Cell Type-specific Transcription Research Articles

From genetic variants to diseases: the role of LACC1 in T cells

Abstract Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with the expression of particular genes. But how these eQTLs directly affect gene expression and in what cell types of the immune system they act remain largely unknown. One hypothesis is that functional eQTLs in the cis-regulatory elements may affect cell type-specific transcription factor binding, thus reducing gene expression and even contributing to the cause of autoimmune and inflammatory diseases. Previously, we identified T cell-specific eQTLs of a gene known as laccase domain containing 1 (LACC1), which are GWAS hits for Crohn’s disease. Four of them are located in the LACC1 promoter region in the same haploblock. Direct association of disease-risk variants with lower LACC1 expression was confirmed by comparing pre-mRNA quantity of the different alleles in LACC1 heterozygous human CD4 T cells. We further validated the functional role of the eQTLs using CRISPR Cas9-mediated base-pair editing. LACC1 function has been analyzed primarily in macrophages and shown to affect their function, but the GWAS hits do not affect expression in macrophages. Moreover, in our in vitro studies, human CD4 T cells with LACC1 knockdown showed altered metabolic programming and proliferation, which may contribute to inflammatory diseases. Overall, our study provides an excellent model for analyzing and explaining how genetic variants contribute to diseases by influencing gene expression in specific cell types and changing cell functions.

Cell Type-Specific Transcriptional Control of Gsk3β in the Developing Mammalian Neocortex.

Temporal control of neurogenesis is central for the development and evolution of species-specific brain architectures. The balance between progenitor expansion and neuronal differentiation is tightly coordinated by cell-intrinsic and cell-extrinsic cues. Wnt signaling plays pivotal roles in the proliferation and differentiation of neural progenitors in a temporal manner. However, regulatory mechanisms that adjust intracellular signaling amplitudes according to cell fate progression remain to be elucidated. Here, we report the transcriptional controls of Gsk3β, a critical regulator of Wnt signaling, in the developing mouse neocortex. Gsk3β expression was higher in ventricular neural progenitors, while it gradually declined in differentiated neurons. We identified active cis-regulatory module (CRM) of Gsk3β that responded to cell type-specific transcription factors, such as Sox2, Sox9, and Neurogenin2. Furthermore, we found extensive conservation of the CRM among mammals but not in non-mammalian amniotes. Our data suggest that a mammalian-specific CRM drives the cell type-specific activity of Gsk3β to fine tune Wnt signaling, which contributes to the tight control of neurogenesis during neocortical development.

TADreg: a versatile regression framework for TAD identification, differential analysis and rearranged 3D genome prediction

Background/AimIn higher eukaryotes, the three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression, DNA repair and DNA replication regulations. Alteration of 3D organization, in particular topologically associating domains (TADs), is detrimental to the organism and can give rise to a broad range of diseases such as cancers.MethodsHere, we propose a versatile regression framework which not only identifies TADs in a fast and accurate manner, but also detects differential TAD borders across conditions for which few methods exist, and predicts 3D genome reorganization after chromosomal rearrangement. Moreover, the framework is biologically meaningful, has an intuitive interpretation and is easy to visualize.Result and conclusionThe novel regression ranks among top TAD callers. Moreover, it identifies new features of the genome we called TAD facilitators, and that are enriched with specific transcription factors. It also unveils the importance of cell-type specific transcription factors in establishing novel TAD borders during neuronal differentiation. Lastly, it compares favorably with the state-of-the-art method for predicting rearranged 3D genome.

An essential function for autocrine hedgehog signaling in epithelial proliferation and differentiation in the trachea.

ABSTRACTThe tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.

Spatzie: an R package for identifying significant transcription factor motif co-enrichment from enhancer-promoter interactions.

Genomic interactions provide important context to our understanding of the state of the genome. One question is whether specific transcription factor interactions give rise to genome organization. We introduce spatzie, an R package and a website that implements statistical tests for significant transcription factor motif cooperativity between enhancer–promoter interactions. We conducted controlled experiments under realistic simulated data from ChIP-seq to confirm spatzie is capable of discovering co-enriched motif interactions even in noisy conditions. We then use spatzie to investigate cell type specific transcription factor cooperativity within recent human ChIA-PET enhancer–promoter interaction data. The method is available online at https://spatzie.mit.edu.

