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.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Cell type-specific novel long non-coding RNA and circular RNA in the BLUEPRINT hematopoietic transcriptomes atlas.

Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/.

Bacterial infection remodels the DNA methylation landscape of human dendritic cells.

DNA methylation is an epigenetic mark thought to be robust to environmental perturbations on a short time scale. Here, we challenge that view by demonstrating that the infection of human dendritic cells (DCs) with a live pathogenic bacteria is associated with rapid and active demethylation at thousands of loci, independent of cell division. We performed an integrated analysis of data on genome-wide DNA methylation, histone mark patterns, chromatin accessibility, and gene expression, before and after infection. We found that infection-induced demethylation rarely occurs at promoter regions and instead localizes to distal enhancer elements, including those that regulate the activation of key immune transcription factors. Active demethylation is associated with extensive epigenetic remodeling, including the gain of histone activation marks and increased chromatin accessibility, and is strongly predictive of changes in the expression levels of nearby genes. Collectively, our observations show that active, rapid changes in DNA methylation in enhancers play a previously unappreciated role in regulating the transcriptional response to infection, even in nonproliferating cells.

Transcriptional, epigenetic and metabolic signatures in cardiometabolic syndrome defined by extreme phenotypes.

BACKGROUND: This work is aimed at improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis by generating a multi-omic disease signature. METHODS/RESULTS: We combined classic plasma biochemistry and plasma biomarkers with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (morbidly obese and lipodystrophy) and lean individuals to identify the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells. Our analyses showed that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, 6 months after bariatric surgery, revealed the loss of the abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurred via the establishment of novel gene expression landscapes. NETosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention. CONCLUSIONS: We showed that the morbidly obese and lipodystrophy groups, despite some differences, shared a common cardiometabolic syndrome signature. We also showed that this could be used to discriminate, amongst the normal population, those individuals with a higher likelihood of presenting with the disease, even when not displaying the classic features.

Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System.

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-pointTM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Structural study of BSA/poly(ethylene glycol) lipid conjugate complexes.

In this work we report the structural characteristics of bovine serum albumin/poly(ethylene glycol) lipid conjugate (BSA/PEG(2000)-PE) complexes under physiological conditions (37 degrees C and pH 7.4) for particular fractions of BSA to PEG-lipid concentration, c(BSA)/c(PEG)(2000)-PE. Ultraviolet fluorescence spectroscopy (UV) results shown that PEG(2000)-PE is associated to BSA, leading to protein unfolding for fixed c(BSA) = 0.01 wt % and variable c(PEG)(2000)-PE = 0.0015-0.6 wt %. Tryptophan groups on the BSA surface are in contact with the PEG-lipid at c(PEG)(2000)-PE = 0.0015 wt %, while they are exposed to water at c(PEG)(2000)-PE > 0.0015 wt %. Dynamic and static light scattering (DLS and SLS) and small-angle neutron scattering (SANS) point out the existence of individual BSA/PEG-lipid complexes in the system for fixed c(BSA) = 1 wt % and variable c(PEG)(2000)-PE = 0.15-2 wt %. DLS shows that there is only one BSA molecule per protein/PEG-lipid complex, while SLS shows that the PEG-lipid associates to the BSA without promoting aggregation between adjacent protein/polymer-lipid conjugate complexes. SANS was used to show that BSA/PEG(2000)-PE complexes adopt an oblate ellipsoidal shape. Partially unfolded BSA is contained in the core of the oblate ellipsoid, which is surrounded by an external shell containing the PEG(2000)-PE.

Chromosome contacts in activated T cells identify autoimmune disease candidate genes.

BACKGROUND: Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4+ T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes. RESULTS: Within 4 h, activation of CD4+ T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach. CONCLUSIONS: Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.

Platelet function is modified by common sequence variation in megakaryocyte super enhancers.

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.