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Mimitou, E.

Publications and source records attributed to Mimitou, E..

8 recordsLinked to original sources

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

Type 1 interferon remodels normal and neoplastic hematopoiesis in human

Inflammation perturbs evolutionary dynamics of hematopoietic stem cell (HSC) clones in clonal hematopoiesis and myeloid neoplasms. We studied HSCs, progenitors and immune cells from patients with myeloproliferative neoplasm (MPN) at baseline and following interferon- (IFN) treatment, the only MPN therapy to deplete clonal stem cells. We focused on essential thrombocythemia, an informative model of early-phase neoplastic hematopoiesis. We integrated somatic genotyping, transcriptomes, immunophenotyping, and chromatin accessibility across single cells. IFN simultaneously activated HSCs into two polarized states, a lymphoid progenitor expansion associated with an anti-inflammatory state and an IFN-specific inflammatory granulocytic progenitor (IGP) state derived directly from HSCs. The augmented lymphoid differentiation balanced the typical MPN-induced myeloid bias, associated with normalized blood counts. Clonal fitness upon IFN exposure was due to resistance of clonal stem cells to differentiate into IGPs. These results support a paradigm wherein inflammation perturbs clonal dynamics by HSC induction into the precipitous IGP differentiation program. One-Sentence SummaryInflammation accelerates clonal evolution by driving stem cell differentiation into an alternate interferon--induced progenitor state.

cancer biology↗

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

New technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization. However, the sparsity of these measurements and the challenge of integrating multiple binding maps represent significant challenges. Here we introduce scCUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.

genomics↗

Discovery of target genes and pathways of blood trait loci using pooled CRISPR screens and single cell RNA sequencing

The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects in cis and trans. By leveraging biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptome sequencing, we discovered target genes of noncoding variants for blood trait loci. The closest gene was often the target gene, but this was not always the case. We also identified trans-effects networks of noncoding variants when cis target genes encoded transcription factors, such as GFI1B and NFE2. We observed that GFI1B trans-target genes were enriched for GFI1B binding sites and fine-mapped GWAS variants, and expressed in human bone marrow progenitor cells, suggesting that GFI1B acts as a master regulator of blood traits. This platform will enable massively parallel assays to catalog the target genes of human noncoding variants in both cis and trans.

genomics↗

Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens

The expression of inhibitory immune checkpoint molecules such as PD-L1 is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here we apply ECCITE-seq, a technology which combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1, and leverage our multi-modal data to identify both transcriptional and post-transcriptional modes of regulation. In particular, we discover that the kelch-like protein KEAP1 and the transcriptional activator NRF2, mediate levels of PD-L1 upregulation after IFN{gamma} stimulation. Our results identify a novel mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multi-modal single-cell perturbation screens.

genomics↗

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. In this study, we investigated the response of oligo-conjugated antibodies towards four variables: Concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background and reduces costs. Background signal can account for a major fraction of the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved. Impact statementOligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.

genomics↗

Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

Rapid technological progress in the recent years has allowed the high-throughput interrogation of different types of biomolecules from single cells. Combining several of these readouts into integrated multi-omic assays is essential to comprehensively understand and model cellular processes. Here, we report the development of Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and sgRNAs. We demonstrate the use of ECCITE-seq to directly and efficiently capture sgRNA molecules and measure their effects on gene expression and protein levels, opening the possibility of performing high throughput single cell CRISPR screens with multimodal readout using existing libraries and commonly used vectors. Finally, by utilizing the combined phenotyping of clonotype and cell surface markers in immune cells, we apply ECCITE to study a lymphoma sample to discriminate cells and define molecular signatures of malignant cells within a heterogeneous population.

genomics↗

S1-seq assay for mapping processed DNA ends

During meiosis, the specialized cell division giving rise to gametes, numerous DNA doublestrand breaks (DSBs) are introduced at multiple places throughout the genome by the topoisomerase-like protein Spo11. Homologous recombination, a highly-conserved DSB repair pathway, is employed for their repair and ensures the formation of chiasmata and the proper segregation of homologous chromosomes. In the initial steps of recombination, end resection takes place, wherein Spo11 is endonucleolytically released and the 5'-terminal strands of each DSB are exonucleolytically processed, exposing the ssDNA necessary to identify a homologous repair template. DNA removed by DSB processing is reconstituted by DNA synthesis, which copies genetic information from the intact homologous template. We developed a next-generation sequencing assay, termed S1-seq, to study DNA end resection genome-wide at high spatial resolution during yeast meiotic recombination. The assay relies on the fact that removal of the ssDNA tails of resected DSBs marks the position where resection stopped. Molecular features of resection are revealed by sequencing of these ssDNA-to-dsDNA junctions and comparison to high-resolution Spo11 DSB maps. We describe the experimental and computational methods for S1-seq as applied to meiosis in the SK1 strain of budding yeast Saccharomyces cerevisiae, and discuss how it can also be applied to map DSBs and recombination intermediates.

molecular biology↗