Cell Identity Genes Research Articles

KNUCKLES Regulates Floral Meristem Termination by Controlling Auxin Distribution and Cytokinin Activity.

The termination of floral meristem (FM) activity is essential for the normal development of reproductive floral organs. During this process, KNUCKLES (KNU), a C2H2-type zinc finger protein, crucially regulates FM termination by directly repressing the expression of both the stem cell identity gene WUSCHEL (WUS) and the stem cell marker gene CLAVATA3 (CLV3) to abolish the WUS-CLV3 feedback loop required for FM maintenance. In addition, phytohormones auxin and cytokinin are involved in FM regulation. However, whether KNU modulates auxin and cytokinin activities for FM determinacy control remains unclear. Here, we show that the auxin distribution and the cytokinin activity mediated by KNU in Arabidopsis (Arabidopsis thaliana) promote the termination of FM during stage 6 of flower development. Mutation of KNU leads to altered distribution of auxin and cytokinin in the FM of a stage 6 floral bud. Moreover, KNU directly represses the auxin transporter gene PIN-FORMED1 (PIN1) and the cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE7 (IPT7) via mediating H3K27me3 deposition on these two loci to regulate auxin and cytokinin activities. Our study presents a molecular regulatory network that elucidates how the transcriptional repressor KNU integrates and modulates the activities of auxin and cytokinin, thus securing the timed FM termination.

Evidence for dual roles of histone H3 lysine 4 in antagonizing Polycomb group function and promoting target gene expression.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the on/off state of cell identity genes such as the Hox factors. Polycomb group complexes repress target genes, whereas trithorax group complexes are required for their expression. Although trithorax and its orthologs function as methyltransferases specific to histone H3 lysine 4 (H3K4), there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo. Using histone gene replacement in Drosophila, we provide evidence of two key roles for replication-dependent histone H3.2K4 in Polycomb target gene control. First, we found that H3.2K4 mutants mimic H3.2K4me3 in antagonizing methyltransferase activity of the PRC2 Polycomb group complex. Second, we found that H3.2K4 is also required for proper activation of Polycomb targets. We conclude that H3.2K4 directly regulates Polycomb target gene expression.

ScPharm: Identifying Pharmacological Subpopulations of Single Cells for Precision Medicine in Cancers.

Intratumour heterogeneity significantly hinders the efficacy of anticancer therapies. Compared with drug perturbation experiments, which yield pharmacological data at the bulk cell level, single-cell RNA sequencing (scRNA-seq) technology provides a means to capture molecular heterogeneity at single-cell resolution. Here, scPharm is introduced, a computational framework that integrates pharmacological profiles with scRNA-seq data to identify pharmacological subpopulations of cells within a tumour and prioritize tailored drugs. scPharm uses the normalized enrichment scores (NESs) determined from gene set enrichment analysisto assess the distribution of cell identity genes in drug response-determined gene lists. Based on the strong correlation between the NES and drug response at single-cell resolution, scPharm successfully identified the sensitive subpopulations in ER-positive and HER2-positive human breast cancer tissues, revealed dynamic changes in the resistant subpopulation of human PC9 cells treated with erlotinib,and expanded itsability to a mouse model. Its superior performance and computational efficiency are confirmed through comparative evaluations with other single-cell prediction tools. Additionally, scPharm predicted combination drug strategies by gauging compensation or booster effects between drugs and evaluated drug toxicity in healthy cells in the tumour microenvironment. Overall, scPharm provides a novel approach for precision medicine in cancers by revealing therapeutic heterogeneity at single-cell resolution.

Dysregulation of zebrin-II cell subtypes in the cerebellum is a shared feature across polyglutamine ataxia mouse models and patients.

Spinocerebellar ataxia type 7 (SCA7) is a genetic neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. Purkinje cells (PCs) are central to the pathology of ataxias, but their low abundance in the cerebellum underrepresents their transcriptomes in sequencing assays. To address this issue, we developed a PC enrichment protocol and sequenced individual nuclei from mice and patients with SCA7. Single-nucleus RNA sequencing in SCA7-266Q mice revealed dysregulation of cell identity genes affecting glia and PCs. Specifically, genes marking zebrin-II PC subtypes accounted for the highest proportion of DEGs in symptomatic SCA7-266Q mice. These transcriptomic changes in SCA7-266Q mice were associated with increased numbers of inhibitory synapses as quantified by immunohistochemistry and reduced spiking of PCs in acute brain slices. Dysregulation of zebrin-II cell subtypes was the predominant signal in PCs of SCA7-266Q mice and was associated with the loss of zebrin-II striping in the cerebellum at motor symptom onset. We furthermore demonstrated zebrin-II stripe degradation in additional mouse models of polyglutamine ataxia and observed decreased zebrin-II expression in the cerebella of patients with SCA7. Our results suggest that a breakdown of zebrin subtype regulation is a shared pathological feature of polyglutamine ataxias.

