Understanding the temporal dynamics of T-cell transcription is crucial for insights into immune cell function and development. In this study, we show the features of the Timer-of-Cell-Kinetics-and-Activity (Tocky) system, which enables analysis of temporal dynamics of cell activities and differentiation, leveraging Fluorescent Timer protein, which spontaneously changes its emission spectrum from blue to red fluorescence in known kinetics, as reporters. The current study examines the properties of the Tocky system, highlighting the Timer-Angle approach, which is a core algorithm of Tocky analysis and converts Timer Blue and Red fluorescence into Timer Angle and Intensity by trigonometric transformation. Importantly, Tocky analyzes time-related events within individual cells by the two phases of measurements, distinguishing between (1) the temporal sequence of cellular activities and differentiation within the time domain, and (2) the transcription frequency within the frequency domain. The transition from time measurement to frequency analysis, particularly at the Persistent locus that bridges these domains, highlights that system\'s unique property in what is measured and analyzed by Tocky. Intriguingly, the sustained transcriptional activities observed in cells at the Persistent locus may have unique biological features as demonstrated in activated regulatory T-cells (Treg) and pathogenic T-cells, respectively, using Foxp3-Tocky and Nr4a3-Tocky models. In conclusion, the Tocky system can provide crucial data for advancing our understanding of T-cell dynamics and function.

(1) Currently, the survival prognosis for patients with relapsed and refractory acute myeloid leukemia (R/R AML) is extremely poor. Therefore, the exploration of novel drugs is imperative to enhance the prognosis of patients with R/R AML. The therapeutic efficacy and mechanism of Chidamide, a novel epigenetic regulatory drug, in the treatment of R/R AML remain unclear.
The mechanism of action of Chidamide has been explored in various AML cell lines through various methods such as cell apoptosis, cell cycle analysis, high-throughput transcriptome sequencing, gene silencing, and xenograft models.
Here, we have discovered that chidamide potently induces apoptosis, G0/G1 phase arrest, and mitochondrial membrane potential depolarization in R/R AML cells, encompassing both primary cells and cell lines. Through RNA-seq analysis, we further revealed that chidamide epigenetically regulates the upregulation of differentiation-related pathways while suppressing those associated with cell replication and cell cycle progression. Notably, our screening identified NR4A3 as a key suppressor gene whose upregulation by chidamide leads to P21-dependent cell cycle arrest in the G0/G1 phase.
We have discovered a novel epigenetic regulatory mechanism of chidamide in the treatment of relapsed and refractory acute myeloid leukemia (R/R AML).

BACKGROUND: Atrial cardiomyopathy (ACM) is responsible for atrial fibrillation (AF) and thromboembolic events. Diabetes mellitus (DM) is an important risk factor for ACM. However, the potential mechanism between ACM and DM remains elusive.
METHODS: Atrial tissue samples were obtained from patients diagnosed with AF or sinus rhythm (SR) to assess alterations in NR4A3 expression, and then two distinct animal models were generated by subjecting Nr4a3-/- mice and WT mice to a high-fat diet (HFD) and Streptozotocin (STZ), while db/db mice were administered AAV9-Nr4a3 or AAV9-ctrl. Subsequently, in vivo and in vitro experiments were conducted to assess the impact of NR4A3 on diabetes-induced atrial remodeling through electrophysiological, biological, and histological analyses. RNA sequencing (RNA-seq) and metabolomics analysis were employed to unravel the downstream mechanisms.
RESULTS: The expression of NR4A3 was significantly decreased in atrial tissues of both AF patients and diabetic mice compared to their respective control groups. NR4A3 deficiency exacerbated atrial hypertrophy and atrial fibrosis, and increased susceptibility to pacing-induced AF. Conversely, overexpression of NR4A3 alleviated atrial structural remodeling and reduced AF induction rate. Mechanistically, we confirmed that NR4A3 improves mitochondrial energy metabolism and reduces oxidative stress injury by preserving the transcriptional expression of Sdha, thereby exerting a protective influence on atrial remodeling induced by diabetes.
CONCLUSIONS: Our data confirm that NR4A3 plays a protective role in atrial remodeling caused by diabetes, so it may be a new target for treating ACM.
BACKGROUND: This study was supported by the major research program of National Natural Science Foundation of China (NSFC) No: 82370316 (to Q-S. W.), No. 81974041 (to Y-P. W.), and No. 82270447 (to Y-P. W.) and Fundation of Shanghai Hospital Development Center (No. SHDC2022CRD044 to Q-S. W.).

