Mutational scanning identified amino acids of the CLOCK exon 19-domain essential for circadian rhythms.

AIM: In the mammalian circadian clock, the CLOCK/BMAL1 heterodimer binds to E-box enhancer elements in the promoters of its target genes to activate transcription. The classical Clock mice, the first circadian mouse mutant discovered, are behaviourally arrhythmic. In this mutant, CLOCK lacks a 51 amino acid domain corresponding to exon 19 (CLOCKΔ19), which is required for normal transactivation. While the importance of this CLOCK domain for circadian rhythms is well established, the exact molecular mechanism is still unclear. METHODS: Using CRISPR/Cas9 technology, we created a CLOCK knockout - CLOCK rescue system in human circadian reporter cells and performed systematic mutational scanning to assess the functionality of individual amino acids within the CLOCK exon 19-domain. RESULTS: CLOCK knockout cells were arrhythmic, and circadian rhythms could be rescued by introducing wild-type CLOCK, but not CLOCKΔ19. In addition, we identified several residues, whose mutation failed to rescue rhythms in CLOCK knockout cells. Many of these are part of the hydrophobic binding interface of the predicted dimer of the CLOCK exon 19-domain. CONCLUSION: Our data not only indicate that CLOCK/BMAL1 oligomerization mediated by the exon 19-domain is important for circadian dynamics but also suggest that the exon 19-domain provides a platform for binding coactivators and repressors, which in turn is required for normal circadian rhythms.

Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation.

The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.

Natural genetic variation of the cardiac transcriptome in non-diseased donors and patients with dilated cardiomyopathy.

BACKGROUND: Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases. RESULTS: Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts. CONCLUSIONS: RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

High salt reduces the activation of IL-4- and IL-13-stimulated macrophages.

A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt-induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis.

histoneHMM: Differential analysis of histone modifications with broad genomic footprints.

BACKGROUND: ChIP-seq has become a routine method for interrogating the genome-wide distribution of various histone modifications. An important experimental goal is to compare the ChIP-seq profiles between an experimental sample and a reference sample, and to identify regions that show differential enrichment. However, comparative analysis of samples remains challenging for histone modifications with broad domains, such as heterochromatin-associated H3K27me3, as most ChIP-seq algorithms are designed to detect well defined peak-like features. RESULTS: To address this limitation we introduce histoneHMM, a powerful bivariate Hidden Markov Model for the differential analysis of histone modifications with broad genomic footprints. histoneHMM aggregates short-reads over larger regions and takes the resulting bivariate read counts as inputs for an unsupervised classification procedure, requiring no further tuning parameters. histoneHMM outputs probabilistic classifications of genomic regions as being either modified in both samples, unmodified in both samples or differentially modified between samples. We extensively tested histoneHMM in the context of two broad repressive marks, H3K27me3 and H3K9me3, and evaluated region calls with follow up qPCR as well as RNA-seq data. Our results show that histoneHMM outperforms competing methods in detecting functionally relevant differentially modified regions. CONCLUSION: histoneHMM is a fast algorithm written in C++ and compiled as an R package. It runs in the popular R computing environment and thus seamlessly integrates with the extensive bioinformatic tool sets available through Bioconductor. This makeshistoneHMM an attractive choice for the differential analysis of ChIP-seq data. Software is available from http://histonehmm.molgen.mpg.de .

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.

Natural variation of histone modification and its impact on gene expression in the rat genome.

Histone modifications are epigenetic marks that play fundamental roles in many biological processes including the control of chromatin-mediated regulation of gene expression. Little is known about interindividual variability of histone modification levels across the genome and to what extent they are influenced by genetic variation. We annotated the rat genome with histone modification maps, identified differences in histone trimethyl-lysine levels among strains, and described their underlying genetic basis at the genome-wide scale using ChIP-seq in heart and liver tissues in a panel of rat recombinant inbred and their progenitor strains. We identified extensive variation of histone methylation levels among individuals and mapped hundreds of underlying cis- and trans-acting loci throughout the genome that regulate histone methylation levels in an allele-specific manner. Interestingly, most histone methylation level variation was trans-linked and the most prominent QTL identified influenced H3K4me3 levels at 899 putative promoters throughout the genome in the heart. Cis- acting variation was enriched in binding sites of distinct transcription factors in heart and liver. The integrated analysis of DNA variation together with histone methylation and gene expression levels showed that histoneQTLs are an important predictor of gene expression and that a joint analysis significantly enhanced the prediction of gene expression traits (eQTLs). Our data suggest that genetic variation has a widespread impact on histone trimethylation marks that may help to uncover novel genotype-phenotype relationships.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats.

