The phosphatase PAC1 acts as a T cell suppressor and attenuates host antitumor immunity.

Cancer cells subvert immune surveillance through inhibition of T cell effector function. Elucidation of the mechanism of T cell dysfunction is therefore central to cancer immunotherapy. Here, we report that dual specificity phosphatase 2 (DUSP2; also known as phosphatase of activated cells 1, PAC1) acts as an immune checkpoint in T cell antitumor immunity. PAC1 is selectively upregulated in exhausted tumor-infiltrating lymphocytes and is associated with poor prognosis of patients with cancer. PAC1hi effector T cells lose their proliferative and effector capacities and convert into exhausted T cells. Deletion of PAC1 enhances immune responses and reduces cancer susceptibility in mice. Through activation of EGR1, excessive reactive oxygen species in the tumor microenvironment induce expression of PAC1, which recruits the Mi-2ß nucleosome-remodeling and histone-deacetylase complex, eventually leading to chromatin remodeling of effector T cells. Our study demonstrates that PAC1 is an epigenetic immune regulator and highlights the importance of targeting PAC1 in cancer immunotherapy.

Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIß (Topo IIß), and knockdown of Topo IIß attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.

Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.

The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Targeted Enhancer Activation by a Subunit of the Integrator Complex.

The control of cell fate is an epigenetic process initiated by transcription factors (TFs) that recognize DNA motifs and recruit activator complexes and transcriptional machineries to chromatin. Lineage specificity is thought to be provided solely by TF-motif pairing, while the recruited activators are passive. Here, we show that INTS13, a subunit of the Integrator complex, operates as monocytic/macrophagic differentiation factor. Integrator is a general activator of transcription at coding genes and is required for eRNA maturation. Here, we show that INTS13 functions as an independent sub-module and targets enhancers through Early Growth Response (EGR1/2) TFs and their co-factor NAB2. INTS13 binds poised monocytic enhancers eliciting chromatin looping and activation. Independent depletion of INTS13, EGR1, or NAB2 impairs monocytic differentiation of cell lines and primary human progenitors. Our data demonstrate that Integrator is not functionally homogeneous and has TF-specific regulatory potential, revealing a new enhancer regulatory axis that controls myeloid differentiation.

Isogenic pairs of induced-pluripotent stem-derived endothelial cells identify DYRK1A/PPARG/EGR1 pathway is responsible for Down syndrome-associated pulmonary hypertension.

Down syndrome (DS) is the most prevalent chromosomal disorder associated with a higher incidence of pulmonary arterial hypertension (PAH). The dysfunction of vascular endothelial cells (ECs) is known to cause pulmonary arterial remodeling in PAH, although the physiological characteristics of ECs harboring trisomy 21 (T21) are still unknown. In this study, we analyzed the human vascular ECs by utilizing the isogenic pairs of T21-induced pluripotent stem cells (iPSCs) and corrected disomy 21 (cDi21)-iPSCs. In T21-iPSC-derived ECs, apoptosis and mitochondrial reactive oxygen species (mROS) were significantly increased, and angiogenesis and oxygen consumption rate (OCR) were significantly impaired as compared with cDi21-iPSC-derived ECs. The RNA-sequencing identified that EGR1 on chromosome 5 was significantly upregulated in T21-ECs. Both EGR1 suppression by siRNA and pharmacological inhibitor could recover the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Alternately, the study also revealed that DYRK1A was responsible to increase EGR1 expression via PPARG suppression, and that chemical inhibition of DYRK1A could restore the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Finally, we demonstrated that EGR1 was significantly upregulated in the pulmonary arterial ECs from lung specimens of a patient with DS and PAH. In conclusion, DYRK1A/PPARG/EGR1 pathway could play a central role for the pulmonary EC functions and thus be associated with the pathogenesis of PAH in DS.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma.

The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription.

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.

The guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

Metformin inhibits nerve growth factor-induced sympathetic neuron differentiation through p35/CDK5 inhibition.

The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.

5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase promotes pulmonary arterial smooth muscle cell proliferation via the Ras signaling pathway.

