Identification of regulatory links between transcription and RNA processing with long-read sequencing.

We present a detailed protocol for sequencing full-length mRNA isoforms using the Oxford nanopore long-read sequencing technology. We describe steps for poly(A) RNA isolation, library preparation, and cDNA size selection. We then detail procedures for sequencing and processing and a computational framework to identify exon couplings and assign mRNA 5' ends and 3' ends to each other. Our approach enables the identification of links between transcription initiation and co-transcriptional RNA processing events. For complete details on the use and execution of this protocol, please refer to Alfonso-Gonzalez et al.1.

Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

Genome-wide cooperation of EMT transcription factor ZEB1 with YAP and AP-1 in breast cancer.

Invasion, metastasis and therapy resistance are the major cause of cancer-associated deaths, and the EMT-inducing transcription factor ZEB1 is a crucial stimulator of these processes. While work on ZEB1 has mainly focused on its role as a transcriptional repressor, it can also act as a transcriptional activator. To further understand these two modes of action, we performed a genome-wide ZEB1 binding study in triple-negative breast cancer cells. We identified ZEB1 as a novel interactor of the AP-1 factors FOSL1 and JUN and show that, together with the Hippo pathway effector YAP, they form a transactivation complex, predominantly activating tumour-promoting genes, thereby synergising with its function as a repressor of epithelial genes. High expression of ZEB1, YAP, FOSL1 and JUN marks the aggressive claudin-low subtype of breast cancer, indicating the translational relevance of our findings. Thus, our results link critical tumour-promoting transcription factors: ZEB1, AP-1 and Hippo pathway factors. Disturbing their molecular interaction may provide a promising treatment option for aggressive cancer types.

RELACS nuclei barcoding enables high-throughput ChIP-seq.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. Current barcoding strategies aim to improve assay throughput and scalability but intense sample handling and lack of standardization over cell types, cell numbers and epitopes hinder wide-spread use in the field. Here, we present a barcoding method to enable high-throughput ChIP-seq using common molecular biology techniques. The method, called RELACS (restriction enzyme-based labeling of chromatin in situ) relies on standardized nuclei extraction from any source and employs chromatin cutting and barcoding within intact nuclei. Barcoded nuclei are pooled and processed within the same ChIP reaction, for maximal comparability and workload reduction. The innovative barcoding concept is particularly user-friendly and suitable for implementation to standardized large-scale clinical studies and scarce samples. Aiming to maximize universality and scalability, RELACS can generate ChIP-seq libraries for transcription factors and histone modifications from hundreds of samples within three days.

Epigenetic dynamics of monocyte-to-macrophage differentiation.

BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. RESULTS: Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. CONCLUSIONS: In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.

Standardizing chromatin research: a simple and universal method for ChIP-seq.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.

Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

RATIONALE: In chronic heart failure, increased adrenergic activation contributes to structural remodeling and altered gene expression. Although adrenergic signaling alters histone modifications, it is unknown, whether it also affects other epigenetic processes, including DNA methylation and its recognition. OBJECTIVE: The aim of this study was to identify the mechanism of regulation of the methyl-CpG-binding protein 2 (MeCP2) and its functional significance during cardiac pressure overload and unloading. METHODS AND RESULTS: MeCP2 was identified as a reversibly repressed gene in mouse hearts after transverse aortic constriction and was normalized after removal of the constriction. Similarly, MeCP2 repression in human failing hearts resolved after unloading by a left ventricular assist device. The cluster miR-212/132 was upregulated after transverse aortic constriction or on activation of α1- and ß1-adrenoceptors and miR-212/132 led to repression of MeCP2. Prevention of MeCP2 repression by a cardiomyocyte-specific, doxycycline-regulatable transgenic mouse model aggravated cardiac hypertrophy, fibrosis, and contractile dysfunction after transverse aortic constriction. Ablation of MeCP2 in cardiomyocytes facilitated recovery of failing hearts after reversible transverse aortic constriction. Genome-wide expression analysis, chromatin immunoprecipitation experiments, and DNA methylation analysis identified mitochondrial genes and their transcriptional regulators as MeCP2 target genes. Coincident with its repression, MeCP2 was removed from its target genes, whereas DNA methylation of MeCP2 target genes remained stable during pressure overload. CONCLUSIONS: These data connect adrenergic activation with a microRNA-MeCP2 epigenetic pathway that is important for cardiac adaptation during the development and recovery from heart failure.

Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.

Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model.

BACKGROUND: Epidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear. METHODS: To investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs. RESULTS: Exposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation. CONCLUSIONS: Our results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.

Thy-1 (CD90) regulates the extravasation of leukocytes during inflammation.

Human Thy-1 (CD90) has been shown to mediate adhesion of inflammatory cells to activated microvascular endothelial cells via interaction with Mac-1 (CD11b/CD18) in vitro. Since there are no data showing the physiological relevance of Thy-1 for the recruitment of inflammatory cells in vivo, different inflammation models were investigated in Thy-1-deficient mice and littermate controls. In thioglycollate-induced peritonitis, the number of neutrophils and monocytes was significantly diminished in Thy-1-deficient mice. During acute lung inflammation, the extravasation of eosinophils and monocytes into the lung was significantly reduced in Thy-1-deficient mice. Moreover, during chronic lung inflammation, the influx of eosinophils and monocytes was also strongly decreased. These effects were independent of Thy-1 expression on T cells, as shown by the transplantation of WT BM into the Thy-1-deficient mice. In spite of the strong Thy-1 expression on T cells in the chimeric mice, the extravasation of the inflammatory cells in these mice was significantly diminished, compared to control mice. Finally, the altered number and composition of infiltrating leukocytes in Thy-1-deficient mice modified the chemokine/cytokine and protease expression at the site of inflammation. In conclusion, Thy-1 is involved in the control of inflammatory cell recruitment and, thus, also in conditioning the inflammatory microenvironment.

Exposure to mycotoxins increases the allergic immune response in a murine asthma model.

RATIONALE: Epidemiological studies have shown that indoor molds are associated with increased prevalence and exacerbation of respiratory symptoms and asthma. Mycotoxins, secondary metabolites of molds, may contribute to these effects. OBJECTIVES: To investigate the adjuvant activity of mycotoxins on allergic airway inflammation. METHODS: Balb/c mice were exposed via the airways to gliotoxin and via the intestine to patulin, sensitized with ovalbumin (OVA), and then analyzed in acute and chronic murine asthma models. In addition, the effect of mycotoxin exposure on dendritic cell (DC) function was investigated using murine bone marrow-derived DCs. MEASUREMENTS AND MAIN RESULTS: Exposure of mice to both mycotoxins enhanced dose-dependently airway hyperreactivity, eosinophilic lung inflammation, and OVA-specific IgE serum levels compared with mice that received only the antigen. These findings correlated with increased Th2 cytokine levels and decreased IFN-gamma production. Long-term mycotoxin exposure exacerbated chronic airway inflammation and airway remodeling. In vitro or in vivo mycotoxin exposure inhibited IL-12 production in maturing DCs and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. Mycotoxin exposure enhanced OVA-induced lung lipid peroxidation and moderately increased isoprostane levels in naive mice. Treatment of mycotoxin-exposed DCs with the antioxidants N-acetylcysteine or glutathione ethyl ester restored IL-12 secretion and pretreatment of exposed mice with N-acetylcysteine prevented the mycotoxin-induced increase of airway inflammation and AHR. CONCLUSIONS: Our results demonstrate that gliotoxin and patulin increase the allergic immune response in mice by modulating the Th1/Th2 balance via direct effects on IL-12 secretion in DCs and by inducing oxidative stress.

Detection and regulation of cationic amino acid transporters in healthy and diseased ocular surface.

PURPOSE: To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions. METHODS: Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR. RESULTS: Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-alpha and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only. CONCLUSIONS: These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.