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.

PVT1 interacts with polycomb repressive complex 2 to suppress genomic regions with pro-apoptotic and tumour suppressor functions in multiple myeloma.

Multiple myeloma is a heterogeneous hematological disease that originates from the bone marrow and is characterized by the monoclonal expansion of malignant plasma cells. Despite novel therapies, multiple myeloma remains clinically challenging. A common feature among patients with poor prognosis is the increased activity of the epigenetic silencer EZH2, which is the catalytic subunit of the PRC2. Interestingly, the recruitment of PRC2 lacks sequence specificity and, to date, the molecular mechanisms that define which genomic locations are destined for PRC2-mediated silencing remain unknown. The presence of a long non-coding RNA (lncRNA)-binding pocket on EZH2 suggests that lncRNA could potentially mediate PRC2 recruitment to specific genomic regions. Here, we coupled RNA immunoprecipitation sequencing, RNA-sequencing and chromatin immunoprecipitation-sequencing analysis of human multiple myeloma primary cells and cell lines to identify potential lncRNA partners to EZH2. We found that the lncRNA plasmacytoma variant translocation 1 (PVT1) directly interacts with EZH2 and is overexpressed in patients with a poor prognosis. Moreover, genes predicted to be targets of PVT1 exhibited H3K27me3 enrichment and were associated with pro-apoptotic and tumor suppressor functions. In fact, PVT1 inhibition independently promotes the expression of the PRC2 target genes ZBTB7C, RNF144A and CCDC136. Altogether, our work suggests that PVT1 is an interacting partner in PRC2-mediated silencing of tumor suppressor and pro-apoptotic genes in multiple myeloma, making it a highly interesting potential therapeutic target.

JAK2V617F leads to intrinsic changes in platelet formation and reactivity in a knock-in mouse model of essential thrombocythemia.

The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.

Quantification and Profiling of Early and Late Differentiation Stage T Cells in Mantle Cell Lymphoma Reveals Immunotherapeutic Targets in Subsets of Patients.

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue (n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (TH) cells and cytotoxic (TC) cells of early (CD57-) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. TC and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ TC-cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL.

Environmentally induced DNA methylation is inherited across generations in an aquatic keystone species.

Transgenerational inheritance of environmentally induced epigenetic marks can have significant impacts on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. We used whole-genome bisulfite sequencing of individual water fleas (Daphnia magna) to assess whether environmentally induced DNA methylation is transgenerationally inherited. Genetically identical females were exposed to one of three natural stressors, or a de-methylating drug, and their offspring were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) in F1 individuals whose mothers were exposed to a natural stressor. Roughly half of these environmentally induced DMPs persisted until generation F4. In contrast, treatment with the drug demonstrated that pervasive hypomethylation upon exposure is reset almost completely after one generation. These results suggest that environmentally induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of freshwater communities.

Genetic associations at regulatory phenotypes improve fine-mapping of causal variants for 12 immune-mediated diseases.

The resolution of causal genetic variants informs understanding of disease biology. We used regulatory quantitative trait loci (QTLs) from the BLUEPRINT, GTEx and eQTLGen projects to fine-map putative causal variants for 12 immune-mediated diseases. We identify 340 unique loci that colocalize with high posterior probability (≥98%) with regulatory QTLs and apply Bayesian frameworks to fine-map associations at each locus. We show that fine-mapping credible sets derived from regulatory QTLs are smaller compared to disease summary statistics. Further, they are enriched for more functionally interpretable candidate causal variants and for putatively causal insertion/deletion (INDEL) polymorphisms. Finally, we use massively parallel reporter assays to evaluate candidate causal variants at the ITGA4 locus associated with inflammatory bowel disease. Overall, our findings suggest that fine-mapping applied to disease-colocalizing regulatory QTLs can enhance the discovery of putative causal disease variants and enhance insights into the underlying causal genes and molecular mechanisms.

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.

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.

Critical parameters from a generalized multifractal analysis at the Anderson transition.

We propose a generalization of multifractal analysis that is applicable to the critical regime of the Anderson localization-delocalization transition. The approach reveals that the behavior of the probability distribution of wave function amplitudes is sufficient to characterize the transition. In combination with finite-size scaling, this formalism permits the critical parameters to be estimated without the need for conductance or other transport measurements. Applying this method to high-precision data for wave function statistics obtained by exact diagonalization of the three-dimensional Anderson model, we estimate the critical exponent ν=1.58±0.03.

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.

Corrigendum: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

This corrects the article DOI: 10.1038/ncomms11208.

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.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

An atlas of genetic influences on human blood metabolites.

Genome-wide association scans with high-throughput metabolic profiling provide unprecedented insights into how genetic variation influences metabolism and complex disease. Here we report the most comprehensive exploration of genetic loci influencing human metabolism thus far, comprising 7,824 adult individuals from 2 European population studies. We report genome-wide significant associations at 145 metabolic loci and their biochemical connectivity with more than 400 metabolites in human blood. We extensively characterize the resulting in vivo blueprint of metabolism in human blood by integrating it with information on gene expression, heritability and overlap with known loci for complex disorders, inborn errors of metabolism and pharmacological targets. We further developed a database and web-based resources for data mining and results visualization. Our findings provide new insights into the role of inherited variation in blood metabolic diversity and identify potential new opportunities for drug development and for understanding disease.

Multifractal analysis with the probability density function at the three-dimensional anderson transition.

The probability density function (PDF) for critical wave function amplitudes is studied in the three-dimensional Anderson model. We present a formal expression between the PDF and the multifractal spectrum f(alpha) in which the role of finite-size corrections is properly analyzed. We show the non-Gaussian nature and the existence of a symmetry relation in the PDF. From the PDF, we extract information about f(alpha) at criticality such as the presence of negative fractal dimensions and the possible existence of termination points. A PDF-based multifractal analysis is shown to be a valid alternative to the standard approach based on the scaling of inverse participation ratios.