Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

A large-scale evaluation of computational protein function prediction.

Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals.

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.

Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.

Evolution of Advanced Chronic Lymphoid Leukemia Unveiled by Single-Cell Transcriptomics: A Case Report.

Genetic and transcriptional heterogeneity of Chronic lymphocytic leukaemia (CLL) limits prevention of disease progression. Longitudinal single-cell transcriptomics represents the state-of-the-art method to profile the disease heterogeneity at diagnosis and to inform about disease evolution. Here, we apply single-cell RNA-seq to a CLL case, sampled at diagnosis and relapse, that was treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) and underwent a dramatic decrease in CD19 expression during disease progression. Computational analyses revealed a major switch in clones' dominance during treatment. The clone that expanded at relapse showed 17p and 3p chromosomal deletions, and up-regulation of pathways related to motility, cytokine signaling and antigen presentation. Single-cell RNA-seq uniquely revealed that this clone was already present at low frequency at diagnosis, and it displays feature of plasma cell differentiation, consistent with a more aggressive phenotype. This study shows the benefit of single-cell profiling of CLL heterogeneity at diagnosis, to identify clones that might otherwise not be recognized and to determine the best treatment options.

Correction to: Functional variation in allelic methylomes underscores a strong genetic contribution and reveals novel epigenetic alterations in the human epigenome.

Following publication of the original article [1], the authors reported an error in Additional file 1.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Functional variation in allelic methylomes underscores a strong genetic contribution and reveals novel epigenetic alterations in the human epigenome.

BACKGROUND: The functional impact of genetic variation has been extensively surveyed, revealing that genetic changes correlated to phenotypes lie mostly in non-coding genomic regions. Studies have linked allele-specific genetic changes to gene expression, DNA methylation, and histone marks but these investigations have only been carried out in a limited set of samples. RESULTS: We describe a large-scale coordinated study of allelic and non-allelic effects on DNA methylation, histone mark deposition, and gene expression, detecting the interrelations between epigenetic and functional features at unprecedented resolution. We use information from whole genome and targeted bisulfite sequencing from 910 samples to perform genotype-dependent analyses of allele-specific methylation (ASM) and non-allelic methylation (mQTL). In addition, we introduce a novel genotype-independent test to detect methylation imbalance between chromosomes. Of the ~2.2 million CpGs tested for ASM, mQTL, and genotype-independent effects, we identify ~32% as being genetically regulated (ASM or mQTL) and ~14% as being putatively epigenetically regulated. We also show that epigenetically driven effects are strongly enriched in repressed regions and near transcription start sites, whereas the genetically regulated CpGs are enriched in enhancers. Known imprinted regions are enriched among epigenetically regulated loci, but we also observe several novel genomic regions (e.g., HOX genes) as being epigenetically regulated. Finally, we use our ASM datasets for functional interpretation of disease-associated loci and show the advantage of utilizing naïve T cells for understanding autoimmune diseases. CONCLUSIONS: Our rich catalogue of haploid methylomes across multiple tissues will allow validation of epigenome association studies and exploration of new biological models for allelic exclusion in the human genome.

Distinct Trends of DNA Methylation Patterning in the Innate and Adaptive Immune Systems.

DNA methylation and the localization and post-translational modification of nucleosomes are interdependent factors that contribute to the generation of distinct phenotypes from genetically identical cells. With 112 whole-genome bisulfite sequencing datasets from the BLUEPRINT Epigenome Project, we analyzed the global development of DNA methylation patterns during lineage commitment and maturation of a range of immune system effector cells and the cancers that arise from them. We show clear trends in methylation patterns that are distinct in the innate and adaptive arms of the human immune system, both globally and in relation to consistently positioned nucleosomes. Most notable are a progressive loss of methylation in developing lymphocytes and the consistent occurrence of non-CG methylation in specific cell types. Cancer samples from the two lineages are further polarized, suggesting the involvement of distinct lineage-specific epigenetic mechanisms. We anticipate broad utility for this resource as a basis for further comparative epigenetic analyses.

Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage.

We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.

Increased DNA methylation variability in type 1 diabetes across three immune effector cell types.

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.

Whole-genome fingerprint of the DNA methylome during human B cell differentiation.

We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.

Rule-based integration of RNA-Seq analyses tools for identification of novel transcripts.

Recent evidences suggest that a substantial amount of genome is transcribed more than that was anticipated, giving rise to a large number of unknown or novel transcripts. Identification of novel transcripts can provide key insights into understanding important cellular functions as well as molecular mechanisms underlying complex diseases like cancer. RNA-Seq has emerged as a powerful tool to detect novel transcripts, which previous profiling techniques failed to identify. A number of tools are available for enabling identification of novel transcripts at different levels. Read mappers such as TopHat, MapSplice, and SOAPsplice predict novel junctions, which are the indicators of novel transcripts. Cufflinks assembles novel transcripts based on alignment information and Oases performs de novo construction of transcripts. A common limitation of all these tools is prediction of sizable number of spurious or false positive (FP) novel transcripts. An approach that integrates information from all above sources and simultaneously scrutinizes FPs to correctly identify authentic novel transcripts of high confidence is proposed.

Anvaya: a workflows environment for automated genome analysis.

Anvaya is a workflow environment for automated genome analysis that provides an interface for several bioinformatics tools and databases, loosely coupled together in a coordinated system, enabling the execution of a set of analyses tools in series or in parallel. It is a client-server workflow environment that has an advantage over existing software as it enables extensive pre & post processing of biological data in an efficient manner. "Anvaya" offers the user, novel functionalities to carry out exhaustive comparative analysis via "custom tools," which are tools with new functionality not available in standard tools, and "built-in PERL parsers," which automate data-flow between tools that hitherto, required manual intervention. It also provides a set of 11 pre-defined workflows for frequently used pipelines in genome annotation and comparative genomics ranging from EST assembly and annotation to phylogenetic reconstruction and microarray analysis. It provides a platform that serves as a single-stop solution for biologists to carry out hassle-free and comprehensive analysis, without being bothered about the nuances involved in tool installation, command line parameters, format conversions required to connect tools and manage/process multiple data sets at a single instance.