Cell Transcriptional Programs Research Articles

FBXO38 is dispensable for PD-1 regulation

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

Polo-like kinase 1 inhibition modulates urinary tract smooth muscle contraction and bladder cell transcriptional programs.

The serine/threonine kinase polo-like kinase 1 (PLK1) is a master regulator of cell proliferation and contraction, but its physiological role in the lower urinary tract is unknown. We utilized transcriptomic programs of human bladder smooth muscle cells (hBSMCs), 3D bladder spheroid viability assays, and human ureterovesical junction contractility measurements to elucidate the impacts of PLK1 inhibition. This work reveals PLK1 reduction with the selective inhibitor TAK-960 (500 nM) suppresses high K+-evoked contractions of human urinary smooth muscle ex vivo while decreasing urothelial cell viability. Transcriptomic analysis of hBSMCs treated with TAK-960 shows modulation of cell cycle and contraction pathways, specifically through altered expression of Cys2/His2-type zinc finger transcription factors. In bladder spheroids, PLK1 inhibition also suppresses smooth muscle contraction protein filamin. Taken together, these findings establish PLK1 is a critical governor of urinary smooth muscle contraction and urothelial proliferation with implications for lower urinary tract disorders. Targeting PLK1 pharmacologically may therefore offer therapeutic potential to ameliorate hypercontractility and aberrant growth. Further elucidation of PLK1 signaling networks promises new insights into pathogenesis and much needed treatment advances for debilitating urinary symptoms.

An endogenous retrovirus regulates tumor-specific expression of the immune transcriptional regulator SP140.

Speckled Protein 140 (SP140) is a chromatin reader with critical roles regulating immune cell transcriptional programs, and SP140 splice variants are associated with immune diseases including Crohn's disease, multiple sclerosis, and chronic lymphocytic leukemia. SP140 expression is currently thought to be restricted to immune cells. However, by analyzing human transcriptomic datasets from a wide range of normal and cancer cell types, we found recurrent cancer-specific expression of SP140, driven by an alternative intronic promoter derived from an intronic endogenous retrovirus (ERV). The ERV belongs to the primate-specific LTR8B family and is regulated by oncogenic mitogen-activated protein kinase (MAPK) signaling. The ERV drives expression of multiple cancer-specific isoforms, including a nearly full-length isoform that retains all the functional domains of the full-length canonical isoform and is also localized within the nucleus, consistent with a role in chromatin regulation. In a fibrosarcoma cell line, silencing the cancer-specific ERV promoter of SP140 resulted in increased sensitivity to interferon-mediated cytotoxicity and dysregulation of multiple genes. Our findings implicate aberrant ERV-mediated SP140 expression as a novel mechanism contributing to immune gene dysregulation in a wide range of cancer cells.

A cell type-specific approach to elucidate the role of miR-96 in inner ear hair cells.

Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes. We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs. Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

Primordial germ cell DNA demethylation and development require DNA translesion synthesis

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Lymph node metastasis regulation by peritumoral tonsillar tissue mitochondria-related pathway activation in oropharyngeal cancer.

Immune-related gene expression profiles of peritumoral tonsillar tissues are modified by oropharyngeal cancer (OPC) nodal status. This study explored immunometabolism and immune cell count alterations in peritumoral tonsillar tissue according to OPC nodal status. Microarray data analysis of 27 peritumoral tonsillar tissue samples, using a newly generated mitochondrial metabolism-related gene set comprised of 948 genes, detected 228 differentially expressed genes (DEGs) (206 up- and 22 downregulated) in metastasis-negative cases compared to metastasis-positive ones. REACTOME pathway analysis of the 206 upregulated genes revealed the Toll-like receptor 4 cascade were most enriched. Immune cell proportion analysis using the CIBERSORTx algorithm revealed a significantly higher rate of naïve B cells, but lower rates of regulatory T cells and resting natural killer cells in metastasis-negative cases. Digital spatial profiling of the 6 OPC tissues detected 9 DEGs in the lymphoid regions, in contrast, no DEGs were identified in tumor regions according to nodal status. Cancer cell nests and pair matched normal epithelia mitochondrial DNA (mtDNA) from 5 OPC tissues were analyzed by next generation sequencing for variant detection. However, no significant mtDNA variation was found. This study identified mitochondria-related immune cell transcriptional programs and immune cell profiles associated with OPC lymphatic spread in peritumoral tonsil tissue, further evaluation of which will elucidate targetable immune mechanisms associated with OPC lymphatic dissemination.