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

An important characteristic of cell differentiation is its stability. Only rarely do cells or their stem cell progenitors change their differentiation pathway. If they do, it is often accompanied by a malfunction such as cancer. A mechanistic understanding of the stability of differentiated states would allow better prospects of alleviating the malfunctioning. However, such complete information is yet elusive. Earlier experiments performed in Xenopus oocytes to address this question suggest that a cell may maintain its gene expression by prolonged binding of cell type-specific transcription factors. Here, using DNA competition experiments, we show that the stability of gene expression in a nondividing cell could be caused by the local entrapment of part of the general transcription machinery in transcriptionally active regions. Strikingly, we found that transcriptionally active and silent forms of the same DNA template can stably coexist within the same nucleus. Both DNA templates are associated with the gene-specific transcription factor Ascl1, the core factor TBP2, and the polymerase II (Pol-II) ser5 C-terminal domain (CTD) phosphorylated form, while Pol-II ser2 CTD phosphorylation is restricted to the transcriptionally dominant template. We discover that the active and silent DNA forms are physically separated in the oocyte nucleus through partition into liquid-liquid phase-separated condensates. Altogether, our study proposes a mechanism of transcriptional regulation involving a spatial entrapment of general transcription machinery components to stabilize the active form of a gene in a nondividing cell.

Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.

An Optimized Protocol for the Mapping of Cell Type-Specific Ribosome-Associated Transcript Isoforms from Small Mouse Brain Regions.

Over the past years, technological advances in transcriptomics provided deep insights into gene expression programs and their role in tissue organization and cellular functions. The isolation of ribosome-associated transcripts is a powerful approach for deep profiling of cell type-specific transcripts, and particularly well-suited for quantitative analysis of transcript isoforms. This method employs conditional ribosome epitope-tagging in genetically defined cell types, followed by affinity-isolation of ribosome-associated mRNAs. Advantages of this approach are twofold: first, the method enables rapid retrieval of mRNAs without tissue dissociation and cell sorting steps. Second, capturing of ribosome-associated mRNAs, enriches for transcripts recruited for active translation, therefore providing an approximation to the cellular translatome. Here, we describe one application of this method for the identification of the transcriptome of excitatory neuronal cells (mitral and tufted cells) of the mouse olfactory bulb, through RiboTag isolation from the vGlut2-IRES-cre mouse line as genetic driver of endogenously tagged ribosome expression.

Integration of high-resolution promoter profiling assays reveals novel, cell type-specific transcription start sites across 115 human cell and tissue types.

Accurate transcription start site (TSS) annotations are essential for understanding transcriptional regulation and its role in human disease. Gene collections such as GENCODE contain annotations for tens of thousands of TSSs, but not all of these annotations are experimentally validated nor do they contain information on cell type–specific usage. Therefore, we sought to generate a collection of experimentally validated TSSs by integrating RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) data from 115 cell and tissue types, which resulted in a collection of approximately 50 thousand representative RAMPAGE peaks. These peaks are primarily proximal to GENCODE-annotated TSSs and are concordant with other transcription assays. Because RAMPAGE uses paired-end reads, we were then able to connect peaks to transcripts by analyzing the genomic positions of the 3′ ends of read mates. Using this paired-end information, we classified the vast majority (37 thousand) of our RAMPAGE peaks as verified TSSs, updating TSS annotations for 20% of GENCODE genes. We also found that these updated TSS annotations are supported by epigenomic and other transcriptomic data sets. To show the utility of this RAMPAGE rPeak collection, we intersected it with the NHGRI/EBI genome-wide association study (GWAS) catalog and identified new candidate GWAS genes. Overall, our work shows the importance of integrating experimental data to further refine TSS annotations and provides a valuable resource for the biological community.

Localization of IL-1α Responsive Dermal Populations in an Ex Vivo Human Model of Wound Healing

While playing a critical role in skin wound healing, the inflammatory phase of this process is poorly understood. To gain a better understanding of the inflammatory phase of wound healing, we developed an ex vivo skin culture model of skin injury-induced inflammation. Previous work in our laboratory showed ex vivo culture of human skin induces an interleukin 1 alpha (IL-1α)-dependent response characterized by increased transcript and protein levels for the inflammatory cytokines/chemokines, IL-6, CXCL1, and CSF3. However, the cellular sources of these factors in ex vivo cultured human skin have not been determined. Prior work with ex vivo cultured mouse skin and single cell RNA sequencing suggested fibroblasts and endothelial cells were potential cellular sources for these inflammatory mediators. The current studies used spatial transcriptomics analysis of ex vivo cultured human skin to localize the IL-1α target cell populations/skin tissue regions that produce IL-6, CXCL1 and CSF3. The Visium Gene Expression Solution platform (10x Genomics Inc.) was used to generate spatial transcriptomics data from skin specimens preserved immediately after biopsy or after skin culture for 24 hours. Loupe Browser version 5.1.0 (10x Genomics Inc) was used for data analysis to identify and characterize cell populations/regions expressing IL6, CXCL1, and CSF3 and associated differentially expressed genes (including cell type-specific transcripts). Notably, these IL-1α-induced transcripts were localized to the parent dermis region cluster. Analysis of subclusters in the dermal region showed differential expression of these inflammatory transcripts in regions enriched with either or both fibroblast and endothelial cell specific-type markers. Potential novel markers of this inflammatory response, like SOD2, were identified and warrant future investigation. Subsequent studies in identifying the targets of IL-1α in skin inflammation is called for, as they may lead to better understanding of this processes in wound healing and better clinical outcomes.

