Selective requirement for polycomb repressor complex 2 in the generation of specific hypothalamic neuronal subtypes.

The hypothalamus displays staggering cellular diversity, chiefly established during embryogenesis by the interplay of several signalling pathways and a battery of transcription factors. However, the contribution of epigenetic cues to hypothalamus development remains unclear. We mutated the polycomb repressor complex 2 gene Eed in the developing mouse hypothalamus, which resulted in the loss of H3K27me3, a fundamental epigenetic repressor mark. This triggered ectopic expression of posteriorly expressed regulators (e.g. Hox homeotic genes), upregulation of cell cycle inhibitors and reduced proliferation. Surprisingly, despite these effects, single cell transcriptomic analysis revealed that most neuronal subtypes were still generated in Eed mutants. However, we observed an increase in glutamatergic/GABAergic double-positive cells, as well as loss/reduction of dopamine, hypocretin and Tac2-Pax6 neurons. These findings indicate that many aspects of the hypothalamic gene regulatory flow can proceed without the key H3K27me3 epigenetic repressor mark, but points to a unique sensitivity of particular neuronal subtypes to a disrupted epigenomic landscape.

Cytocipher determines significantly different populations of cells in single-cell RNA-seq data.

MOTIVATION: Identification of cell types using single-cell RNA-seq is revolutionizing the study of multicellular organisms. However, typical single-cell RNA-seq analysis often involves post hoc manual curation to ensure clusters are transcriptionally distinct, which is time-consuming, error-prone, and irreproducible. RESULTS: To overcome these obstacles, we developed Cytocipher, a bioinformatics method and scverse compatible software package that statistically determines significant clusters. Application of Cytocipher to normal tissue, development, disease, and large-scale atlas data reveals the broad applicability and power of Cytocipher to generate biological insights in numerous contexts. This included the identification of cell types not previously described in the datasets analysed, such as CD8+ T cell subtypes in human peripheral blood mononuclear cells; cell lineage intermediate states during mouse pancreas development; and subpopulations of luminal epithelial cells over-represented in prostate cancer. Cytocipher also scales to large datasets with high-test performance, as shown by application to the Tabula Sapiens Atlas representing >480 000 cells. Cytocipher is a novel and generalizable method that statistically determines transcriptionally distinct and programmatically reproducible clusters from single-cell data. AVAILABILITY AND IMPLEMENTATION: The software version used for this manuscript has been deposited on Zenodo (https://doi.org/10.5281/zenodo.8089546), and is also available via github (https://github.com/BradBalderson/Cytocipher).

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

ChIP-R: Assembling reproducible sets of ChIP-seq and ATAC-seq peaks from multiple replicates.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the primary protocol for detecting genome-wide DNA-protein interactions, and therefore a key tool for understanding transcriptional regulation. A number of factors, including low specificity of antibody and cellular heterogeneity of sample, may cause "peak" callers to output noise and experimental artefacts. Statistically combining multiple experimental replicates from the same condition could significantly enhance our ability to distinguish actual transcription factor binding events, even when peak caller accuracy and consistency of detection are compromised. We adapted the rank-product test to statistically evaluate the reproducibility from any number of ChIP-seq experimental replicates. We demonstrate over a number of benchmarks that our adaptation "ChIP-R" (pronounced 'chipper') performs as well as or better than comparable approaches on recovering transcription factor binding sites in ChIP-seq peak data. We also show ChIP-R extends to evaluate ATAC-seq peaks, finding reproducible peak sets even at low sequencing depth. ChIP-R decomposes peaks across replicates into "fragments" which either form part of a peak in a replicate, or not. We show that by re-analysing existing data sets, ChIP-R reconstructs reproducible peaks from fragments with enhanced biological enrichment relative to current strategies.

Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure.

Immune cells cycle between a resting and an activated state. Their metabolism is tightly linked to their activation status and, consequently, functions. Ag recognition induces T lymphocyte activation and proliferation and acquisition of effector functions that require and depend on cellular metabolic reprogramming. Likewise, recognition of pathogen-associated molecular patterns by monocytes and macrophages induces changes in cellular metabolism. As obligate intracellular parasites, viruses manipulate the metabolism of infected cells to meet their structural and functional requirements. For example, HIV-induced changes in immune cell metabolism and redox state are associated with CD4(+) T cell depletion, immune activation, and inflammation. In this review, we highlight how HIV modifies immunometabolism with potential implications for cure research and pathogenesis of comorbidities observed in HIV-infected patients, including those with virologic suppression. In addition, we highlight recently described key methods that can be applied to study the metabolic dysregulation of immune cells in disease states.

Systematic analysis of the transcriptional landscape of melanoma reveals drug-target expression plasticity.

Metastatic melanoma originates from melanocytes of the skin. Melanoma metastasis results in poor treatment prognosis for patients and is associated with epigenetic and transcriptional changes that reflect the developmental program of melanocyte differentiation from neural crest stem cells. Several studies have explored melanoma transcriptional heterogeneity using microarray, bulk and single-cell RNA-sequencing technologies to derive data-driven models of the transcriptional-state change which occurs during melanoma progression. No study has systematically examined how different models of melanoma progression derived from different data types, technologies and biological conditions compare. Here, we perform a cross-sectional study to identify averaging effects of bulk-based studies that mask and distort apparent melanoma transcriptional heterogeneity; we describe new transcriptionally distinct melanoma cell states, identify differential co-expression of genes between studies and examine the effects of predicted drug susceptibilities of different cell states between studies. Importantly, we observe considerable variability in drug-target gene expression between studies, indicating potential transcriptional plasticity of melanoma to down-regulate these drug targets and thereby circumvent treatment. Overall, observed differences in gene co-expression and predicted drug susceptibility between studies suggest bulk-based transcriptional measurements do not reliably gauge heterogeneity and that melanoma transcriptional plasticity is greater than described when studies are considered in isolation.