Super-Enhancer Dysregulation in Rhabdoid Tumor Cells Is Regulated by the SWI/SNF ATPase BRG1.

Mutations in the SWI/SNF chromatin remodeling complex occur in ~20% of cancers. In rhabdoid tumors defined by loss of the SWI/SNF subunit SMARCB1, dysregulation of enhancer-mediated gene expression is pivotal in driving oncogenesis. Enhancer dysregulation in this setting is tied to retention of the SWI/SNF ATPase BRG1-which becomes essential in the absence of SMARCB1-but precisely how BRG1 contributes to this process remains unknown. To characterize how BRG1 participates in chromatin remodeling and gene expression in SMARCB1-deficient cells, we performed a genome-wide characterization of the impact of BRG1 depletion in multiple rhabdoid tumor cell lines. We find that although BRG1-regulated open chromatin sites are distinct at the locus level, the biological characteristics of the loci are very similar, converging on a set of thematically related genes and pointing to the involvement of the AP-1 transcription factor. The open chromatin sites regulated by BRG1 colocalize with histone-marked enhancers and intriguingly include almost all super-enhancers, revealing that BRG1 plays a critical role in maintaining super-enhancer function in this setting. These studies can explain the essentiality of BRG1 to rhabdoid tumor cell identity and survival and implicate the involvement of AP-1 as a critical downstream effector of rhabdoid tumor cell transcriptional programs.

Remodeling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO

Myeloperoxidase (MPO) is an enzyme that functions in host defense. MPO is released into the vascular lumen by neutrophils during inflammation and may adhere and subsequently penetrate endothelial cells (ECs) coating vascular walls. We show that MPO enters the nucleus of ECs and binds chromatin independently of its enzymatic activity. MPO drives chromatin decondensation at its binding sites and enhances condensation at neighboring regions. It binds loci relevant for endothelial-to-mesenchymal transition (EndMT) and affects the migratory potential of ECs. Finally, MPO interacts with the RNA-binding factor ILF3 thereby affecting its relative abundance between cytoplasm and nucleus. This interaction leads to change in stability of ILF3-bound transcripts. MPO-knockout mice exhibit reduced number of ECs at scar sites following myocardial infarction, indicating reduced neovascularization. In summary, we describe a non-enzymatic role for MPO in coordinating EndMT and controlling the fate of endothelial cells through direct chromatin binding and association with co-factors.

Chromatin state transitions in the Drosophila intestinal lineage identify principles of cell-type specification

Tissue homeostasis relies on rewiring of stem cell transcriptional programs into those of differentiated cells. Here, we investigate changes in chromatin occurring in a bipotent adult stem cells. Combining mapping of chromatin-associated factors with statistical modeling, we identify genome-wide transitions during differentiation in the adult Drosophila intestinal stem cell (ISC) lineage. Active, stem-cell-enriched genes transition to a repressive heterochromatin protein-1-enriched state more prominently in enteroendocrine cells (EEs) than in enterocytes (ECs), in which the histone H1-enriched Black state is preeminent. In contrast, terminal differentiation genes associated with metabolic functions follow a common path from a repressive, primed, histone H1-enriched Black state in ISCs to active chromatin states in EE and EC cells. Furthermore, we find that lineage priming has an important function in adult ISCs, and we identify histone H1 as a mediator of this process. These data define underlying principles of chromatin changes during adult multipotent stem cell differentiation.

