Search

The alternative use of transcription start sites (TSSs) regulates the production of distinct mRNA isoforms, which have crucial roles in both physiological and pathological contexts. Recent technological advances have provided deeper insights into the role of TSSs in the regulation of co-transcriptional and post-transcriptional RNA processing. We discuss the impact and possible mechanisms of the crosstalk between transcription and co-transcriptional RNA processing, especially 3′-end site selection. Alternative transcription start site usage (ATSS) is a widespread regulatory strategy that enables genes to choose between multiple genomic loci for initiating transcription. This mechanism is tightly controlled during development and is often altered in disease states. In this review, we examine the growing evidence highlighting a role for transcription start sites (TSSs) in the regulation of mRNA isoform selection during and after transcription. We discuss how the choice of transcription initiation sites influences RNA processing and the importance of this crosstalk for cell identity and organism function. We also speculate on possible mechanisms underlying the integration of transcriptional and post-transcriptional processes. Alternative transcription start site usage (ATSS) is a widespread regulatory strategy that enables genes to choose between multiple genomic loci for initiating transcription. This mechanism is tightly controlled during development and is often altered in disease states. In this review, we examine the growing evidence highlighting a role for transcription start sites (TSSs) in the regulation of mRNA isoform selection during and after transcription. We discuss how the choice of transcription initiation sites influences RNA processing and the importance of this crosstalk for cell identity and organism function. We alsospeculate on possible mechanisms underlying the integration of transcriptional and post-transcriptional processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding how cell identity is encoded by the genome and acquired during differentiation is a central challenge in cell biology. I have developed a theoretical framework called EnhancerNet, which models the regulation of cell identity through the lens of transcription factor-enhancer interactions. I demonstrate that autoregulation in these interactions imposes a constraint on the model, resulting in simplified dynamics that can be parameterized from observed cell identities. Despite its simplicity, EnhancerNet recapitulates a broad range of experimental observations on cell identity dynamics, including enhancer selection, cell fate induction, hierarchical differentiation through multipotent progenitor states and direct reprogramming by transcription factor overexpression. The model makes specific quantitative predictions, reproducing known reprogramming recipes and the complex haematopoietic differentiation hierarchy without fitting unobserved parameters. EnhancerNet provides insights into how new cell types could evolve and highlights the functional importance of distal regulatory elements with dynamic chromatin in multicellular evolution. [ABSTRACT FROM AUTHOR]

Abstract The incidence of esophageal adenocarcinoma (EAC) has surged by 600% in recent decades, with a dismal 5-year survival rate of just 15%. Barrett’s esophagus (BE), affecting about 2% of the population, raises the risk of EAC by 40-fold. Despite this, the transcriptomic changes during the BE to EAC progression remain unclear. Our study addresses this gap through comprehensive transcriptomic profiling to identify key mRNA signatures and genomic alterations, such as gene fusions. We performed RNA-sequencing on BE and EAC tissues from 8 individuals, followed by differential gene expression, pathway and network analysis, and gene fusion prediction. We identified mRNA changes during the BE-to-EAC transition and validated our results with single-cell RNA-seq datasets. We observed upregulation of keratin family members in EAC and confirmed increased levels of keratin 14 (KRT14) using immunofluorescence. More differentiated BE marker genes are downregulated during progression to EAC, suggesting undifferentiated BE subpopulations contribute to EAC. We also identified several gene fusions absent in paired BE and normal esophagus but present in EAC. Our findings are critical for the BE-to-EAC transition and have the potential to promote early diagnosis, prevention, and improved treatment strategies for EAC.

Polycomb proteins are a fundamental repressive system that plays crucial developmental roles by orchestrating cell-type-specific transcription programs that govern cell identity. Direct alterations of Polycomb activity are indeed implicated in human pathologies, including developmental disorders and cancer. General Polycomb repression is coordinated by three distinct activities that regulate the deposition of two histone post-translational modifications: tri-methylation of histone H3 lysine 27 (H3K27me3) and histone H2A at lysine 119 (H2AK119ub1). These activities exist in large and heterogeneous multiprotein ensembles consisting of common enzymatic cores regulated by heterogeneous non-catalytic modules composed of a large number of accessory proteins with diverse biochemical properties. Here, we have analyzed the current molecular knowledge, focusing on the functional interaction between the core enzymatic activities and their regulation mediated by distinct accessory modules. This provides a comprehensive analysis of the molecular details that control the establishment and maintenance of Polycomb repression, examining their underlying coordination and highlighting missing information and emerging new features of Polycomb-mediated transcriptional control. The Polycomb machinery is a major repressive system essential for development, stem cell, and tissue homeostasis directly involved in several human pathologies. Tamburri et al. explored the molecular complexity by which different core enzymatic activities and regulatory modules coordinate to establish and maintain repressive domains to control cell transcriptional identities. [ABSTRACT FROM AUTHOR]

The incidence of esophageal adenocarcinoma (EAC) has surged by 600% in recent decades, with a dismal 5-year survival rate of just 15%. Barrett's esophagus (BE), affecting about 2% of the population, raises the risk of EAC by 40-fold. Despite this, the transcriptomic changes during the BE to EAC progression remain unclear. Our study addresses this gap through comprehensive transcriptomic profiling to identify key mRNA signatures and genomic alterations, such as gene fusions. We performed RNA-sequencing on BE and EAC tissues from 8 individuals, followed by differential gene expression, pathway and network analysis, and gene fusion prediction. We identified mRNA changes during the BE-to-EAC transition and validated our results with single-cell RNA-seq datasets. We observed upregulation of keratin family members in EAC and confirmed increased levels of keratin 14 (KRT14) using immunofluorescence. More differentiated BE marker genes are downregulated during progression to EAC, suggesting undifferentiated BE subpopulations contribute to EAC. We also identified several gene fusions absent in paired BE and normal esophagus but present in EAC. Our findings are critical for the BE-to-EAC transition and have the potential to promote early diagnosis, prevention, and improved treatment strategies for EAC. [ABSTRACT FROM AUTHOR]