Single-Cell Regulatory Network Inference and Clustering Identifies Cell-Type Specific Expression Pattern of Transcription Factors in Mouse Sciatic Nerve.

Advances in single-cell RNA sequencing technologies and bioinformatics methods allow for both the identification of cell types in a complex tissue and the large-scale gene expression profiling of various cell types in a mixture. In this report, we analyzed a single-cell RNA sequencing (scRNA-seq) dataset for the intact adult mouse sciatic nerve and examined cell-type specific transcription factor expression and activity during peripheral nerve homeostasis. In total, we identified 238 transcription factors expressed in nine different cell types of intact mouse sciatic nerve. Vascular smooth muscle cells have the lowest number of transcription factors expressed with 17 transcription factors identified. Myelinating Schwann cells (mSCs) have the highest number of transcription factors expressed, with 61 transcription factors identified. We created a cell-type specific expression map for the identified 238 transcription factors. Our results not only provide valuable information about the expression pattern of transcription factors in different cell types of adult peripheral nerves but also facilitate future studies to understand the function of key transcription factors in the peripheral nerve homeostasis and disease.

Linking GATA2 Deficiency to Dysregulated Innate Immune Signaling

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivo system, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/- E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8 in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnf expression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4 efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnf and Ccl3 expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnf and Ccl3 expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Differential transcript usage analysis of bulk and single-cell RNA-seq data with DTUrtle.

MotivationEach year, the number of published bulk and single-cell RNA-seq datasets is growing exponentially. Studies analyzing such data are commonly looking at gene-level differences, while the collected RNA-seq data inherently represents reads of transcript isoform sequences. Utilizing transcriptomic quantifiers, RNA-seq reads can be attributed to specific isoforms, allowing for analysis of transcript-level differences. A differential transcript usage (DTU) analysis is testing for proportional differences in a gene’s transcript composition, and has been of rising interest for many research questions, such as analysis of differential splicing or cell-type identification.ResultsWe present the R package DTUrtle, the first DTU analysis workflow for both bulk and single-cell RNA-seq datasets, and the first package to conduct a ‘classical’ DTU analysis in a single-cell context. DTUrtle extends established statistical frameworks, offers various result aggregation and visualization options and a novel detection probability score for tagged-end data. It has been successfully applied to bulk and single-cell RNA-seq data of human and mouse, confirming and extending key results. In addition, we present novel potential DTU applications like the identification of cell-type specific transcript isoforms as biomarkers.Availability and implementationThe R package DTUrtle is available at https://github.com/TobiTekath/DTUrtle with extensive vignettes and documentation at https://tobitekath.github.io/DTUrtle/.Supplementary information Supplementary data are available at Bioinformatics online.

Methods for the Differential Analysis of Hi-C Data.

The 3D organization of chromatin within the nucleus enables dynamic regulation and cell type-specific transcription of the genome. This is true at multiple levels of resolution: on a large scale, with chromosomes occupying distinct volumes (chromosome territories); at the level of individual chromatin fibers, which are organized into compartmentalized domains (e.g., Topologically Associating Domains-TADs), and at the level of short-range chromatin interactions between functional elements of the genome (e.g., enhancer-promoter loops).The widespread availability of Chromosome Conformation Capture (3C)-based high-throughput techniques has been instrumental in advancing our knowledge of chromatin nuclear organization. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D interactions, as it is theoretically able to detect any pair of restriction fragments connected as a result of ligation by proximity.This chapter will illustrate how to compare the chromatin interactome in different experimental conditions, starting from pre-computed Hi-C contact matrices, how to visualize the results, and how to correlate the observed variations in chromatin interaction strength with changes in gene expression.