The extrafollicular B cell response is a hallmark of childhood idiopathic nephrotic syndrome

The efficacy of the B cell-targeting drug rituximab (RTX) in childhood idiopathic nephrotic syndrome (INS) suggests that B cells may be implicated in disease pathogenesis. However, B cell characterization in children with INS remains limited. Here, using single-cell RNA sequencing, we demonstrate that a B cell transcriptional program poised for effector functions represents the major immune perturbation in blood samples from children with active INS. This transcriptional profile was associated with an extrafollicular B cell response marked by the expansion of atypical B cells (atBCs), marginal zone-like B cells, and antibody-secreting cells (ASCs). Flow cytometry of blood from 13 children with active INS and 24 healthy donors confirmed the presence of an extrafollicular B cell response denoted by the expansion of proliferating RTX-sensitive extrafollicular (CXCR5–) CD21low T-bet+ CD11c+ atBCs and short-lived T-bet+ ASCs in INS. Together, our study provides evidence for an extrafollicular origin for humoral immunity in active INS.

Loss of CFTR function in macrophages alters the cell transcriptional program and delays lung resolution of inflammation.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the CF Transmembrane-conductance Regulator (CFTR) gene. The most severe pathologies of CF occur in the lung, manifesting as chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite increasing knowledge of the CF primary defect and the resulting clinical sequelae, the relationship between the CFTR loss of function and the neutrophilic inflammation remains incompletely understood. Here, we report that loss of CFTR function in macrophages causes extended lung inflammation. After intratracheal inoculation with Pseudomonas aeruginosa, mice with a macrophage-specific Cftr-knockout (Mac-CF) were able to mount an effective host defense to clear the bacterial infection. However, three days post-inoculation, Mac-CF lungs demonstrated significantly more neutrophil infiltration and higher levels of inflammatory cytokines, suggesting that Mac-CF mice had a slower resolution of inflammation. Single-cell RNA sequencing revealed that absence of CFTR in the macrophages altered the cell transcriptional program, affecting the cell inflammatory and immune responses, antioxidant system, and mitochondrial respiration. Thus, loss of CFTR function in macrophages influences cell homeostasis, leading to a dysregulated cellular response to infection that may exacerbate CF lung disease.

AIM2 promotes TH17 cells differentiation by regulating RORγt transcription activity

AIM2 is an interferon-inducible HIN-200 protein family member and is well-documented for its roles in innate immune responses as a DNA sensor. Recent studies have highlighted AIM2's function on regulatory Tcells (Treg) and follicular Tcells (Tfh). However, its involvement in Th17 cell differentiation remains unclear. This study reveals that AIM2 promotes Th17 cell differentiation. AIM2 deficiency decreases IL-17A production and downregulates key Th17 associated proteins (RORγt, IL-1R1, IL-23R). AIM2 is located in the nucleus of Th17 cells, where it interacts with RORγt, enhancing its binding to the Il17a promoter. The absence of AIM2 hinders naive CD4 Tcells from differentiating into functional Th17 cells and from inducing colitis in Rag1-/- mice. This study uncovers AIM2's role as a regulator of Th17 cell transcriptional programming, highlighting its potential as a therapeutic target for Th17 cell-mediated inflammatory diseases.

Dynamic 3D genome reorganization during senescence: defining cell states through chromatin.