Background: The human epididymis is poorly studied due to the lack of availability of tissue samples. Our understanding of its structure and function depends upon anatomical and histological observations of archived material. Objectives: Here we used single‐cell RNA sequencing (scRNA‐seq) technologies to elucidate the identity of cells within the human efferent ducts (EDs) and compared them to caput epididymis cells. We also compared the cellularity of primary tissues with those of 2D and 3D (organoid) culture models used for functional studies. Materials and methods: Human epididymis tissue was dissected to separate different anatomical regions and digested to release single cells for processing on the 10X Genomics Chromium platform. Primary human epididymis epithelial (HEE) cells and HEE organoids were grown as described previously and subjected to scRNA‐seq. scRNA‐seq data were processed by standard bioinformatics pipelines and used for comparative analysis. Results: We define the cell types in the EDs which include specialized epithelial cells, connective tissue stromal cells, vascular endothelial cells, smooth muscle cells, and immune cells, but lack basal cells that are seen in the caput epididymis. Furthermore, we identify a sub‐population of epithelial cells which have marker genes found in the bladder and urothelium. Comparative genomics analysis of the 2D and 3D culture models shows cellular identities adapted to the culture environment while still maintaining similarity to the primary tissue. Discussion: Our data suggest that EDs are lined with a transitional epithelium, which like the urothelium is able to stretch and contract depending on luminal volume. This is consistent with its primary role in seminal fluid resorption and sperm concentration. Moreover, we describe the cellularity of models to study the human epididymis epithelium in vitro. Conclusion: Single‐cell RNA‐seq data from the human epididymis make a valuable contribution to our understanding of this highly specialized organ. [ABSTRACT FROM AUTHOR]

Introduction: Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) provide valuable insights into the cellular states of kidney cells. However, the annotation of cell types often requires extensive domain expertise and time-consuming manual curation, limiting scalability and generalizability. To facilitate this process, we tested the performance of five supervised classification methods for automatic cell type annotation. Results: We analyzed publicly available sc/snRNA-seq datasets from five expert-annotated studies, comprising 62,120 cells from 79 kidney biopsy samples. Datasets were integrated by harmonizing cell type annotations across studies. Five different supervised machine learning algorithms (support vector machines, random forests, multilayer perceptrons, k-nearest neighbors, and extreme gradient boosting) were applied to automatically annotate cell types using four training datasets and one testing dataset. Performance metrics, including accuracy (F1 score) and rejection rates, were evaluated. All five machine learning algorithms demonstrated high accuracies, with a median F1 score of 0.94 and a median rejection rate of 1.8 %. The algorithms performed equally well across different datasets and successfully rejected cell types that were not present in the training data. However, F1 scores were lower when models trained primarily on scRNA-seq data were tested on snRNA-seq data. Conclusions: Despite limitations including the number of biopsy samples, our findings demonstrate that machine learning algorithms can accurately annotate a wide range of adult kidney cell types in scRNA-seq/snRNA-seq data. This approach has the potential to standardize cell type annotation and facilitate further research on cellular mechanisms underlying kidney disease.

Adipose-derived stem cells (ADSCs) play an important role in the differential capacity for excess energy storage between upper body abdominal (ABD) adipose tissue (AT) and lower body gluteofemoral (GF) AT. We cultured ADSCs from subcutaneous ABD AT and GF AT isolated from eight women with differential body fat distribution and performed single-cell RNA sequencing. Six populations of ADSCs were identified and segregated according to their anatomical origin. The three ADSC subpopulations in GF AT were characterized by strong cholesterol/fatty acid (FA) storage and proliferation signatures. The two ABD subpopulations, differentiated by higher expression of committed preadipocyte marker genes, were set apart by differential expression of extracellular matrix and ribosomal genes. The last population, identified in both depots, was similar to smooth muscle cells and when individually isolated and cultured in vitro they differentiated less than the other subpopulations. This work provides important insight into the use of ADSC as an in vitro model of adipogenesis and suggests that specific subpopulations of GF-ADSCs contribute to the more robust capacity for GF-AT to expand and grow compared with ABD-AT in women. NEW & NOTEWORTHY: Identification of distinct subpopulations of adipose-derived stem cells (ADSCs) in upper body abdominal subcutaneous (ABD) and lower body gluteofemoral subcutaneous (GF) adipose tissue depots. In ABD-ADSCs, subpopulations are more committed to adipocyte lineage. GF-ADSC subpopulations are enriched for genes involved in lipids and cholesterol metabolism. Similar depot differences were found in stem cell population identified in freshly isolated stoma vascular fraction. The repertoire of ADSCs subpopulations was different in apple-shaped versus pear-shaped women. [ABSTRACT FROM AUTHOR]

Abstract Precision in the establishment and maintenance of cellular identities is crucial for the development of multicellular organisms and requires tight regulation of gene expression. While extensive research has focused on understanding cell type-specific gene activation, the complex mechanisms underlying the transcriptional repression of alternative fates are not fully understood. Here, we provide an overview of the repressive mechanisms involved in cell fate regulation. We discuss the molecular machinery responsible for suppressing alternative fates and highlight the crucial role of sequence-specific transcription factors (TFs) in this process. Depletion of these TFs can result in unwanted gene expression and increased cellular plasticity. We suggest that these TFs recruit cell type-specific repressive complexes to their cis-regulatory elements, enabling them to modulate chromatin accessibility in a context-dependent manner. This modulation effectively suppresses master regulators of alternative fate programs and their downstream targets. The modularity and dynamic behavior of these repressive complexes enables a limited number of repressors to canalize and maintain major and minor cell fate decisions at different stages of development.