Integrating Single-Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation.

The dermis and epidermis, crucial structural layers of the skin, encompass appendages, hair follicles (HFs), and intricate cellular heterogeneity. However, an integrated spatiotemporal transcriptomic atlas of embryonic skin has not yet been described and would be invaluable for studying skin-related diseases in humans. Here, single-cell and spatial transcriptomic analyses are performed on skin samples of normal and hairless fetal pigs across four developmental periods. The cross-species comparison of skin cells illustrated that the pig epidermis is more representative of the human epidermis than mice epidermis. Moreover, Phenome-wide association study analysis revealed that the conserved genes between pigs and humans are strongly associated with human skin-related diseases. In the epidermis, two lineage differentiation trajectories describe hair follicle (HF) morphogenesis and epidermal development. By comparing normal and hairless fetal pigs, it is found that the hair placode (Pc), the most characteristic initial structure in HFs, arises from progenitor-like OGN+/UCHL1+ cells. These progenitors appear earlier in development than the previously described early Pc cells and exhibit abnormal proliferation and migration during differentiation in hairless pigs. The study provides a valuable resource for in-depth insights into HF development, which may serve as a key reference atlas for studying human skin disease etiology using porcine models.

Robust mapping of spatiotemporal trajectories and cell-cell interactions in healthy and diseased tissues.

Spatial transcriptomics (ST) technologies generate multiple data types from biological samples, namely gene expression, physical distance between data points, and/or tissue morphology. Here we developed three computational-statistical algorithms that integrate all three data types to advance understanding of cellular processes. First, we present a spatial graph-based method, pseudo-time-space (PSTS), to model and uncover relationships between transcriptional states of cells across tissues undergoing dynamic change (e.g. neurodevelopment, brain injury and/or microglia activation, and cancer progression). We further developed a spatially-constrained two-level permutation (SCTP) test to study cell-cell interaction, finding highly interactive tissue regions across thousands of ligand-receptor pairs with markedly reduced false discovery rates. Finally, we present a spatial graph-based imputation method with neural network (stSME), to correct for technical noise/dropout and increase ST data coverage. Together, the algorithms that we developed, implemented in the comprehensive and fast stLearn software, allow for robust interrogation of biological processes within healthy and diseased tissues.

Spatial transcriptomic analysis of Sonic hedgehog medulloblastoma identifies that the loss of heterogeneity and promotion of differentiation underlies the response to CDK4/6 inhibition.

BACKGROUND: Medulloblastoma (MB) is a malignant tumour of the cerebellum which can be classified into four major subgroups based on gene expression and genomic features. Single-cell transcriptome studies have defined the cellular states underlying each MB subgroup; however, the spatial organisation of these diverse cell states and how this impacts response to therapy remains to be determined. METHODS: Here, we used spatially resolved transcriptomics to define the cellular diversity within a sonic hedgehog (SHH) patient-derived model of MB and show that cells specific to a transcriptional state or spatial location are pivotal for CDK4/6 inhibitor, Palbociclib, treatment response. We integrated spatial gene expression with histological annotation and single-cell gene expression data from MB, developing an analysis strategy to spatially map cell type responses within the hybrid system of human and mouse cells and their interface within an intact brain tumour section. RESULTS: We distinguish neoplastic and non-neoplastic cells within tumours and from the surrounding cerebellar tissue, further refining pathological annotation. We identify a regional response to Palbociclib, with reduced proliferation and induced neuronal differentiation in both treated tumours. Additionally, we resolve at a cellular resolution a distinct tumour interface where the tumour contacts neighbouring mouse brain tissue consisting of abundant astrocytes and microglia and continues to proliferate despite Palbociclib treatment. CONCLUSIONS: Our data highlight the power of using spatial transcriptomics to characterise the response of a tumour to a targeted therapy and provide further insights into the molecular and cellular basis underlying the response and resistance to CDK4/6 inhibitors in SHH MB.

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.

Glucose metabolism regulates T cell activation, differentiation, and functions.

The adaptive immune system is equipped to eliminate both tumors and pathogenic microorganisms. It requires a series of complex and coordinated signals to drive the activation, proliferation, and differentiation of appropriate T cell subsets. It is now established that changes in cellular activation are coupled to profound changes in cellular metabolism. In addition, emerging evidence now suggest that specific metabolic alterations associated with distinct T cell subsets may be ancillary to their differentiation and influential in their immune functions. The "Warburg effect" originally used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis for their growth is a key process that sustain T cell activation and differentiation. Here, we review how different aspects of metabolism in T cells influence their functions, focusing on the emerging role of key regulators of glucose metabolism such as HIF-1α. A thorough understanding of the role of metabolism in T cell function could provide insights into mechanisms involved in inflammatory-mediated conditions, with the potential for developing novel therapeutic approaches to treat these diseases.