Cellular senescence, a cell state characterized by growth arrest and insensitivity to growth stimulatory hormones, is accompanied by a massive change in chromatin organization. Senescence can be induced by a range of physiological signals and pathological stresses and was originally thought to be an irreversible state, implicated in normal development, wound healing, tumor suppression and aging. Recently cellular senescence was shown to be reversible in some cases, with exit being triggered by the modulation of the cell's transcriptional program by the four Yamanaka factors, the suppression of p53 or H3K9me3, PDK1, and/or depletion of AP-1. Coincident with senescence reversal are changes in chromatin organization, most notably the loss of senescence-associated heterochromatin foci (SAHF) found in oncogene-induced senescence. In addition to fixed-cell imaging, chromatin conformation capture and multi-omics have been used to examine chromatin reorganization at different spatial resolutions during senescence. They identify determinants of SAHF formation and other key features that differentiate distinct types of senescence. Not surprisingly, multiple factors, including the time of induction, the type of stress experienced, and the type of cell involved, influence the global reorganization of chromatin in senescence. Here we discuss how changes in the three-dimensional organization of the genome contribute to the regulation of transcription at different stages of senescence. In particular, the distinct contributions of heterochromatin- and lamina-mediated interactions, changes in gene expression, and other cellular control mechanisms are discussed. We propose that high-resolution temporal and spatial analyses of the chromatin landscape during senescence will identify early markers of the different senescence states to help guide clinical diagnosis.

Organization of the human intestine at single-cell resolution

The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.

Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4β7 in conventional T cells while preserving α4β7 in regulatory T cells, with findings suggesting increased β1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4β7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Single-nucleus and bulk RNA sequencing reveal cellular and transcriptional mechanisms underlying lipid dynamics in high marbled pork

Pork is the most consumed meat in the world, and its quality is associated with human health. Intramuscular fat (IMF) deposition (also called marbling) is a key factor positively correlated with various quality traits and lipo-nutritional values of meat. However, the cell dynamics and transcriptional programs underlying lipid deposition in highly marbled meat are still unclear. Here, we used Laiwu pigs with high (HLW) or low (LLW) IMF contents to explore the cellular and transcriptional mechanisms underlying lipid deposition in highly-marbled pork by single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing. The HLW group had higher IMF contents but less drip loss than the LLW group. Lipidomics results revelled the changes of overall lipid classes composition (e.g., glycerolipids including triglycerides, diglycerides, and monoglycerides; sphingolipids including ceramides and monohexose ceramide significantly increased) between HLW and LLW groups. SnRNA-seq revealed nine distinct cell clusters, and the HLW group had a higher percentage of adipocytes (1.40% vs. 0.17%) than the LLW group. We identified 3 subpopulations of adipocytes, including PDE4D+/PDE7B+ (in HLW and LLW), DGAT2+/SCD+ (mostly in HLW) and FABP5+/SIAH1+ cells (mostly in HLW). Moreover, we showed that fibro/adipogenic progenitors could differentiate into IMF cells and contribute to 43.35% of adipocytes in mice. In addition, RNA-seq revealed different genes involved in lipid metabolism and fatty acid elongation. Our study provides new insights into the cellular and molecular signatures of marbling formation; such knowledge may facilitate the development of new strategies to increase IMF deposition and the lipo-nutritional quality of high marbled pork.

Clonally Expanded Cytotoxic CD8 +T cells Target Citrullinated Antigens In ACPA+ Rheumatoid Arthritis

Abstract The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8 +T cells have been described in RA, their role in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8 +T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB +CD8 +subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK +CD8 +memory subpopulation comprises of smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB +CD8 +T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB +CD8 +cells are present in RA synovium. These findings suggest that cytotoxic CD8 +T cells targeting citrullinated antigens have a role in contributing to synovitis and joint tissue destruction in ACPA+ RA. The study was supported by following grants: National Institutes of Health grant R01 AR063676, U19 AI110491, R01 AR078268, and Janssen Biotech, Inc.