Precision in the establishment and maintenance of cellular identities is crucial for the development of multicellular organisms and requires tight regulation of gene expression. While extensive research has focused on understanding cell type-specific gene activation, the complex mechanisms underlying the transcriptional repression of alternative fates are not fully understood. Here, we provide an overview of the repressive mechanisms involved in cell fate regulation. We discuss the molecular machinery responsible for suppressing alternative fates and highlight the crucial role of sequence-specific transcription factors (TFs) in this process. Depletion of these TFs can result in unwanted gene expression and increased cellular plasticity. We suggest that these TFs recruit cell type-specific repressive complexes to their cis-regulatory elements, enabling them to modulate chromatin accessibility in a context-dependent manner. This modulation effectively suppresses master regulators of alternative fate programs and their downstream targets. The modularity and dynamic behavior of these repressive complexes enables a limited number of repressors to canalize and maintain major and minor cell fate decisions at different stages of development. This Review discusses the key role of transcriptional repression in cell fate regulation. It provides an overview of epigenetic mechanisms and transcription factors suppressing undesired gene expression or alternative cell fates and discusses the implications for cellular reprogramming. [ABSTRACT FROM AUTHOR]

Deep and comprehensive molecular analyses have started to pinpoint the genetic and epigenetic differences between α- and β-cells that could underlie the mechanisms defining their identity. The α-cell transcriptome is altered under different diabetic conditions, resulting in decreased expression of some α-cell-enriched genes alongside increased expression of some β-cell-enriched genes, but without reprogramming into insulin (INS)+ cells or activation of other key β-cell-specific genes. Multiple genetic and chemical methods have been used to reprogram mouse α-cells into INS-secreting cells in vivo , highlighting a difference in plasticity between embryonic and adult α-cells. Human α-cells have also been reprogrammed in vitro using PDX1 plus MAFA overexpression into INS-secreting cells that have reduced immunogenicity. Systematic molecular dissection of the mechanisms controlling α-cell reprogramming using an array of different in vitro and in vivo techniques will be necessary to develop efficient diabetes therapies. β-Cell replacement by in situ reprogramming of non-β-cells is a promising diabetes therapy. Following the observation that near-total β-cell ablation in adult mice triggers the reprogramming of pancreatic α-, δ-, and γ-cells into insulin (INS)-producing cells, recent studies are delving deep into the mechanisms controlling adult α-cell identity. Systematic analyses of the α-cell transcriptome and epigenome have started to pinpoint features that could be crucial for maintaining α-cell identity. Using different transgenic and chemical approaches, significant advances have been made in reprogramming α-cells in vivo into INS-secreting cells in mice. The recent reprogramming of human α-cells in vitro is an important step forward that must now be complemented with a comprehensive molecular dissection of the mechanisms controlling α-cell identity. [ABSTRACT FROM AUTHOR]

Epigenetic modifications, including DNA methylation and histone post-translational modifications, intricately regulate gene expression patterns by influencing DNA accessibility and chromatin structure in higher organisms. These modifications are heritable, are independent of primary DNA sequences, undergo dynamic changes during development and differentiation, and are frequently disrupted in human diseases. The reversibility of epigenetic modifications makes them promising targets for therapeutic intervention and drugs targeting epigenetic regulators (e.g., tazemetostat, targeting the H3K27 methyltransferase EZH2) have been applied in clinical therapy for multiple cancers. The NSD family of H3K36 methyltransferase enzymes—including NSD1 (KMT3B), NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1)—are now receiving drug development attention, with the exciting advent of an NSD2 inhibitor (KTX-1001) advancing to Phase I clinical trials for relapsed or refractory multiple myeloma. NSD proteins recognize and catalyze methylation of histone lysine marks, thereby regulating chromatin integrity and gene expression. Multiple studies have implicated NSD proteins in human disease, noting impacts from translocations, aberrant expression, and various dysfunctional somatic mutations. Here, we review the biological functions of NSD proteins, epigenetic cooperation related to NSD proteins, and the accumulating evidence linking these proteins to developmental disorders and tumorigenesis, while additionally considering prospects for the development of innovative epigenetic therapies.

Abstract Introduction Single-cell (SC) gene expression analysis is crucial to dissect the complex cellular heterogeneity of solid tumors, which is one of the main obstacles for the development of effective cancer treatments. Such tumors typically contain a mixture of cells with aberrant genomic and transcriptomic profiles affecting specific sub-populations that might have a pivotal role in cancer progression, whose identification eludes bulk RNA-sequencing approaches. We present scMuffin, an R package that enables the characterization of cell identity in solid tumors on the basis of a various and complementary analyses on SC gene expression data. Results scMuffin provides a series of functions to calculate qualitative and quantitative scores, such as: expression of marker sets for normal and tumor conditions, pathway activity, cell state trajectories, Copy Number Variations, transcriptional complexity and proliferation state. Thus, scMuffin facilitates the combination of various evidences that can be used to distinguish normal and tumoral cells, define cell identities, cluster cells in different ways, link genomic aberrations to phenotypes and identify subtle differences between cell subtypes or cell states. We analysed public SC expression datasets of human high-grade gliomas as a proof-of-concept to show the value of scMuffin and illustrate its user interface. Nevertheless, these analyses lead to interesting findings, which suggest that some chromosomal amplifications might underlie the invasive tumor phenotype and the presence of cells that possess tumor initiating cells characteristics. Conclusions The analyses offered by scMuffin and the results achieved in the case study show that our tool helps addressing the main challenges in the bioinformatics analysis of SC expression data from solid tumors.