Dual PPAR-a/g enhances beta cell identity, preserving islet structure in HF-fed mice.

The pancreas suffers from lipotoxicity, which threatens the survival of pancreatic islets. Dual PPAR-alpha(a)/gamma(g) agonism is a promising method for treating type 2 diabetes. This study evaluated the effects of single PPAR-a and PPAR-g or their combined activation on pancreatic islet remodeling, beta-cell proliferation, identity, and maintenance in an experimental obesity model. Fifty three-month-old mice, randomly divided to receive the control (C) or high-fat (HF) diet for ten weeks, were then redivided for a four-week treatment: C, HF, HF-a (received the PPAR-a agonist), HF-g (PPAR-g agonist pioglitazone), and HF-d (PPAR-a/g agonists). The HF group was overweight, had oral glucose intolerance, showed a proinflammatory adipokine profile, exhibited increased alpha and beta cell masses, and islet gene expression compatible with compromised beta cell proliferation and favored dedifferentiation. All treatments reduced body weight, mitigated oral glucose intolerance, and produced an anti-inflammatory adipokine profile, which rescued islet cytoarchitecture, and beta cell function. Principal component analysis (PCA) revealed a shift in the antiapoptotic gene Bcl2 and beta cell proliferation genes (Pax4 and Neurog3) in HF-a. Conversely, HF-g and HF-d benefited from the upregulation of genes related to beta cell function (Fgf21, Glut2, and Glp1r), identity, and maintenance (Pdx1, Neurod1, Mafa, and Nkx6.1). The HF mice were glucose intolerant, showing islet hypertrophy and low beta cell identity-related genes. In contrast, PPAR activation rescued islet structure, and PCA showed that the PPAR-a/g combination was the most effective treatment because it favored beta cell function, identity, and maintenance-related genes, halting the T2DM spectrum in diet-induced obese mice.

Detecting expressed genes in cell populations at the single-cell level with scGeneXpress.

Determining whether genes are expressed or not remains a challenge in single-cell RNAseq experiments due to their different expression spectra, which are influenced by genetics, the microenvironment and gene length. Current approaches for addressing this issue fail to provide a comprehensive landscape of expressed genes, since they neglect the inherent differences in the expression ranges and distributions of genes. Here, we present scGeneXpress, a method for detecting expressed genes in cell populations of single-cell RNAseq samples based on gene-specific reference distributions. We demonstrate that scGeneXpress accurately detects expressed cell markers and identity genes in 34 human and mouse tissues and can be employed to improve differential expression analysis of single-cell RNAseq data.

Single-cell RNA-seq analysis of rat molars reveals cell identity and driver genes associated with dental mesenchymal cell differentiation

BackgroundThe molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge.ResultsHere, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar.ConclusionsOur results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.

Machine Learning Uncovers Vascular Endothelial Cell Identity Genes by Expression Regulation Features in Single Cells.

Deciphering cell identity genes is pivotal to understanding cell differentiation, development, and many diseases involving cell identity dysregulation. Here, we introduce SCIG, a machine-learning method to uncover cell identity genes in single cells. In alignment with recent reports that cell identity genes are regulated with unique epigenetic signatures, we found cell identity genes exhibit distinctive genetic sequence signatures, e.g., unique enrichment patterns of cis-regulatory elements. Using these genetic sequence signatures, along with gene expression information from single-cell RNA-seq data, enables SCIG to uncover the identity genes of a cell without a need for comparison to other cells. Cell identity gene score defined by SCIG surpassed expression value in network analysis to uncover master transcription factors regulating cell identity. Applying SCIG to the human endothelial cell atlas revealed that the tissue microenvironment is a critical supplement to master transcription factors for cell identity refinement. SCIG is publicly available at https://github.com/kaifuchenlab/SCIG , offering a valuable tool for advancing cell differentiation, development, and regenerative medicine research.

Single-nucleus chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry.

