints have been proposed as a new class of non-invasive human cancer biomarkers. In this study, we compared equine sarcoid (ES) disease-specific serum and whole blood miRNA fingerprints and correlated them to miRNA expression in sarcoid tissue. After high throughput sequencing, miRNA differential expression analysis between six ES-affected and five control horses was carried out in serum and whole blood using a DESeq algorithm, accounting for the influence of hemolysis and the white blood cell count. Target gene, pathway prediction and enrichment analyses were conducted using TarBase, mirPath and GeneCodis. After exclusion of 4 hemolyzed out of a total of 11 serum samples, 9 miRNAs were found to be differentially expressed in serum of ES vs control horses. In whole blood, all 11 samples showed normal white blood cell counts and 19 miRNAs were found to be differentially expressed. A total of 2/9 serum and 7/19 whole blood differentially expressed miRNAs were also highly expressed at the tissue level and their predicted target genes were associated with cancer pathways. Serum and whole blood miRNA expression allowed discrimination between ES and control horses and merits further validation in a larger study cohort. The use of whole blood might be superior because it has higher miRNA content and is less influenced by pre-analytical variables compared to serum. Concurrent dysregulation of single miRNAs in tissue and blood suggests a possible biological function of circulating miRNAs.

Differential Expression of Serum MicroRNAs Supports CD4⁺ T Cell Differentiation into Th2/Th17 Cells in Severe Equine Asthma.

MicroRNAs (miRNAs) regulate post-transcriptional gene expression and may be exported from cells via exosomes or in partnership with RNA-binding proteins. MiRNAs in body fluids can act in a hormone-like manner and play important roles in disease initiation and progression. Hence, miRNAs are promising candidates as biomarkers. To identify serum miRNA biomarkers in the equine model of asthma we investigated small RNA derived from the serum of 34 control and 37 asthmatic horses. These samples were used for next generation sequencing, novel miRNA identification and differential miRNA expression analysis. We identified 11 significantly differentially expressed miRNAs between case and control horses: eca-miR-128, eca-miR-744, eca-miR-197, eca-miR-103, eca-miR-107a, eca-miR-30d, eca-miR-140-3p, eca-miR-7, eca-miR-361-3p, eca-miR-148b-3p and eca-miR-215. Pathway enrichment using experimentally validated target genes of the human homologous miRNAs showed a significant enrichment in the regulation of epithelial-to-mesenchymal transition (key player in airway remodeling in asthma) and the phosphatidylinositol (3,4,5)-triphosphate (PIP3) signaling pathway (modulator of CD4⁺ T cell maturation and function). Downregulated miR-128 and miR-744 supports a Th2/Th17 type immune response in severe equine asthma.

Optimized methods for extracting circulating small RNAs from long-term stored equine samples.

Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.

Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse.

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre- and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses.

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.

Impaired Cell Cycle Regulation in a Natural Equine Model of Asthma.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

The transcriptome of equine peripheral blood mononuclear cells.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.