Mitochondrial genetics through the lens of single-cell multi-omics.

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease.

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

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility.

Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called 'mitochondrial single-cell assay for transposase-accessible chromatin with sequencing' (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3-4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery.

BTBBCL6 dimers as building blocks for reversible drug-induced protein oligomerization.

Here, we characterize the BTB domain of the transcription factor BCL6 (BTBBCL6) as a small-molecule-controlled, reversible oligomerization switch, which oligomerizes upon BI-3802 treatment and de-oligomerizes upon addition of BI-3812. We show that the magnitude of oligomerization can be controlled in vitro by BI-3802 concentration and exposure time. In cellular models, exposure to BI-3802/BI-3812 can drive multiple cycles of foci formation consisting of BTBBCL6 fused to EGFP, which are not degraded due to the lack of a degron. We generated an epidermal growth factor receptor (EGFR)-BTBBCL6 fusion. Treatment with BI-3802, as an ON switch, induced EGFR-BTBBCL6 phosphorylation and activation of downstream effectors, which could in part be reversed by the addition of BI-3812, as an OFF switch. Finally, BI-3802-induced oligomerization of the EGFR-BTBBCL6 fusion enhanced proliferation of an EGF-dependent cell line in absence of EGF. These results demonstrate the successful application of small-molecule-induced, reversible oligomerization as a switch for synthetic biology.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium.

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.