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk.

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.

Detection of arthritis-susceptibility loci, including Ncf1, and variable effects of the major histocompatibility complex region depending on genetic background in rats.

OBJECTIVE: To characterize the arthritis-modulating effects of 3 non-major histocompatibility complex (MHC) quantitative trait loci (QTLs) in rat experimental arthritis in the disease-resistant E3 strain, and to investigate the disease-modulating effects of the MHC region (RT1) in various genetic backgrounds. METHODS: A congenic fragment containing Ncf1 along with congenic fragments containing the strongest remaining loci, Pia5/Cia3 and Pia7/Cia13 on chromosome 4, were transferred from the arthritis-susceptible DA strain into the background of the completely resistant E3 strain. The arthritis-regulatory potential of the transferred alleles was evaluated by comparing the susceptibility to experimental arthritis in congenic rats with that in E3 rats. The RT1(u) haplotype from the E3 strain was transferred into the susceptible DA strain (RT1(av1)), and various F(1) and F(2) hybrids were generated to assess the effects of RT1 on arthritis susceptibility. RESULTS: The DA allele of Ncf1 did not break the arthritis resistance of the E3 rats, although it led to enhanced autoimmune B cell responses, as indicated by significantly elevated levels of anticollagen antibodies in congenic rats. Introgressing Pia5 and Pia7 loci on chromosome 4 broke the resistance to arthritis, and the MHC locus on chromosome 20 in DA rats enhanced arthritis when RT1 interacted with E3 genes. CONCLUSION: The findings in these congenic lines confirm the existence of 3 major QTLs that regulate the severity of arthritis and are sufficient to induce the transformation of a completely arthritis-resistant rat strain into an arthritis-susceptible strain. This study also reveals a dramatic difference in the arthritis-regulatory potential of the rat MHC depending on genetic background, suggesting that strong epistatic interactions occur between MHC and non-MHC genes.

Positional cloning of the Igl genes controlling rheumatoid factor production and allergic bronchitis in rats.

Rheumatoid factors (RF), autoantibodies that bind the Fc region of IgG, are one of the major diagnostic marker in rheumatoid arthritis (RA) but occur with lower frequency also in other infectious and inflammatory conditions. Through positional cloning of the previously described quantitative trait locus (QTL) Rf1 in congenic and advanced intercrossed rats, we identified the Ig lambda light chain locus as a locus that regulates the production of RF in rats. The congenic rats produce RF-Ig lambda and have significant higher levels of RF-IgG and RF-IgM in serum, while the DA rat has an impaired RF production and does not produces RF-Ig lambda. Thus, we could investigate the role of RF in pristane-induced arthritis (PIA) as well as ovalbumin-induced airway inflammation. We show that there was no difference in the development and severity of PIA between congenic and parental DA rats, suggesting that RF using lambda light chains have no impact on PIA. However, the RF producing congenic rats developed a more severe airway inflammation as indicated in the significantly increased number of eosinophils in bronchoalveolar lavage fluid as well as total IgE in serum. In addition, RF congenic rats had a significantly enhanced immune response toward OVA due to increased OVA-Igk but not OVA-Igl antibodies, suggesting a possible involvement of RF in the regulation of the humoral immune response.

DA rats from two colonies differ genetically and in their arthritis susceptibility.

The arthritis-susceptible DA rat is one of the most commonly used rat strains for genetic linkage analysis and is instrumental for the identification of many genetic loci. Even though DA rats were kept as inbred lines at different institutes and suppliers, it became obvious that the various breeding stocks differed genetically. To be able to compare the results from different linkage studies it is very import to verify the genetic background of the substrains used in those studies. We performed a genetic and phenotypic analysis of two DA substrains, DA/ZtmRhd and DA/OlaHsd, and found several genetic differences. One of the allelic differences between the DA/ZtmRhd and the DA/OlaHsd strain was located at rat chromosome 3, a 17-Mb large fragment, including the peak marker of a previously identified quantitative trait locus (QTL) for collagen-induced arthritis, Cia11. In addition, the substrains exhibited a significant difference in the susceptibility to pristane-induced arthritis (PIA) and disease severity of collagen-induced arthritis and PIA. However, by generating and testing a congenic line, we could demonstrate that phenotypic differences were not due to the contaminating fragment on chromosome 3. Nevertheless, we conclude that DA substrains show distinct genetic differences and caution should be taken when comparing arthritis data from different DA substrains.