Single-nucleus analysis allows robust cell-type classification and helps to establish relationships between chromatin accessibility and cell-type-specific gene expression. Here, using samples from 92 women of several genetic ancestries, we developed a comprehensive chromatin accessibility and gene expression atlas of the breast tissue. Integrated analysis revealed ten distinct cell types, including three major epithelial subtypes (luminal hormone sensing, luminal adaptive secretory precursor (LASP) and basal-myoepithelial), two endothelial and adipocyte subtypes, fibroblasts, T cells, and macrophages. In addition to the known cell identity genes FOXA1 (luminal hormone sensing), EHF and ELF5 (LASP), TP63 and KRT14 (basal-myoepithelial), epithelial subtypes displayed several uncharacterized markers and inferred gene regulatory networks. By integrating breast epithelial cell gene expression signatures with spatial transcriptomics, we identified gene expression and signaling differences between lobular and ductal epithelial cells and age-associated changes in signaling networks. LASP cells and fibroblasts showed genetic ancestry-dependent variability. An estrogen receptor-positive subpopulation of LASP cells with alveolar progenitor cell state was enriched in women of Indigenous American ancestry. Fibroblasts from breast tissues of women of African and European ancestry clustered differently, with accompanying gene expression differences. Collectively, these data provide a vital resource for further exploring genetic ancestry-dependent variability in healthy breast biology.

Dual roles of histone H3 lysine-4 in antagonizing Polycomb group function and promoting target gene expression.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo . Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

Assessing the Comparative Effectiveness of Upregulation of Beta Cell Identity Genes and Downregulation of Senescence-Associated Markers for Senescence Reversal: A Research Protocol

Assessing the Comparative Effectiveness of Upregulation of Beta Cell Identity Genes and Downregulation of Senescence-Associated Markers for Senescence Reversal: A Research Protocol

Determinants of Chromatin Organization in Aging and Cancer-Emerging Opportunities for Epigenetic Therapies and AI Technology.

This review article critically examines the pivotal role of chromatin organization in gene regulation, cellular differentiation, disease progression and aging. It explores the dynamic between the euchromatin and heterochromatin, coded by a complex array of histone modifications that orchestrate essential cellular processes. We discuss the pathological impacts of chromatin state misregulation, particularly in cancer and accelerated aging conditions such as progeroid syndromes, and highlight the innovative role of epigenetic therapies and artificial intelligence (AI) in comprehending and harnessing the histone code toward personalized medicine. In the context of aging, this review explores the use of AI and advanced machine learning (ML) algorithms to parse vast biological datasets, leading to the development of predictive models for epigenetic modifications and providing a framework for understanding complex regulatory mechanisms, such as those governing cell identity genes. It supports innovative platforms like CEFCIG for high-accuracy predictions and tools like GridGO for tailored ChIP-Seq analysis, which are vital for deciphering the epigenetic landscape. The review also casts a vision on the prospects of AI and ML in oncology, particularly in the personalization of cancer therapy, including early diagnostics and treatment optimization for diseases like head and neck and colorectal cancers by harnessing computational methods, AI advancements and integrated clinical data for a transformative impact on healthcare outcomes.

Senescent adipocytes and type 2 diabetes - current knowledge and perspective concepts.

Among civilization diseases, the number of individuals suffering from type 2 diabetes (T2DM) is expected to increase to more than a billion in less than 20 years, which is associated with, e.g., populational aging, poor diet, sedentary lifestyle, genetic predispositions, and immunological factors. T2DM affects many organs and is characterized by insulin resistance, high glucose levels, and adipocyte dysfunction, which are related to senescence. Although this type of cellular aging has beneficial biological functions, it can also act unfavorable since senescent adipocytes resist apoptosis, enhance cytokine secretion, downregulate cell identity genes, and acquire the senescence-associated secretory phenotype that renders a more oxidative environment. Opposing T2DM is possible via a wide variety of senotherapies, including senolytics and senomorphics; nevertheless, further research is advised to expand therapeutic possibilities and benefits. Consequences that ought to be deeply researched include secretory phenotype, chronic inflammation, increasing insulin resistance, as well as impairment of adipogenesis and functioning of adipocyte cells. Herein, despite reviewing T2DM and fat tissue senescence, we summarized the latest adipocyte-related anti-diabetes solutions and suggested further research directions.