BACKGROUND: Nor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive.
METHODS: We used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy.
RESULTS: Here, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals.
CONCLUSIONS: Our study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

This study aimed to reveal the specific role of early growth response protein 1 (EGR1) and nuclear receptor 4A3 (NR4A3) in nucleus pulposus cells (NPCs) and the related molecular mechanism and to identify a new strategy for treating intervertebral disc degeneration (IVDD). Bioinformatics analysis was used to explore and predict IVDD-related differentially expressed genes, and chromatin immunoprecipitation sequencing (ChIP-seq) revealed NR4A3 as the EGR1 target gene. An in vitro NPC model induced by tributyl hydrogen peroxide (TBHP) and a rat model induced by fibrous ring acupuncture were established. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining, immunofluorescence staining, and flow cytometry were used to detect the effects of EGR1 and NR4A3 knockdown and overexpression on NPC apoptosis and the expression of extracellular matrix (ECM) anabolism-related proteins. Interactions between EGR1 and NR4A3 were analyzed via ChIP-qPCR and dual luciferase assays. EGR1 and NR4A3 expression levels were significantly higher in severely degenerated discs (SDD) than in mildly degenerated discs (MDD), indicating that these genes are important risk factors in IVDD progression. ChIP-seq and RNA-seq revealed NR4A3 as a direct downstream target of EGR1, and this finding was verified by ChIP-qPCR and dual luciferase reporter experiments. Remarkably, the rescue experiments showed that EGR1 promotes TBHP-induced NPC apoptosis and impairs ECM anabolism, dependent on elevated NR4A3 expression. In summary, the EGR1-NR4A3 axis mediates the progression of NPC apoptosis and ECM impairment and is a potential therapeutic target in IVDD.

Despite extensive regulatory T cell (Treg) research, fundamental questions on in vivo dynamics remain to be answered. The current study aims to dissect several interwoven concepts in Treg biology, highlighting the \'self-reactivity\' of Treg and their counterparts, namely naturally-arising memory-phenotype T-cells, as a key mechanism to be exploited by a human retroviral infection. We propose the novel key concept, Periodic T cell receptor (TCR)-signalled T-cells, capturing self-reactivity in a quantifiable manner using the Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky) technology. Periodic and brief TCR signals in self-reactive T-cells contrast with acute TCR signals during inflammation. Thus, we propose a new two-axis model for T-cell activation by the two types of TCR signals or antigen recognition, elucidating how Foxp3 expression and acute TCR signals actively regulate Periodic TCR-signalled T-cells. Next, we highlight an underappreciated branch of immunological research on Human T-cell Leukemia Virus type 1 (HTLV-1) that precedes Treg studies, illuminating the missing link between the viral infection, CD25, and Foxp3. Based on evidence by single-cell analysis, we show how the viral infection exploits the regulatory mechanisms for T-cell activation and suggests a potential role of periodic TCR signalling in infection and malignant transformation. In conclusion, the new perspectives and models in this study provide a working framework for investigating Treg within the self-reactive T-cell spectrum, expected to advance understanding of HTLV-1 infection, cancer, and immunotherapy strategies for these conditions.

The Grey allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the Syntaxin 17 gene. A recent study demonstrated that the most common allele at the Grey locus (G3) involves three tandem copies of this sequence, whilst a more rare allele (G2) has two tandem copies and the wild-type allele (G1) only one copy. The G3 allele is causing fast greying and high incidence of skin melanoma, whereas the G2 allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented in melanoma tissue from Grey horses. Functional studies showed that this intronic sequence acts as a weak melanocyte-specific enhancer that becomes substantially stronger by the copy number expansion. The Grey mutation is associated with upregulated expression of both Syntaxin 17 and the neighbouring NR4A3 gene in Grey horse melanomas. It is still an open question which of these genes is most important for the phenotypic effects or if causality is due to the combined effect of upregulation of both genes. Interestingly, RNAseq data in the Human Protein Atlas give support for a possible role of NR4A3 because it is particularly upregulated in human skin cancer, and it belongs to a cluster of genes associated with skin cancer and melanin biosynthesis. The Grey mutation and its association with melanoma provide a possibility to study the path to tumour development in numerous Grey horses carrying exactly the same predisposing mutation.

