Highlights d A genetic, functional, and structural analysis of mammalian Mediator is provided d Contacts between a conserved core and the tail impact mMED-Pol II interaction d Loss of non-essential mMED subunits affects promoters linked to multiple enhancers d Cohesin is required to tether regulatory DNA; mMED and Pol II are not

The development of therapeutic strategies to combat immune-associated diseases requires the molecular mechanisms of human Th17 cell differentiation to be fully identified and understood. To investigate transcriptional control of Th17 cell differentiation, we used primary human CD4 T cells in small interfering RNA (siRNA)-mediated gene silencing and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) to identify both the early direct and indirect targets of STAT3. The integrated dataset presented in this study confirms that STAT3 is critical for transcriptional regulation of early human Th17 cell differentiation. Additionally, we found that a number of SNPs from loci associated with immune-mediated disorders were located at sites where STAT3 binds to induce Th17 cell specification. Importantly, introduction of such SNPs alters STAT3 binding in DNA affinity precipitation assays. Overall, our study provides important insights for modulating Th17-mediated pathogenic immune responses in humans.

SUMMARY Naïve CD4+ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hrs of in vitro polarization toward T helper-1 (Th1) and T helper-2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic associated single nucleotide polymorphisms (SNPs) suggests a role for distal regulatory elements in disease etiology.

Combined with TCR stimuli, extracellular cytokine signals initiate the differentiation of naive CD4+ T cells into specialized effector T-helper (Th) and regulatory T (Treg) cell subsets. The lineage specification and commitment process occurs through the combinatorial action of multiple transcription factors (TFs) and epigenetic mechanisms that drive lineage-specific gene expression programs. In this article, we review recent studies on the transcriptional and epigenetic regulation of distinct Th cell lineages. Moreover, we review current study linking immune disease-associated single-nucleotide polymorphisms with distal regulatory elements and their potential role in the disease etiology.

Early regulators of disease may increase understanding disease mechanisms, and serve as markers for pre-symptomatic diagnosis and treatment. However, early regulators are difficult to identify because patients generally present after they are symptomatic. We hypothesized that early regulators of T-cell associated diseases could be found by identifying upstream transcription factors (TFs) in T-cell differentiation, and by prioritizing hub TFs that were enriched for disease associated This analytical strategy to identify early regulators of disease by combining gene regulatory networks with GWAS may be generally applicable for functional and clinical studies of early disease development.

Motivation: Gene expression profiling using RNA-seq is a powerful technique for screening RNA species’ landscapes and their dynamics in an unbiased way. While several advanced methods exist for differential expression analysis of RNA-seq data, proper tools to anal.yze RNA-seq time-course have not been proposed.Results: In this study, we use RNA-seq to measure gene expression during the early human T helper 17 (Th17) cell differentiation and T-cell activation (Th0). To quantify Th17-specific gene expression dynamics, we present a novel statistical methodology, DyNB, for analyzing time-course RNA-seq data. We use non-parametric Gaussian processes to model temporal correlation in gene expression and combine that with negative binomial likelihood for the count data. To account for experiment-specific biases in gene expression dynamics, such as differences in cell differentiation efficiencies, we propose a method to rescale the dynamics between replicated measurements. We develop an MCMC sampling method to make inference of differential expression dynamics between conditions. DyNB identifies several known and novel genes involved in Th17 differentiation. Analysis of differentiation efficiencies revealed consistent patterns in gene expression dynamics between different cultures. We use qRT-PCR to validate differential expression and differentiation efficiencies for selected genes. Comparison of the results with those obtained via traditional timepoint-wise analysis shows that time-course analysis together with time rescaling between cultures identifies differentially expressed genes which would not otherwise be detected.Availability: An implementation of the proposed computational methods will be available at http://research.ics.aalto.fi/csb/software/Contact: tarmo.aijo@aalto.fi or harri.lahdesmaki@aalto.fiSupplementary information: Supplementary data are available at Bioinformatics online.

Th17 cells play an essential role in the pathogenesis of autoimmune and inflammatory diseases. Most of our current understanding on Th17 cell differentiation relies on studies carried out in mice, whereas the molecular mechanisms controlling human Th17 cell differentiation are less well defined. In this study, we identified gene expression changes characterizing early stages of human Th17 cell differentiation through genome-wide gene expression profiling. CD4 ؉ cells isolated from umbilical cord blood were used to determine detailed kinetics of gene expression after initiation of Th17 differentiation with IL1␤, IL6, and TGF␤. The differential expression of selected candidate genes was further validated at protein level and analyzed for specificity in initiation of Th17 compared with initiation of other Th subsets, namely Th1, Th2, and iTreg. This first genome-wide profiling of transcriptomics during the induction of human Th17 differentiation provides a starting point for defining gene regulatory networks and identifying new candidates regulating Th17 differentiation in humans. (Blood. 2012;119(23):e151-e160) IntroductionNaive CD4 ϩ T cells differentiate into functionally distinct lineages in response to environmental cues and interaction with APCs. The nature of invading pathogens determines the cytokine environment in which the cognate Ag recognition by TCR takes place, subsequently influencing the phenotype of differentiating CD4 ϩ Th cells. Classically, presentation of intra-or extracellular pathogens to naive T cells leads to either a Th1 response or a Th2 response, respectively. 1 Today, new functionally distinct subtypes of CD4 ϩ have been identified. 2 Since the original identification of IL17-secreting T cells, 3 further research has led to the definition of an effector Th17 cell lineage. [4][5][6] Shortly after these findings, Th17 cells were characterized also in humans by using peripheral blood T cells and T-cell clones derived from gut tissue of patients having Crohn disease. [7][8][9] Human Th17 cells express CCR6, CCR4, IL23R, and CD161 on their cell membrane. 9,10 The characteristic cytokine secreted by these cells is IL17A (also referred to as IL17). IL17A stimulates the secretion of wide range of proinflammatory chemokines and cytokines. As its receptor is widely expressed, many cells of the immune system as well as other cell types can respond to it. 11 In addition to IL17A, cytokines IL17F, IFN␥, IL22, and IL26 have been shown to be secreted by human Th17 cells. 7 Proper function of Th17 cells is needed for eradication of extracellular bacterial and fungal infections. 11 CD4 ϩ cells isolated from peripheral or cord blood have been used to examine Th17 polarization in human. In several studies, differentiation of naive cells from peripheral blood has not succeeded, or the IL17A production has been markedly less efficient than detected by memory cells. IL17A secretion of polarized cord blood cells is also modest. 12 Human Th17 cells have also been shown to originate from CD161 ϩ precursor cells...

BackgroundAlthough genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). lncRNAs are known to show more cell-type specificity than protein-coding genes.MethodsWe aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AIDs which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, natural killer (NK) cells, B cells, memory T cells, naive CD4+ and naive CD8+ T cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells).ResultsWe show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory T and CD8+ T cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved.ConclusionsThe observed enrichment of lncRNA transcripts in AID loci implies lncRNAs play an important role in AID etiology and suggests that lncRNA genes should be studied in more detail to interpret GWAS findings correctly. The co-expression results strongly support a model in which the lncRNA and protein-coding genes function together in the same pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0088-0) contains supplementary material, which is available to authorized users.