Discovery of a molecular clock that controls CD8+ T cell function and exhaustion

Abstract During cancer and chronic viral infections, the persistence of antigen progressively causes CD8+ T cells to differentiate into a dysfunctional PD1+ “exhausted” state, with reduced production of inflammatory cytokines relative to effector cells that form during acute infections. Antigen and costimulation signals activate kinase cascades to induce distinct T cell transcription programs, but how T cells distinguish acute and chronic signals to program the exhausted or effector transcriptional states remains poorly understood. We found that members of the protein kinase C (PKC) family function together as a “molecular clock,” sensing acute or chronic agonism to drive distinct transcriptional programs. Continuous stimulation of PKC induces many features of T cell exhaustion, including a loss of production of the cytokines IFNγ and TNF, upregulation of inhibitory receptors and TOX, and altered expression of the proteins in the AP-1 family. Mechanistically, CD8+ T cells express several different PKC proteins, and chronic agonism of PKC leads to degradation of multiple family members and selective maintenance of only one PKC protein, PKC-η. This “PKC switch” alters downstream signaling to support the transcriptional reprogramming of T cells into a terminally exhausted state. In summary, continuous signaling through PKCs causes changes in the output from these kinases initially at the protein level, driving transcriptional changes downstream of PKC targets in the AP-1 transcription factor family and thus allowing further widespread transcriptional and functional changes that characterize T cell exhaustion. Supported by a Damon Runyon Cancer Research Fellowship (DRG-2358-19) and an NIH training grant, T32-CA009370.

Class switched memory B cell development and transcriptional programming is regulated by EZH2

Abstract The most effective vaccines involve the development of antigen specific memory B cells (MBCs). MBCs make up a heterogenous population derived from germinal center (GC) dependent or independent processes, resulting in primarily immunoglobulin class switched and non-class switched MBCs, respectively. Class switched IgG MBCs are programmed to become plasma cells following a reinfection and non-class switched MBCs are programmed to seed secondary germinal centers. It is not clearly defined what epigenetic factors influence MBC differentiation and programming. EZH2 is a histone methyltransferase that catalyzes H3K27me3, resulting in gene repression. EZH2 has been shown to regulate multiple aspects of B cell differentiation, including development, germinal centers, and plasma cells; however, it is unknown if EZH2 regulates MBC development. To study EZH2, multiple knockout models have been established where EZH2 is conditionally deleted (cEZH2-KO). Here we used the influenza (PR8) model to determine the kinetics of antigen-specific MBC differentiation following a live infection in cEZH2-KO mice. We show that antigen positive MBCs are altered when EZH2 is deleted at days 14 and 39 post infection. Of the MBC subsets, IgG+, CD73+, and DP (PDL2+CD80+) MBCs were reliant on EZH2 for development but not IgM MBCs. These data indicate that extrafollicular/non-GC derived MBC differentiate in an EZH2-independent manner which reveals a distinct gene profile for extrafollicular/non-GC derived MBCs. EZH2 is necessary for repression of key gene regulators which determines the fate of class switched and non-switched MBCs. Overall, this model highlights the role of EZH2-dependent programming regarding MBC development and function. Supported by R01 AI148471 from NIH/NIAID to CDS

Mapping the gene space at single-cell resolution with gene signal pattern analysis

Abstract In single-cell RNA sequencing analysis, several computational methods have been developed to map the cellular state space, but little has been done to map the gene space. A mapping that preserves gene-gene relationships within the dataset is particularly useful for characterizing cellular heterogeneity within cell types, where boundaries between cell subpopulations are often unclear or even arbitrary. Here, we present gene signal pattern analysis, a new paradigm for analyzing single cells. We build a cell-cell graph and design a dictionary of diffusion wavelets, capturing a multiscale view of the cell space. We then transform genes by the dictionary and learn a reduced gene representation. Given the gap in prior research for this problem, we design nine alternative strategies and three benchmarks for evaluating preservation of gene-gene relationships, all of which are outperformed by diffusion wavelet-transformed signals. We also define, calculate, and evaluate localization, a key property of a gene signal on the cellular graph. We demonstrate the utility of gene signal pattern analysis on T cells from a mouse model of peripheral tolerance in skin. The gene space mapping reveals a continuum of gene signals characterized by T cell subtypes and transcriptional programs related to effector function and proliferation. Furthermore, we built a multiscale manifold of 48 melanoma patient samples, demonstrating the ability of our method to characterize differences between responders and non-responders to checkpoint immunotherapy. Together, we show gene signal pattern analysis, through methodology from graph signal processing, spectral graph theory, and machine learning, represents an avenue for future research in scRNA-seq analysis. Gruber Foundation