A recent report by Chervova, Molliex, et al. shows redundant functions for the transcription factors (TFs) ESRRB and NR5A2 as mitotic bookmarkers in mouse embryonic stem (ES) cells. These occupy some of their target sites in mitotic chromatin, ensuring their robust reactivation after cell division, including markers and regulators of pluripotency. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Recent single-cell transcriptome analyses have defined the molecular correlates of mouse proprioceptor subtypes innervating different muscles and end-organ receptors. Proprioceptor muscle subtypes present distinct molecular profiles according to the identity of the muscle they innervate and express gene programs involved in the control of target selectivity. Transcriptional profiling of proprioceptor receptor subtypes reveals up to seven molecularly distinct muscle spindle afferents, but only a single class of Golgi tendon organs, suggesting a higher need for precisely calibrated spindle feedback. Proprioceptor muscle-type identity becomes apparent concomitantly with muscle innervation, while receptor-type appears to be defined after end-organ innervation, indicating a differential reliance on intrinsic and extrinsic signals. The developmental dynamic of molecular identities suggests that proprioceptors undergo plastic changes in gene expression that reflect the needs of their different functional states. Proprioception, the sense of body position in space, has a critical role in the control of posture and movement. Aside from skin and joint receptors, the main sources of proprioceptive information in tetrapods are mechanoreceptive end organs in skeletal muscle: muscle spindles (MSs) and Golgi tendon organs (GTOs). The sensory neurons that innervate these receptors are divided into subtypes that detect discrete aspects of sensory information from muscles with different biomechanical functions. Despite the importance of proprioceptive neurons in motor control, the developmental mechanisms that control the acquisition of their distinct functional properties and positional identity are not yet clear. In this review, we discuss recent findings on the development of mouse proprioceptor subtypes and challenges in defining them at the molecular and functional level. [ABSTRACT FROM AUTHOR]

Introduction: Single-cell (SC) gene expression analysis is crucial to dissect the complex cellular heterogeneity of solid tumors, which is one of the main obstacles for the development of effective cancer treatments. Such tumors typically contain a mixture of cells with aberrant genomic and transcriptomic profiles affecting specific sub-populations that might have a pivotal role in cancer progression, whose identification eludes bulk RNA-sequencing approaches. We present scMuffin, an R package that enables the characterization of cell identity in solid tumors on the basis of a various and complementary analyses on SC gene expression data. Results: scMuffin provides a series of functions to calculate qualitative and quantitative scores, such as: expression of marker sets for normal and tumor conditions, pathway activity, cell state trajectories, Copy Number Variations, transcriptional complexity and proliferation state. Thus, scMuffin facilitates the combination of various evidences that can be used to distinguish normal and tumoral cells, define cell identities, cluster cells in different ways, link genomic aberrations to phenotypes and identify subtle differences between cell subtypes or cell states. We analysed public SC expression datasets of human high-grade gliomas as a proof-of-concept to show the value of scMuffin and illustrate its user interface. Nevertheless, these analyses lead to interesting findings, which suggest that some chromosomal amplifications might underlie the invasive tumor phenotype and the presence of cells that possess tumor initiating cells characteristics. Conclusions: The analyses offered by scMuffin and the results achieved in the case study show that our tool helps addressing the main challenges in the bioinformatics analysis of SC expression data from solid tumors. [ABSTRACT FROM AUTHOR]

Peripheral immune cells play important roles in the maintenance of systemic and microenvironmental hemostasis. Measurements of circulating blood cells by single-cell RNA sequencing (scRNA-seq) were proposed as one of the routine measures in the clinical biochemistry of haematology. Out of translational challenges, defining precise identities of cell subsets and states is more difficult, due to the complexity of immune cell development, location, regulation, function, and metabolism. It is also a challenge to precisely interpret the clinical significance and impact of each cell identity marker gene panel (ciMGPs). ciMGPs have the potential to advance the understanding of systemic responses to the disease, identify disease-specific biomarkers and define cell heterogeneity. Recently, a large number of peripheral cell subsets and expanding/activating states have been identified and validated for use in the fast developments in clinical single-cell biomedicine. Defining the specificity, measurability and repeatability of cell subsets/states is important for the translation of peripheral scRNA-seq in clinical haematology and biochemistry. The development of standard operating procedures and the performance of clinical trials in large populations of various ages, diseases, and therapies will promote the clinical translation of ciMGPs to measures. Thus, defining cell subset/state identities will provide the multi-dimensional and comprehensive readouts of systemic immune cells, the precision monitoring of immune dynamics, and a deeper understanding of the disease and response to therapy [ABSTRACT FROM AUTHOR]

The role of serum response factor (Srf), a central mediator of actin dynamics and mechanical signaling, in cell identity regulation is debated to be either a stabilizer or a destabilizer. We investigated the role of Srf in cell fate stability using mouse pluripotent stem cells. Despite the fact that serum-containing cultures yield heterogeneous gene expression, deletion of Srf in mouse pluripotent stem cells leads to further exacerbated cell state heterogeneity. The exaggerated heterogeneity is detectible not only as increased lineage priming but also as the developmentally earlier 2C-like cell state. Thus, pluripotent cells explore more variety of cellular states in both directions of development surrounding naïve pluripotency, a behavior that is constrained by Srf. These results support that Srf functions as a cell state stabilizer, providing rationale for its functional modulation in cell fate intervention and engineering. [ABSTRACT FROM AUTHOR]

During development and in several diseases, endothelial cells (EC) can undergo complete endothelial-to-mesenchymal transition (EndoMT or EndMT) to generate endothelial-derived mesenchymal cells. Emerging evidence suggests that ECs can also undergo a partial EndoMT to generate cells with intermediate endothelial- and mesenchymal-character. This partial EndoMT event is transient, reversible, and supports both developmental and pathological angiogenesis. Here, we discuss possible regulatory mechanisms that may control the EndoMT program to dictate whether cells undergo complete or partial mesenchymal transition, and we further consider how these pathways might be targeted therapeutically in cancer.