Hypoxic-ischemic brain injury induces metabolic dysfunction that ultimately leads to neuronal cell death. Astrocytes, a type of glial cell, play a key role in brain metabolism; however, their response to hypoxic-ischemic brain injury is not fully understood. Microglia were removed from murine primary mixed glial cultures to enrich astrocytes. Next, we explored genes whose expression is altered following oxygen-glucose deprivation using a microarray. Microarray analysis revealed that the expression of Nr4a1 and Nr4a3 is markedly increased in astrocyte-enriched cultures after 15 h of oxygen-glucose deprivation. The expression of both Nr4a1 and Nr4a3 was regulated by HIF-1α. At the protein level, NR4A1 was translocated from the nucleus to the cytoplasm following oxygen-glucose deprivation and co-localized with mitochondria in apoptotic cells; however, its localization was restored to the nucleus after reoxygenation. Oxygen-glucose deprivation causes an increase in NR4A1 mRNA in astrocytes as well as its nuclear to cytoplasmic transfer. Furthermore, reoxygenation enhances NR4A1 transcription and promotes its nuclear translocation.

Recurrent gene fusions have been observed in epithelioid and myxoid variants of uterine leiomyosarcoma. PGR::NR4A3 fusions were recently described in a subset of epithelioid leiomyosarcomas exhibiting rhabdoid morphology. In this study, we sought to expand the clinical, morphologic, immunohistochemical, and genetic features of gynecologic leiomyosarcomas harboring NR4A3 rearrangements with PGR and novel fusion partners. We identified 9 gynecologic leiomyosarcomas harboring PGR::NR4A3, CARMN::NR4A3, ACTB::NR4A3, and possible SLCO5A1::NR4A3 fusions by targeted RNA sequencing. Tumors frequently affected premenopausal women, involving the uterine corpus, uterine cervix, or pelvis. All were similarly characterized by lobules of monomorphic epithelioid and/or spindled cells arranged in sheets, cords, trabeculae, and micro- and macrocysts associated with abundant myxoid matrix and hemorrhage, creating labyrinth-like or pulmonary edema-like architecture. Myogenic differentiation with frequent estrogen receptor and progesterone receptor staining and no CD10 expression characterized all tumors. All cases showed high NR4A3 RNA expression levels and NOR1 (NR4A3) nuclear staining similar to salivary gland acinic cell carcinomas and a subset of extraskeletal myxoid chondrosarcomas harboring NR4A3 rearrangements. NOR1 (NR4A3) immunohistochemistry may serve as a useful diagnostic marker of NR4A3 fusion-positive gynecologic leiomyosarcomas.

Hypercapnia occurs when the partial pressure of carbon dioxide (CO2) in the blood exceeds 45 mmHg. Hypercapnia is associated with several lung pathologies and is transcriptionally linked to suppression of immune and inflammatory signalling through poorly understood mechanisms. Here we propose Orphan Nuclear Receptor Family 4A (NR4A) family members NR4A2 and NR4A3 as potential transcriptional regulators of the cellular response to hypercapnia in monocytes. Using a THP-1 monocyte model, we investigated the sensitivity of NR4A family members to CO2 and the impact of depleting NR4A2 and NR4A3 on the monocyte response to buffered hypercapnia (10% CO2) using RNA-sequencing. We observed that NR4A2 and NR4A3 are CO2-sensitive transcription factors and that depletion of NR4A2 and NR4A3 led to reduced CO2-sensitivity of mitochondrial and heat shock protein (Hsp)-related genes, respectively. Several CO2-sensitive genes were, however, refractory to depletion of NR4A2 and NR4A3, indicating that NR4As regulate certain elements of the cellular response to buffered hypercapnia but that other transcription factors also contribute. Bioinformatic analysis of conserved CO2-sensitive genes implicated several novel putative CO2-sensitive transcription factors, of which the ETS Proto-Oncogene 1 Transcription Factor (ETS-1) was validated to show increased nuclear expression in buffered hypercapnia. These data give significant insights into the understanding of immune responses in patients experiencing hypercapnia.