Sleep loss drives acetylcholine- and somatostatin interneuron–mediated gating of hippocampal activity to inhibit memory consolidation

Sleep loss disrupts consolidation of hippocampus-dependent memory. To characterize effects of learning and sleep loss, we quantified activity-dependent phosphorylation of ribosomal protein S6 (pS6) across the dorsal hippocampus of mice. We find that pS6 is enhanced in dentate gyrus (DG) following single-trial contextual fear conditioning (CFC) but is reduced throughout the hippocampus after brief sleep deprivation (SD; which disrupts contextual fear memory [CFM] consolidation). To characterize neuronal populations affected by SD, we used translating ribosome affinity purification sequencing to identify cell type-specific transcripts on pS6 ribosomes (pS6-TRAP). Cell type-specific enrichment analysis revealed that SD selectively activated hippocampal somatostatin-expressing (Sst+) interneurons and cholinergic and orexinergic hippocampal inputs. To understand the functional consequences of SD-elevated Sst+ interneuron activity, we used pharmacogenetics to activate or inhibit hippocampal Sst+ interneurons or cholinergic input from the medial septum. The activation of either cell population was sufficient to disrupt sleep-dependent CFM consolidation by gating activity in granule cells. The inhibition of either cell population during sleep promoted CFM consolidation and increased S6 phosphorylation among DG granule cells, suggesting their disinhibition by these manipulations. The inhibition of either population across post-CFC SD was insufficient to fully rescue CFM deficits, suggesting that additional features of sleeping brain activity are required for consolidation. Together, our data suggest that state-dependent gating of DG activity may be mediated by cholinergic input and local Sst+ interneurons. This mechanism could act as a sleep loss-driven inhibitory gate on hippocampal information processing.

ANANSE: an enhancer network-based computational approach for predicting key transcription factors in cell fate determination.

Proper cell fate determination is largely orchestrated by complex gene regulatory networks centered around transcription factors. However, experimental elucidation of key transcription factors that drive cellular identity is currently often intractable. Here, we present ANANSE (ANalysis Algorithm for Networks Specified by Enhancers), a network-based method that exploits enhancer-encoded regulatory information to identify the key transcription factors in cell fate determination. As cell type-specific transcription factors predominantly bind to enhancers, we use regulatory networks based on enhancer properties to prioritize transcription factors. First, we predict genome-wide binding profiles of transcription factors in various cell types using enhancer activity and transcription factor binding motifs. Subsequently, applying these inferred binding profiles, we construct cell type-specific gene regulatory networks, and then predict key transcription factors controlling cell fate transitions using differential networks between cell types. This method outperforms existing approaches in correctly predicting major transcription factors previously identified to be sufficient for trans-differentiation. Finally, we apply ANANSE to define an atlas of key transcription factors in 18 normal human tissues. In conclusion, we present a ready-to-implement computational tool for efficient prediction of transcription factors in cell fate determination and to study transcription factor-mediated regulatory mechanisms. ANANSE is freely available at https://github.com/vanheeringen-lab/ANANSE.

Abstract 2681: Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms

Abstract Co-detection of RNA and protein can greatly expand the data output from a single specimen, providing critical information such as the source of secreted proteins (e.g. cytokines) or cell type specific transcript levels. MultiOmyx is a proprietary immunofluorescence (IF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section. RNAscope Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. Combination of MultiOmyx IF with RNAscope Multiplex ISH would therefore provide a novel and powerful platform to co-detect multiple RNA and protein markers in a single slide/sample. However, the RNAscope Multiplex assay includes a protease pretreatment step, which may compromise downstream antibody-antigen interaction and thereby the IF signal. This study is a validation of the sensitivity, specificity, reproducibility, and repeatability of an integrated MultiOmyx IF and RNAscope ISH assay. Depending on the context, cytokines interleukin-10 (IL10) and interferon gamma (IFNγ) have both been shown to either induce immunosuppression and favor tumor growth or promote an anti-tumor response. The mechanisms and cues determining the pro- or anti-tumor activity of IL10 and IFNγ are still poorly understood. Spatiotemporal characterization of IFNG and IL10 is therefore critical to help define the dynamic relationship of cytokines and the immune system within the tumor microenvironment. Therefore, for validation of the integrated MultiOmyx-RNAscope platform, RNA ISH markers for IL10 and IFNγ were combined with the MultiOmyx 12 marker tumor infiltrating lymphocyte (TIL) panel (CD3, CD4, CD8, CD20, CD68, CD56, CD45RO, PD-1, PD-L1, CTLA4, FOXP3, and tumor marker PanCK) on FFPE human NSCLC samples. This combined MultiOmyx-RNAscope workflow was performed for three individual runs using triplicate NSCLC samples for each run. Intensity and cell classification for each ISH and TIL marker was quantified using the proprietary MultiOmyx Analytics pipeline. The results demonstrate that the integrated assay maintains sensitivity and specificity of the TIL IF markers in the integrated workflow when benchmarked to an IF alone workflow. Furthermore, these results can be used to characterize expression of IL10 and IFNγ within immune cell subsets represented by the TIL IF panel (e.g. T cells, NK cells, macrophages). This integrated approach can be used to spatially correlate the distribution of cytokine and immune cell expression within the tumor microenvironment. Therefore, the RNAscope-MultiOmyx IF assay provides a robust and powerful platform to simultaneously co-detect RNA with protein IF in a single specimen. Citation Format: Courtney Todorov, Maricel Gozo, Harry Nunns, Erinn Parnell, Judy Kuo, Eric Leones, Mate Nagy, Qingyan Au. Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2681.