Cell identity is specified by a core transcriptional regulatory circuitry (CoRC), typically limited to a small set of interconnected cell‐specific transcription factors (TFs). By mining global hepatic TF regulons, we reveal a more complex organization of the transcriptional regulatory network controlling hepatocyte identity. We show that tight functional interconnections controlling hepatocyte identity extend to non‐cell‐specific TFs beyond the CoRC, which we call hepatocyte identity (Hep‐ID)CONNECT TFs. Besides controlling identity effector genes, Hep‐IDCONNECT TFs also engage in reciprocal transcriptional regulation with TFs of the CoRC. In homeostatic basal conditions, this translates into Hep‐IDCONNECT TFs being involved in fine tuning CoRC TF expression including their rhythmic expression patterns. Moreover, a role for Hep‐IDCONNECT TFs in the control of hepatocyte identity is revealed in dedifferentiated hepatocytes where Hep‐IDCONNECT TFs are able to reset CoRC TF expression. This is observed upon activation of NR1H3 or THRB in hepatocarcinoma or in hepatocytes subjected to inflammation‐induced loss of identity. Our study establishes that hepatocyte identity is controlled by an extended array of TFs beyond the CoRC. Synopsis: Hepatocyte identity is controlled by interconnected transcription factors (TFs) extending beyond the core regulatory TF network (CoRC). The extended network comprises thyroid hormone receptor beta whose activation resets CoRC TFs expression and hepatocyte identity in dedifferentiated hepatocytes. Hepatocyte identity TFs of the CoRC are tightly connected to a large set of non‐hepatocyte‐specific TFs that we call Hep‐IDCONNECT TFsHep‐IDCONNECT TFs fine tune hepatocyte identity TF expression in homeostatic basal conditionsIn dedifferentiated hepatocytes, Hep‐IDCONNECT TFs can reset hepatocyte identity as shown for the thyroid hormone receptor beta whose activation induces expression of the CoRC TFs in hepatocarcinoma and in hepatocytes subjected to inflammation‐induced loss of identity. [ABSTRACT FROM AUTHOR]

Peripheral immune cells play important roles in the maintenance of systemic and microenvironmental hemostasis. Measurements of circulating blood cells by single‐cell RNA sequencing (scRNA‐seq) were proposed as one of the routine measures in clinical biochemistry of hematology. Out of translational challenges, defining precise identities of cell subsets and states is more difficult, due to the complexity of immune cell development, location, regulation, function, and metabolism. It is also a challenge to precisely interpret clinical significance and impact of each cell identity marker gene panel (ciMGPs). ciMGPs have potential to advance the understanding of systemic responses of the disease, identify disease‐specific biomarkers, and to define cell heterogeneity. Recently, a large number of peripheral cell subsets and expending/activating states have been identified and validated for use in the fast developments in clinical single cell biomedicine. Defining specificity, measurability, and repeatability of cell subsets/states is important for translation of peripheral scRNA‐seq in clinical hematology and biochemistry. The development of standard operating procedure and performance of clinical trials in large populations at various ages, diseases, and therapies will promote the clinical translation of ciMGPs to measures. Thus, defining cell subset/state identities will provide the multi‐dimensional and comprehensive readouts of systemic immune cells, the precision monitoring of immune dynamics, and deeper‐understanding of the disease and response to therapy. [ABSTRACT FROM AUTHOR]

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells. [ABSTRACT FROM AUTHOR]

DNA methylation (5mC) is an essential epigenetic mark associated with transcriptional silencing. The role of 5mC in transcriptional repression is well established for a few hundred genes through methylation of their promoters. Yet, whether 5mC contributes more broadly to gene expression is an important open question. 5mC removal has recently been associated with the activation of enhancers, opening the possibility that 5mC may globally contribute to the expression of genes defining cell identities. Here, we will review the evidence and molecular mechanisms that link 5mC with the activity of enhancers. We will discuss the spread and amplitude of the potential gene expression changes controlled by 5mC at enhancers, and how these may contribute to the determination of cell identities during development. [ABSTRACT FROM AUTHOR]

Chromatin regulators contribute to the maintenance of the germline transcriptional program. In the absence of SET-2, the Caenorhabditis elegans homolog of the SET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational loss of germline identity, leading to sterility. To identify transcriptional signatures associated with progressive loss of fertility, we performed expression profiling of set-2 mutant germlines across generations. We identify a subset of genes whose misexpression is first observed in early generations, a step we refer to as priming; their misexpression then further progresses in late generations, as animals reach sterility. Analysis of misregulated genes shows that down-regulation of germline genes, expression of somatic transcriptional programs, and desilencing of the X-chromosome are concurrent events leading to loss of germline identity in both early and late generations. Upregulation of transcription factor LIN-15B, the C/EBP homolog CEBP-1, and TGF-β pathway components strongly contribute to loss of fertility, and RNAi inactivation of cebp-1 and TGF-β/Smad signaling delays the onset of sterility, showing they individually contribute to maintenance of germ cell identity. Our approach therefore identifies genes and pathways whose misexpression actively contributes to the loss of germ cell fate. More generally, our data shows how loss of a chromatin regulator in one generation leads to transcriptional changes that are amplified over subsequent generations, ultimately leading to loss of appropriate cell fate.

Summary: Characterizing cell identity in complex tissues such as the human retina is essential for studying its development and disease. While retinal organoids derived from pluripotent stem cells have been widely used to model development and disease of the human retina, there is a lack of studies that have systematically evaluated the molecular and cellular fidelity of the organoids derived from various culture protocols in recapitulating their in vivo counterpart. To this end, we performed an extensive meta-atlas characterization of cellular identities of the human eye, covering a wide range of developmental stages. The resulting map uncovered previously unknown biomarkers of major retinal cell types and those associated with cell-type-specific maturation. Using our retinal-cell-identity map from the fetal and adult tissues, we systematically assessed the fidelity of the retinal organoids in mimicking the human eye, enabling us to comprehensively benchmark the current protocols for retinal organoid generation.