Abstract 4879: Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic lymphoid structures that form in chronically inflamed non-lymphoid tissue. The presence of TLSs in tumors is largely associated with favorable outcomes among patients and also exhibit positive associations with response to immune checkpoint blockade. Although mainly comprised of lymphocytes (B cells and T cells), they are considered a separate entity from lymphocyte aggregates within tissue. Lymphocyte aggregates are fairly routine to identify in H&E stained tissue sections, but delineating TLSs from simple lymphocyte aggregates is subjective and not clearly defined. Additionally, the definition of TLSs is only recently being elucidated, so a clear molecular definition of both TLSs and lymphocyte aggregates is needed. To address this problem, we leveraged open-source spatial transcriptomics (ST) data from 17 samples across 7 cancer types. High resolution H&E images were manually evaluated for lymphocyte aggregates by identifying dense pockets of lymphocytic-appearing nuclei. Suspected aggregates were detected in 10 of 17 samples. We then applied a visium spot-level signature scoring method for 4 well published TLS signatures. Of the spots identified, 62% had enhanced signature expression for at least 2 of the signatures, although most of the incorrect annotations came from the lone gastric cancer sample. Differential expression between a TLS-sig- suspected lymphoid aggregate and a TLS-sig+ region not identified by H&E enriched primarily for B cell identity genes, suggesting B-cell-rich TLSs may not always appear as lymphoid aggregates in histology. [AG1] Strikingly, ST revealed strongly positive enhancements in the TLS signatures, where no annotations were made, and no obvious lymphoid aggregation was present, suggesting manual annotation from H&E is inadequate for identifying TLSs. Together, these results demonstrate that literature derived TLS signatures can correctly identify lymphoid aggregates as TLSs and that cellular and molecular differences between standard lymphoid aggregates and TLSs may be smaller than is currently accepted. Citation Format: Jordan Krull, Anjali Byappanahalli, Yi Jiang, Andrew Gunderson, Qin Ma. Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4879.

Coactivator condensation drives cardiovascular cell lineage specification.

During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.

A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer

Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Regulatory architecture of cell identity genes and housekeeping genes.

Gene regulation and chromosome architecture are intimately linked. Genes with prominent roles in cell identity are often regulated by clusters of enhancer elements. By contrast, a recent study shows housekeeping genes are often regulated through clustering of promoters. We discuss here new regulatory insights for these two types of genes.

Abstract 15295: Myocardin Condensation Drives Cell Lineage Specification During Cardiogenesis

During cardiovascular development, cells make switch-like decisions to activate the expression of new gene programs leading to lineage specification. The mechanisms underlying these decisive choices remain unclear. Myocardin is a cardiomyocyte and smooth muscle cell specific transcriptional coactivator, which is necessary and important for activating cardiovascular genes. We show that Myocardin activates lineage-specific gene programs by concentration-dependent and switch-like formation of nuclear condensates. By modeling the natural changes in Myocardin concentration during development, coupled with quantitative fluorescence microscopy, single-cell resolution reporter assays, and cellular differentiation assays, we demonstrate that condensate formation is directly linked to transcriptional activation and cardiovascular lineage specification. During cardiomyocyte and smooth muscle cell differentiation, the formation of Myocardin condensates precedes activation of cell identity genes and these condensates are present at sites of cell identity gene transcription. Myocardin condensates form, activate gene expression, and specify cell state at critical concentration thresholds, dependent upon the C-terminal disordered region of Myocardin. Disrupting condensate formation by manipulating the sequence of this region abolishes gene activation, which can be rescued by replacing this region with condensate-forming disordered regions from other unrelated proteins. Our work demonstrates that Myocardin condensation at critical concentrations enables switch-like changes in gene expression programs, providing new insights into cardiomyocyte and smooth muscle cell lineage specification during cardiovascular development.

Targeted perturbation of signaling-driven condensates

Biomolecular condensates have emerged as a major organizational principle in the cell. However, the formation, maintenance, and dissolution of condensates are still poorly understood. Transcriptional machinery partitions into biomolecular condensates at key cell identity genes to activate these. Here, we report a specific perturbation of WNT-activated β-catenin condensates that disrupts oncogenic signaling. We use a live-cell condensate imaging method in human cancer cells to discover FOXO and TCF-derived peptides that specifically inhibit β-catenin condensate formation on DNA, perturb nuclear β-catenin condensates in cells, and inhibit β-catenin-driven transcriptional activation and colorectal cancer cell growth. We show that these peptides compete with homotypic intermolecular interactions that normally drive condensate formation. Using this framework, we derive short peptides that specifically perturb condensates and transcriptional activation of YAP and TAZ in the Hippo pathway. We propose a "monomer saturation" model in which short interacting peptides can be used to specifically inhibit condensate-associated transcription in disease.