Johanna Klughammer

Single-Cell DNA Methylome Sequencing and Bioinformatic Inference of Epigenomic Cell-State Dynamics

Summary Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples.

Specification of Tissue-resident Macrophages During Organogenesis

INTRODUCTION Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also serve tissue-specific purposes: For example, microglia in the central nervous system support neuronal circuit development, Kupffer cells scavenge blood particles and dying red blood cells in the liver, and alveolar macrophages uptake surfactant and remove airborne pollutants and microbes from the airways. Resident macrophage diversity in adult mice is reflected in tissue-specific gene expression profiles, which may be due to responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types. Altogether, the mechanisms responsible for the generation of tissue-resident macrophage diversity remain unclear. RATIONALE Tissue-resident macrophages originate, at least in part, from mesodermal erythro-myeloid progenitors (EMPs) from the yolk sac, which invade the embryo proper at the onset of organogenesis. These tissue-resident macrophages are also self-maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore hypothesized that resident macrophages represent a founding cell type within most organ anlagen. In this model, the generation of macrophage diversity, as observed in the tissues of postnatal mice, may be integral to organogenesis. RESULTS To test this hypothesis and explore the molecular basis of macrophage diversity in mammals, we performed a spatiotemporal analysis of macrophage development in mice, from embryonic day 9 (E9) to 3 weeks after birth. Unbiased single-cell RNA sequencing (RNA-seq) analysis of CD45+ cells, combined with RNA-seq analyses of sorted cell populations, genetic fate mapping, and in situ analyses, revealed that EMPs give rise to a population of premacrophages (pMacs) that colonize the whole embryo from E9.5, as they acquire a core macrophage differentiation program that includes pattern recognition, scavengers, and cytokine receptors. The chemokine receptor Cx3cr1 is up-regulated in pMacs and is important for embryo colonization, which is delayed in Cx3cr1-deficient embryos. Fate mapping of pMacs using a Tnfrsf11a–Cre reporter labels homogeneously fetal and adult tissue-resident macrophages but not HSCs and their progeny. Transcriptional regulators that identify postnatal tissue-resident macrophages in the brain, liver, kidney, skin, and lung were specifically up-regulated immediately after colonization. These dynamic changes mark the onset of diversification into adult macrophages. We identified Id3 as a Kupffer cell–specific transcriptional regulator. Deletion of Id3 in pMacs resulted in Kupffer cell deficiency but did not affect development of microglia and kidney macrophages. CONCLUSION Our study shows that EMP-derived precursors colonize embryonic tissues and simultaneously acquire a full core macrophage program. This is followed by their diversification into tissue-specific macrophages during organogenesis, likely via the expression of distinct sets of transcriptional regulators. These results indicate that differentiation of tissue-resident macrophages is an integral part of organogenesis and identify a spatiotemporal molecular road map for the generation of macrophage diversity in vivo. Our findings provide a conceptual framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to development, homeostasis, and disease pathogenesis in different tissues and will support efforts to differentiate specialized macrophages in vitro. Specification of tissue-resident macrophages. Erythro-myeloid progenitors (EMPs) from the yolk sac colonize the fetal liver and give rise to macrophage precursors (pMacs) that acquire a core macrophage transcriptional program and colonize the embryo from E9.5 in a Cx3cr1-dependent manner (green arrows). Specification of F4/80+ resident macrophages (brown arrows), starting from E10.25, is initiated by the expression of tissue-specific transcriptional regulators. Id3 (red) is important for Kupffer cell development. Transcription factors noted in blue have been shown to be important for the differentiation or the maintenance of the corresponding macrophage subsets. MΦ, macrophage. Tissue-resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro-myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine-receptor–dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Summary Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types

Pancreatic islets of Langerhans contain several specialized endocrine cell types, which are commonly identified by the expression of single marker genes. However, the established marker genes cannot capture the complete spectrum of cellular heterogeneity in human pancreatic islets, and existing bulk transcriptome datasets provide averages across several cell populations. To dissect the cellular composition of the human pancreatic islet and to establish transcriptomes for all major cell types, we performed single‐cell RNA sequencing on 70 cells sorted from human primary tissue. We used this dataset to validate previously described marker genes at the single‐cell level and to identify specifically expressed transcription factors for all islet cell subtypes. All data are available for browsing and download, thus establishing a useful resource of single‐cell expression profiles for endocrine cells in human pancreatic islets.

Pooled CRISPR screening with single-cell transcriptome readout

CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation

Summary Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases.

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Differential DNA Methylation Analysis without a Reference Genome.

Summary Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

Paternal chronic colitis causes epigenetic inheritance of susceptibility to colitis.

Inflammatory bowel disease (IBD) arises by unknown environmental triggers in genetically susceptible individuals. Epigenetic regulation of gene expression may integrate internal and external influences and may thereby modulate disease susceptibility. Epigenetic modification may also affect the germ-line and in certain contexts can be inherited to offspring. This study investigates epigenetic alterations consequent to experimental murine colitis induced by dextran sodium sulphate (DSS), and their paternal transmission to offspring. Genome-wide methylome- and transcriptome-profiling of intestinal epithelial cells (IECs) and sperm cells of males of the F0 generation, which received either DSS and consequently developed colitis (F0DSS), or non-supplemented tap water (F0Ctrl) and hence remained healthy, and of their F1 offspring was performed using reduced representation bisulfite sequencing (RRBS) and RNA-sequencing (RNA-Seq), respectively. Offspring of F0DSS males exhibited aberrant methylation and expression patterns of multiple genes, including Igf1r and Nr4a2, which are involved in energy metabolism. Importantly, DSS colitis in F0DSS mice was associated with decreased body weight at baseline of their F1 offspring, and these F1 mice exhibited increased susceptibility to DSS-induced colitis compared to offspring from F0Ctrl males. This study hence demonstrates epigenetic transmissibility of metabolic and inflammatory traits resulting from experimental colitis.

Parental micronutrient deficiency distorts liver DNA methylation and expression of lipid genes associated with a fatty-liver-like phenotype in offspring

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.