Cell identity is determined by the chromatin structure and profiles of gene expression, which are dependent on chromatin accessibility and DNA methylation of the regions critical for gene expression, such as enhancers and promoters. These epigenetic modifications are required for mammalian development and are essential for the establishment and maintenance of the cellular identity. DNA methylation was once thought to be a permanent repressive epigenetic mark, but systematic analyses in various genomic contexts have revealed a more dynamic regulation than previously thought. In fact, both active DNA methylation and demethylation occur during cell fate commitment and terminal differentiation. To link methylation signatures of specific genes to their expression profiles, we determined the methyl-CpG configurations of the promoters of five genes switched on and off during murine postnatal brain differentiation by bisulfite-targeted sequencing. Here, we report the structure of significant, dynamic, and stable methyl-CpG profiles associated with silencing or activation of the expression of genes during neural stem cell and brain postnatal differentiation. Strikingly, these methylation cores mark different mouse brain areas and cell types derived from the same areas during differentiation. [ABSTRACT FROM AUTHOR]

T cells, with numerous classifications, are vital components of the adaptive immune system, which function to maintain homeostasis to protect against pathogens. With the rapid development of single‐cell RNA sequencing (scRNA‐seq), the specificity and functions of high‐resolution characterizations and identities of T cells are continuously explored and discovered. The exact T cell identities provide new insights for deeply understanding the heterogeneity of T cells and for the identification of previously unrecognized cell subsets. The accuracy and specificity of T cell cluster and annotation are critical and important in scRNA‐seq analyses, even though the characters and numbers of T cell marker gene panels (MGPs) are to be furthermore improved and uncovered. In order to initiate the discussion on identities of T cell subsets/clusters and impacts of identity specificity in the understanding of immune function, the present review systematically summarized the T cell identities of MGPs and functional characteristics of distinct T cell identities in the scRNA‐seq analysis. We also discussed the critical gene differences among panels across T cell subsets, cell functional states, tissue types, and diseases, with a special focus on the significance and potential values of T cell MGP accuracy and specificity in clinical applications. We hope that the precise knowledge of T cell subsets/clusters benefit decision designs and makings of biomarker discoveries and therapeutic strategies to improve the outcomes of patients. [ABSTRACT FROM AUTHOR]

The generation of neuronal diversity involves temporal patterning mechanisms by which a given progenitor sequentially produces multiple cell types. Several parallels are evident between the brain development programs of Drosophila and vertebrates, such as the successive emergence of specific cell types and the use of combinations of transcription factors to specify cell fates. Furthermore, cell-extrinsic cues such as hormones and signaling pathways have also been shown to be regulatory modules of temporal patterning. Recently, transcriptomic and epigenomic studies using large single-cell sequencing datasets have provided insights into the transcriptional dynamics of neurogenesis in the Drosophila and mammalian central nervous systems. We review these commonalities in the specification of neuronal identity and highlight the conserved or convergent strategies of brain development by discussing temporal patterning mechanisms found in flies and vertebrates. [ABSTRACT FROM AUTHOR]

The mammary gland (MG) is composed of three main epithelial lineages, the basal cells (BC), the estrogen receptor (ER) positive luminal cells (ER+ LC), and the ER negative LC (ER− LC). Defining the cell identity of each lineage and how it is modulated throughout the different stages of life is important to understand how these cells function and communicate throughout life. Here, we used transgenic mice specifically labelling ER+ LC combined to cell surface markers to isolate with high purity the 3 distinct cell lineages of the mammary gland and defined their expression profiles and chromatin landscapes by performing bulk RNAseq and ATACseq of these isolated populations in puberty, adulthood and mid-pregnancy. Our analysis identified conserved genes, ligands and transcription factor (TF) associated with a specific lineage throughout life as well as genes, ligands and TFs specific for a particular stage of the MG. In summary, our study identified genes and TF network associated with the identity, function and cell-cell communication of the different epithelial lineages of the MG at different stages of life.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Abstract T cells, with numerous classifications, are vital components of the adaptive immune system, which function to maintain homeostasis to protect against pathogens. With the rapid development of single‐cell RNA sequencing (scRNA‐seq), the specificity and functions of high‐resolution characterizations and identities of T cells are continuously explored and discovered. The exact T cell identities provide new insights for deeply understanding the heterogeneity of T cells and for the identification of previously unrecognized cell subsets. The accuracy and specificity of T cell cluster and annotation are critical and important in scRNA‐seq analyses, even though the characters and numbers of T cell marker gene panels (MGPs) are to be furthermore improved and uncovered. In order to initiate the discussion on identities of T cell subsets/clusters and impacts of identity specificity in the understanding of immune function, the present review systematically summarized the T cell identities of MGPs and functional characteristics of distinct T cell identities in the scRNA‐seq analysis. We also discussed the critical gene differences among panels across T cell subsets, cell functional states, tissue types, and diseases, with a special focus on the significance and potential values of T cell MGP accuracy and specificity in clinical applications. We hope that the precise knowledge of T cell subsets/clusters benefit decision designs and makings of biomarker discoveries and therapeutic strategies to improve the outcomes of patients.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes – evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

