Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Loss of the Vitamin B-12 Transport Protein Tcn2 Results in Maternally Inherited Growth and Developmental Defects in Zebrafish.

BACKGROUND: In humans, vitamin B-12 (cobalamin) transport involves 3 paralogous proteins: transcobalamin, haptocorrin, and intrinsic factor. Zebrafish (Danio rerio) express 3 genes that encode proteins homologous to known B-12 carrier proteins: tcn2 (a transcobalamin ortholog) and 2 atypical ß-domain-only homologs, tcnba and tcnbb. OBJECTIVES: Given the orthologous relation between zebrafish Tcn2 and human transcobalamin, we hypothesized that zebrafish carrying null mutations of tcn2 would exhibit phenotypes consistent with vitamin B-12 deficiency. METHODS: First-generation and second-generation tcn2-/- zebrafish were characterized using phenotypic assessments, metabolic analyses, viability studies, and transcriptomics. RESULTS: Homozygous tcn2-/- fish produced from a heterozygous cross are viable and fertile but exhibit reduced growth, which persists into adulthood. When first-generation female tcn2-/- fish are bred, their offspring exhibit gross developmental and metabolic defects. These phenotypes are observed in all offspring from a tcn2-/- female regardless of the genotype of the male mating partner, suggesting a maternal effect, and can be rescued with vitamin B-12 supplementation. Transcriptome analyses indicate that offspring from a tcn2-/- female exhibit expression profiles distinct from those of offspring from a tcn2+/+ female, which demonstrate dysregulation of visual perception, fatty acid metabolism, and neurotransmitter signaling pathways. CONCLUSIONS: Our findings suggest that the deposition of vitamin B-12 in the yolk by tcn2-/- females may be insufficient to support the early development of their offspring. These data present a compelling model to study the effects of vitamin B-12 deficiency on early development, with a particular emphasis on transgenerational effects and gene-environment interactions.

Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution.

Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility and other factors. Current methods for defining chromatin states cannot measure more than one aspect in a single experiment at single-cell resolution. Here we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), an assay capable of measuring the genome-wide presence of up to three histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq uses recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk-cell and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in human immune cells. We also extend NTT-seq to enable simultaneous profiling of cell surface protein expression and multimodal chromatin states to study cells of the immune system.