Single-Cell Analysis of the Pan-Cancer Immune Microenvironment and scTIME Portal.

Single-cell sequencing opens a new era for the investigation of tumor immune microenvironments (TIME). However, at single-cell resolution, a pan-cancer analysis that addresses the identity and diversity of TIMEs is lacking. Here, we first built a pan-cancer single-cell reference of TIMEs with refined subcell types and recognized new cell type-specific transcription factors. We then presented a pan-cancer view of the common features of the TIME and compared the variation of each immune cell type across patients and tumor types in the aspects of abundance, cell states, and cell communications. We found that the abundance and the cell states of dysfunctional T cells were most variable, whereas those of regulatory T cells were relatively stable. A subset of tumor-associated macrophages (TAM), PLTP + C1QC + TAMs, may regulate the abundance of dysfunctional T cells through cytokine/chemokine signaling. The ligand-receptor communication network of TIMEs was tumor-type specific and dominated by the tumor-enriched immune cells. We additionally developed the single-cell TIME (scTIME) portal (http://scTIME.sklehabc.com) with the scTIME-specific analysis modules and a unified cell annotation. In addition to the immune cell compositions and correlation analysis using refined cell type classifications, the portal also provides cell-cell interaction and cell type-specific gene signature analysis. Our single-cell pan-cancer analysis and scTIME portal will provide more insights into the features of TIMEs, as well as the molecular and cellular mechanisms underlying immunotherapies.

028 Sleep loss disrupts hippocampal memory consolidation via an acetylcholine- and somatostatin interneuron-mediated inhibitory gate

Abstract Introduction Sleep loss profoundly disrupts consolidation of hippocampus-dependent memory. To better characterize effects of learning and sleep loss on the hippocampal circuit, we quantified activity-dependent phosphorylation of ribosomal subunit S6 (pS6) across the dorsal hippocampus of mice. Methods We first measured pS6 throughout the hippocampus after learning (single trial contextual fear conditioning; CFC), and after subsequent sleep or sleep deprivation (SD). To characterize cell populations with activity affected by SD, we used translating ribosome affinity purification (TRAP)-seq to identify cell type-specific transcripts on pS6 ribosomes after SD vs. sleep. We next used pharmacogenetics to mimic the effects of SD, selectively activating hippocampal Sst+ interneurons or cholinergic inputs to hippocampus from the medial septum (MS) while mice slept in the hours following CFC. We also inhibited these neuronal populations to assess effects on memory consolidation. Results We find that pS6 in enhanced in the dentate gyrus (DG) following single-trial CFC, but is reduced throughout the hippocampus after brief SD – a manipulation which disrupts contextual fear memory (CFM) consolidation. Cell type-specific enrichment analysis (CSEA) of these transcripts revealed that hippocampal somatostatin-expressing (Sst+) interneurons, and cholinergic and orexinergic inputs to hippocampus, are selectively activated after SD. We used TRAP targeted to hippocampal Sst+ interneurons to identify cellular mechanisms mediating SD-driven Sst+ interneuron activation. . We find that activation of Sst+ interneurons is sufficient to disrupt CFM consolidation, by gating activity in surrounding pyramidal neurons, while inhibition of Sst+ interneurons enhances memory consolidation. Similarly, pharmacogenetic activation of cholinergic input to hippocampus from the MS disrupted CFM. Inhibition of MS cholinergic neurons promoted CFM consolidation and disinhibited neurons in the DG, increasing pS6 expression among DG granule cells. Conclusion Our data suggest that state-dependent gating of DG activity during SD is mediated by cholinergic input. Together these data provide evidence for an inhibitory gate on hippocampal information processing, which is activated by sleep loss. Support (if any) R01-NS118440 to SJA from NINDS, DP2-MH104119 to SJA from the NIH Director’s Office, and a Human Frontiers Science Program Young Investigator Award

Single cell regulatory landscape of the mouse kidney highlights cellular differentiation programs and disease targets

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development.