Single-cell epigenomic variability reveals functional cancer heterogeneity.

BackgroundCell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.ResultsWe develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.ConclusionSingle-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.Peer reviewe

A Flexible Approach for Highly Multiplexed Candidate Gene Targeted Resequencing

We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies

Induction and transcriptional regulation of the co-inhibitory gene module in T cells

Expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated co-inhibitory receptor expression on CD4+ T cells promotes autoimmunity while sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and cancer1,2. Here, we used RNA and protein expression profiling at single-cell resolution to identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity

Heritability enrichment of specifically expressed genes identifies disease-relevant tissues and cell types.

We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals

chromVAR: Inferring transcription factor-associated accessibility from single-cell epigenomic data

Single cell ATAC-seq (scATAC) yields sparse data that makes application of conventional analysis approaches challenging. We developed chromVAR, an R package for analyzing sparse chromatin accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and enables characterization of known and de novo sequence motifs associated with variation in chromatin accessibility

Protocol for single-cell ATAC sequencing using combinatorial indexing in mouse lung adenocarcinoma

Summary: Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples.For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020)

Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions

Objectives: This study sought to answer two questions: (1) what are the characteristics of young Kenyans aged 18-24 who use contraception obtained at pharmacies, and (2) why are pharmacies appealing sources of contraception? Design and setting: This was a mixed-methods study in one peri-urban part of Kwale County, Kenya. Methods included cross-sectional survey (n=740), six focus group discussions, 18 in-depth interviews and 25 key-informant interviews. Quantitative data analysis identified factors pushing young people to pharmacies for modern contraception versus other sources. Qualitative data analysis identified reasons pharmacies were perceived to be appealing to young clients. Participants: Participants were (1) young people aged 18-24 from the study area, including a subset who had recently purchased contraception from a pharmacy; or (2) pharmacy personnel and pharmacy stakeholders. Results: Among surveyed participants who had ever had sexual intercourse and had used modern contraception at last sexual intercourse, 59% obtained it from a pharmacy. In multivariable analysis, participants who used a condom or emergency contraception as well as those living alone were significantly more likely to get contraception from pharmacies. Pharmacies were valued for their convenience, privacy, non-judgmental and personable staff, service speed, as well as predictable and affordable prices. Conclusions: Our findings indicate a high percentage of young people in Coastal Kenya use pharmacies for contraception. Our inclusion of emergency contraception users partially explains this. Pharmacies were perceived to be everything that health facilities are not: fast, private and non-limiting. Policy-makers should recognise the role of pharmacies as contraception providers and look for opportunities to link pharmacies to the public health system. This would create a network of accessible and appealing contraception services for young people. Keywords: community child health; public health; qualitative research; reproductive medicine