Cell fate specification is essential for every major event of embryogenesis, and subsequent cell maturation ensures individual cell types acquire specialized functions. The mechanisms that regulate cell fate specification have been studied exhaustively, and each technological advance in developmental biology ushers in a new era of studies aimed at uncovering the most fundamental processes by which cells acquire unique identities. What is less appreciated is that mechanisms are in place to ensure cell identity is maintained throughout the life of the organism. The body wall musculature in the Drosophila embryo is a well-established model to study cell fate specification, as each hemisegment in the embryo generates and maintains thirty muscles with distinct identities. Once specified, the thirty body wall muscles fuse with mononucleate muscle precursors that lack a specific identity to form multinucleate striated muscles. Multinucleate body wall muscles do not fuse with each other, which maintains a diversification of muscle cell identities. Here we show the serine/threonine kinase Back seat driver (Bsd) prevents inappropriate muscle fusion to maintain cell identity. Thus, the regulation of cell fusion is one mechanism that maintains cell identity. • Back seat driver (Bsd) prevents myotube fusion. • Bsd regulates ERK activity and kirre expression. • Jumeau acts upstream of Bsd and the FGF receptor heartless. [ABSTRACT FROM AUTHOR]

Cell identity is determined by the transcriptional regulation of a cell‐type‐specific gene group. The Paf1 complex (Paf1C), an RNA polymerase II‐associating factor, is an important transcriptional regulator that not only participates in transcription elongation and termination but also affects transcription‐coupled histone modifications and chromatin organisation. Recent studies have shown that Paf1C is involved in the expression of genes required for self‐renewal and pluripotency in stem cells and tumorigenesis. In this review, we focused on the role of Paf1C as a critical transcriptional regulator in cell fate decisions. Paf1C affects the pluripotency of stem cells by regulating the expression of core transcription factors such as Oct4 and Nanog. In addition, Paf1C directly binds to the promoters or distant elements of target genes, thereby maintaining the pluripotency in embryonic stem cells derived from an early stage of the mammalian embryo. Paf1C is upregulated in cancer stem cells, as compared with that in cancer cells, suggesting that Paf1C may be a target for cancer therapy. Interestingly, Paf1C is involved in multiple developmental stages in Drosophila, zebrafish, mice and even humans, thereby displaying a trend for the correlation between Paf1C and cell fate. Thus, we propose that Paf1C is a critical contributor to cell differentiation, cell specification and its characteristics and could be employed as a therapeutic target in developmental diseases. [ABSTRACT FROM AUTHOR]

Mitosis entails global and dramatic alterations, such as higher-order chromatin organization disruption, concomitant with global transcription downregulation. Cells reliably re-establishing gene expression patterns upon mitotic exit and maintaining cellular identities remain poorly understood. Previous studies indicated that certain transcription factors (TFs) remain associated with individual loci during mitosis and serve as mitotic bookmarkers. However, it is unclear which regulatory factors remain bound to the compacted mitotic chromosomes. We developed formaldehyde-assisted isolation of regulatory elements-coupled mass spectrometry (FAIRE-MS) that combines FAIRE-based open chromatin-associated protein pull-down and mass spectrometry (MS) to quantify the open chromatin-associated proteome during the interphase and mitosis. We identified 189 interphase and mitosis maintained (IM) regulatory factors using FAIRE-MS and found intrinsically disordered proteins and regions (IDP(R)s) are highly enriched, which plays a crucial role in liquid-liquid phase separation (LLPS) and chromatin organization during the cell cycle. Notably, in these IDP(R)s, we identified mitotic bookmarkers, such as CEBPB, HMGB1, and TFAP2A, and several factors, including MAX, HMGB3, hnRNP A2/B1, FUS, hnRNP D, and TIAL1, which are at least partially bound to the mitotic chromosome. Furthermore, it will be essential to study whether these IDP(R)s through LLPS helps cells transit from mitosis to the G1 phase during the cell cycle. [ABSTRACT FROM AUTHOR]

Suspension and imaging cytometry techniques that simultaneously measure hundreds of cellular features are powering a new era of cell biology and transforming our understanding of human tissues and tumors. However, a central challenge remains in learning the identities of unexpected or novel cell types. Cell identification rubrics that could assist trainees, whether human or machine, are not always rigorously defined, vary greatly by field, and differentially rely on cell intrinsic measurements, cell extrinsic tissue measurements, or external contextual information such as clinical outcomes. This challenge is especially acute in the context of tumors, where cells aberrantly express developmental programs that are normally time, location, or cell-type restricted. Well-established fields have contrasting practices for cell identity that have emerged from convention and convenience as much as design. For example, early immunology focused on identifying minimal sets of protein features that mark individual, functionally distinct cells. In neuroscience, features including morphology, development, and anatomical location were typical starting points for defining cell types. Both immunology and neuroscience now aim to link standardized measurements of protein or RNA to informative cell functions such as electrophysiology, connectivity, lineage potential, phospho-protein signaling, cell suppression, and tumor cell killing ability. The expansion of automated, machine-driven methods for learning cell identity has further created an urgent need for a harmonized framework for distinguishing cell identity across fields and technology platforms. Here, we compare practices in the fields of immunology and neuroscience, highlight concepts from each that might work well in the other, and propose ways to implement these ideas to study neural and immune cell interactions in brain tumors and associated model systems. [ABSTRACT FROM AUTHOR]

Roots are a highly organised plant tissue consisting of different cell types with distinct developmental functions defined by cell identity networks. Roots are the target of some of the most devastating diseases and possess a highly effective immune system. The recognition of microbe‐ or plant‐derived molecules released in response to microbial attack is highly important in the activation of complex immunity gene networks. Development and immunity are intertwined, and immunity activation can result in growth inhibition. In turn, by connecting immunity and cell identity regulators, cell types are able to launch a cell type‐specific immunity based on the developmental function of each cell type. By this strategy, fundamental developmental processes of each cell type contribute their most basic functions to drive cost‐effective but highly diverse and, thus, efficient immune responses. This review highlights the interdependence of root development and immunity and how the developmental age of root cells contributes to positive and negative outcomes of development‐immunity cross‐talk. [ABSTRACT FROM AUTHOR]

