Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity.

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here, we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. By integrating patterns of repressive chromatin deposited across diverse cell types with weighted density estimation, TRIAGE-Cluster determines cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method which evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

Integrating single-cell genomics pipelines to discover mechanisms of stem cell differentiation.

Pluripotent stem cells underpin a growing sector that leverages their differentiation potential for research, industry, and clinical applications. This review evaluates the landscape of methods in single-cell transcriptomics that are enabling accelerated discovery in stem cell science. We focus on strategies for scaling stem cell differentiation through multiplexed single-cell analyses, for evaluating molecular regulation of cell differentiation using new analysis algorithms, and methods for integration and projection analysis to classify and benchmark stem cell derivatives against in vivo cell types. By discussing the available methods, comparing their strengths, and illustrating strategies for developing integrated analysis pipelines, we provide user considerations to inform their implementation and interpretation.

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.

Conserved Epigenetic Regulatory Logic Infers Genes Governing Cell Identity.

Determining genes that orchestrate cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive trimethylation of histone H3 at lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, which uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate the performance of TRIAGE in identifying cell-type-specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this paper's transparent peer review process is included in the Supplemental Information.