Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Identification of astrocyte regulators by nucleic acid cytometry.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

MAFG-driven astrocytes promote CNS inflammation.

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA.

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger.

Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre- or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.

Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.

Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

Genetic dissection of chlorate respiration in Pseudomonas stutzeri†PDA reveals syntrophic (per)chlorate reduction.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

Chlorate reduction in Shewanella algae†ACDC is a recently acquired metabolism characterized by gene loss, suboptimal regulation and oxidative stress.

Previous work on respiratory chlorate reduction has biochemically identified the terminal reductase ClrABC and the chlorite detoxifying enzyme Cld. In Shewanella algae ACDC, genes encoding these enzymes reside on composite transposons whose core we refer to as the chlorate reduction composite transposon interior (CRI). To better understand this metabolism in ACDC, we used RNA-seq and proteomics to predict carbon and electron flow during chlorate reduction and posit that formate is an important electron carrier with lactate as the electron donor, but that NADH predominates on acetate. Chlorate-specific transcription of electron transport chain components or the CRI was not observed, but clr and cld transcription was attenuated by oxygen. The major chlorate-specific response related to oxidative stress and was indicative of reactive chlorine species production. A genetic system based on rpsL-streptomycin counter selection was developed to further dissect the metabolism, but ACDC readily lost the CRI via homologous recombination of the composite transposon's flanking insertion sequences. An engineered strain containing a single chromosomal CRI did not grow on chlorate, but overexpression of cld and its neighbouring cytochrome c restored growth. We postulate that the recently acquired CRI underwent copy-number expansion to circumvent insufficient expression of key genes in the pathway.

Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.

Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.

FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics.

Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called 'focused interrogation of cells by nucleic acid detection and sequencing' (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence.

Multiomic single cell sequencing identifies stemlike nature of mixed phenotype acute leukemia.

Despite recent work linking mixed phenotype acute leukemia (MPAL) to certain genetic lesions, specific driver mutations remain undefined for a significant proportion of patients and no genetic subtype is predictive of clinical outcomes. Moreover, therapeutic strategy for MPAL remains unclear, and prognosis is overall poor. We performed multiomic single cell profiling of 14 newly diagnosed adult MPAL patients to characterize the inter- and intra-tumoral transcriptional, immunophenotypic, and genetic landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. Despite this, we find that MPAL blasts express a shared stem cell-like transcriptional profile indicative of high differentiation potential. Patients with the highest differentiation potential demonstrate inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in the most stem-like MPAL cells, is applicable to bulk RNA sequencing data and is predictive of survival in an independent patient cohort, suggesting a potential strategy for clinical risk stratification.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

Fe(II) oxidation is an innate capability of nitrate-reducing bacteria that involves abiotic and biotic reactions.

Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous iron [Fe(II)] coupled to nitrate (NO(3)(-)) reduction, often referred to as nitrate-dependent iron oxidation (NDFO). Very little is known about the biochemistry of NDFO, and though growth benefits have been observed, mineral encrustations and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest that the induction of specific Fe(II) oxidoreductase proteins is not required for NDFO. No upregulated periplasmic or outer membrane redox-active proteins, like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic photoferrotrophs, were observed in proteomic experiments. We demonstrate that while "abiotic" extracellular reactions between Fe(II) and biogenic NO(2)(-)/NO can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic components are essential to initiate extensive NDFO. We present evidence that an organic cosubstrate inhibits NDFO, likely by keeping periplasmic enzymes in their reduced state, stimulating metal efflux pumping, or both, and that growth during NDFO relies on the capacity of a nitrate-reducing bacterium to overcome the toxicity of Fe(II) and reactive nitrogen species. On the basis of our data and evidence in the literature, we postulate that all respiratory nitrate-reducing bacteria are innately capable of catalyzing NDFO. Our findings have implications for a mechanistic understanding of NDFO, the biogeochemical controls on anaerobic Fe(II) oxidation, and the production of NO(2)(-), NO, and N(2)O in the environment.

Multiomic Single Cell Sequencing Identifies Stemlike Nature of Mixed Phenotype Acute Leukemia and Provides Novel Risk Stratification.

Mixed phenotype acute leukemia (MPAL) is a leukemia whose biologic drivers are poorly understood, therapeutic strategy remains unclear, and prognosis is poor. We performed multiomic single cell (SC) profiling of 14 newly diagnosed adult MPAL patients to characterize the immunophenotypic, genetic, and transcriptional landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. However, progressive acquisition of mutations is associated with increased expression of immunophenotypic markers of immaturity. Using SC transcriptional profiling, we find that MPAL blasts express a stem cell-like transcriptional profile distinct from other acute leukemias and indicative of high differentiation potential. Further, patients with the highest differentiation potential demonstrated inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in this cohort, is applicable to bulk RNA sequencing data and was predictive of survival in an independent patient cohort, suggesting utility for clinical risk stratification.

Microfluidics-free single-cell genomics with templated emulsification.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Identification of a perchlorate reduction genomic island with novel regulatory and metabolic genes.

A comparative analysis of the genomes of four dissimilatory (per)chlorate-reducing bacteria has revealed a genomic island associated with perchlorate reduction. In addition to the characterized metabolic genes for perchlorate reductase and chlorite dismutase, the island contains multiple conserved uncharacterized genes possibly involved in electron transport and regulation.