Cellular senescence is implicated in a wide range of physiological and pathological conditions throughout an organism's entire lifetime. In particular, it has become evident that senescence plays a causative role in aging and age-associated disorders. This is not due simply to the loss of function of senescent cells. Instead, the substantial alterations of the cellular activities of senescent cells, especially the array of secretory factors, impact the surrounding tissues or even entire organisms. Such non-cell-autonomous functionality is largely coordinated by tissue-specific genes, constituting a cell fate–determining state. Senescence can be viewed as a gain-of-function phenotype or a process of cell identity shift. Cellular functionality or lineage-specific gene expression is tightly linked to the cell type–specific epigenetic landscape, reinforcing the heterogeneity of senescence across cell types. Here, we aim to define the senescence cellular functionality and epigenetic features that may contribute to the gain-of-function phenotype. [ABSTRACT FROM AUTHOR]

Macrophages are versatile myeloid leukocytes with flexible cellular states to perform diverse tissue functions beyond immunity. This plasticity is however often hijacked by diseases to promote pathology. Scanning kinetics of macrophage states by single-cell transcriptomics and flow cytometry, we observed atopic dermatitis drastically exhausted a resident subtype S1. Characterized by FRβ/CD163 expression, S1 exhibited strong efferocytosis and chemoattracted monocytes and eosinophils. Here we have delineated mechanisms regulating monocyte decision to acquire S1 identity in skin. During M-CSF driven macrophage differentiation in healthy skin, FRβ was expressed via intrinsic control of STAT6 and ALK5 activities, and did not require heterotypic cellular crosstalk. In contrast, CD163 expression required exposure to fibroblastic secretion. This process depended on SHP1 activity and involved STAT5 inactivation. Suppressed STAT5 activity caused CD163 expression and rendered macrophage insensitive to further induction by fibroblasts. Parsing coculture experiments with in silico ligand expression, we identified laminin-α2 and type-V collagen secreted by hypodermal fibroblasts as CD163-driving factors. S1 identity loss in AD followed a stepwise cascade: reduced laminins availability first dampened CD163 expression, IL4 and TGFβ subsequently acted on CD163 lo/- cells to downregulate FRβ. In AD skin, we showed that imitating this fibroblast-macrophage crosstalk with exogenous laminin-211 encouraged monocyte differentiation to S1 macrophages, fostered homeostatic commitment of extravasated eosinophils, and alleviated dermatitis. Hence, we demonstrated that reinforcing a steady-state cue from hypodermal fibroblasts could override maladaptive pressure on macrophage and restored tissue homeostasis., Competing Interests: Declarations. Conflict of interest: KT is a scientific founder, stockholder of and received research funding from StemRIM. ET and YO are employees of StemRIM. All other authors declare they have no competing interests. Human and animal rights: All animal experiments were conducted under the guidelines of and approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine with reference numbers 02-075-003 and 02-075-005. The study did not involve human subjects., (© 2024. The Author(s).)

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28-activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3 + CAR Tregs uniquely acquiring CD40L surface expression and producing IFN-γ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy., Competing Interests: L.M.R.F. is the inventor on a provisional patent based on results from this work, an inventor on provisional and licensed patents on engineered immune cells, and a consultant with Guidepoint Global and McKesson., (© 2024 The Author(s).)

Direct reprogramming involves the conversion of differentiated cell types without returning to an earlier developmental state. Here, we explore how heterogeneity in developmental lineage and maturity of the starting cell population contributes to direct reprogramming using the conversion of murine fibroblasts into neurons. Our hypothesis is that a single lineage of cells contributes to most reprogramming and that a rare elite precursor with intrinsic bias is the source of reprogrammed neurons. We find that nearly all reprogrammed neurons are derived from the neural crest (NC) lineage. Moreover, when rare proliferating NC precursors are selectively ablated, there is a large reduction in the number of reprogrammed neurons. Previous interpretations of this paradigm are that it demonstrates a cell fate conversion across embryonic germ layers (mesoderm to ectoderm). Our interpretation is that this is actually directed differentiation of a neural lineage stem cell in the skin that has intrinsic bias to produce neuronal progeny., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Establishing, maintaining, and removing histone post-translational modifications associated with heterochromatin is critical for shaping genomic structure and function as a cell navigates different stages of development, activity, and disease. Dynamic regulation of the repressive chromatin landscape has been documented in several key cell states - germline cells, activated immune cells, actively replicating, and quiescent cells - with notable variations in underlying mechanisms. Here, we discuss the role of cell states of these diverse contexts in directing and maintaining observed chromatin landscapes. These investigations reveal heterochromatin architectures that are highly responsive to the functional context of a cell's existence and, in turn, their contribution to the cell's stable identity., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aging is often associated with a loss of cell type identity that results in an increase in transcriptional noise in aged tissues. If this phenomenon reflects a fundamental property of aging remains an open question. Transcriptional changes at the cellular level are best detected by single-cell RNA sequencing (scRNAseq). However, the diverse computational methods used for the quantification of age-related loss of cellular identity have prevented reaching meaningful conclusions by direct comparison of existing scRNAseq datasets. To address these issues we created Decibel, a Python toolkit that implements side-to-side four commonly used methods for the quantification of age-related transcriptional noise in scRNAseq data. Additionally, we developed Scallop, a novel computational method for the quantification of membership of single cells to their assigned cell type cluster. Cells with a greater Scallop membership score are transcriptionally more stable. Application of these computational tools to seven aging datasets showed large variability between tissues and datasets, suggesting that increased transcriptional noise is not a universal hallmark of aging. To understand the source of apparent loss of cell type identity associated with aging, we analyzed cell type-specific changes in transcriptional noise and the changes in cell type composition of the mammalian lung. No robust pattern of cell type-specific transcriptional noise alteration was found across aging lung datasets. In contrast, age-associated changes in cell type composition of the lung were consistently found, particularly of immune cells. These results suggest that claims of increased transcriptional noise of aged tissues should be reformulated.