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of monocrotaline (MCT)-induced PAH rat model, hypoxia-induced PAH mouse model, and platelet-derived growth factor (PDGF)-stimulated PASMCs. Inhibition of ATIC attenuated PDGF-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation, and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Furthermore, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factors MYC, early growth response protein 1 (EGR1), and specificity protein 1 (SP1) directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway.NEW & NOTEWORTHY Our findings highlight the important role of ATIC in the PASMC proliferation of pulmonary vascular remodeling through its modulation of the Ras signaling pathway and its regulation by transcription factors MYC, EGR1, and SP1. ATIC's modulation of Ras signal pathway represents a novel mechanism contributing to PAH development.

Muscle memory in humans: evidence for myonuclear permanence and long-term transcriptional regulation after strength training.

The objective of this work was to investigate myonuclear permanence and transcriptional regulation as mechanisms for cellular muscle memory after strength training in humans. Twelve untrained men and women performed 10 weeks of unilateral elbow-flexor strength training followed by 16 weeks of de-training. Thereafter, 10 weeks' re-training was conducted with both arms: the previously trained arm and the contralateral untrained control arm. Muscle biopsies were taken from the trained arm before and after both training periods and from the control arm before and after re-training. Muscle biopsies were analysed for fibre cross-sectional area (fCSA), myonuclei and global transcriptomics (RNA sequencing). During the first training period, myonuclei increased in type 1 (13 ± 17%) and type 2 (33 ± 23%) fibres together with a 30 ± 43% non-significant increase in mixed fibre fCSA (P = 0.069). Following de-training, fCSA decreased in both fibre types, whereas myonuclei were maintained, resulting in 33% higher myonuclear number in previously trained vs. control muscle in type 2 fibres. Furthermore, in the previously trained muscle, three differentially expressed genes (DEGs; EGR1, MYL5 and COL1A1) were observed. Following re-training, the previously trained muscle showed larger type 2 fCSA compared to the control (P = 0.035). However, delta change in type 2 fCSA was not different between muscles. Gene expression was more dramatically changed in the control arm (1338 DEGs) than in the previously trained arm (822 DEGs). The sustained higher number of myonuclei in the previously trained muscle confirms myonuclear accretion and permanence in humans. Nevertheless, because of the unclear effect on the subsequent hypertrophy with re-training, the physiological benefit remains to be determined. KEY POINTS: Muscle memory is a cellular mechanism that describes the capacity of skeletal muscle fibres to respond differently to training stimuli if the stimuli have been previously encountered. This study overcomes past methodological limitations related to the choice of muscles and analytical procedures. We show that myonuclear number is increased after strength training and maintained during de-training. Increased myonuclear number and differentially expressed genes related to muscle performance and development in the previously trained muscle did not translate into a clearly superior responses during re-training. Because of the unclear effect on the subsequent hypertrophy and muscle strength gain with re-training, the physiological benefit remains to be determined.

A whole-brain analysis of functional connectivity and immediate early gene expression reveals functional network shifts after operant learning.

Previous studies of operant learning have addressed neuronal activities and network changes in specific brain areas, such as the striatum, sensorimotor cortex, prefrontal/orbitofrontal cortices, and hippocampus. However, how changes in the whole-brain network are caused by cellular-level changes remains unclear. We, therefore, combined resting-state functional magnetic resonance imaging (rsfMRI) and whole-brain immunohistochemical analysis of early growth response 1 (EGR1), a marker of neural plasticity, to elucidate the temporal and spatial changes in functional networks and underlying cellular processes during operant learning. We used an 11.7-Tesla MRI scanner and whole-brain immunohistochemical analysis of EGR1 in mice during the early and late stages of operant learning. In the operant training, mice received a reward when they pressed left and right buttons alternately, and were punished with a bright light when they made a mistake. A group of mice (n = 22) underwent the first rsfMRI acquisition before behavioral sessions, the second acquisition after 3 training-session-days (early stage), and the third after 21 training-session-days (late stage). Another group of mice (n = 40) was subjected to histological analysis 15 min after the early or late stages of behavioral sessions. Functional connectivity increased between the limbic areas and thalamus or auditory cortex after the early stage of training, and between the motor cortex, sensory cortex, and striatum after the late stage of training. The density of EGR1-immunopositive cells in the motor and sensory cortices increased in both the early and late stages of training, whereas the density in the amygdala increased only in the early stage of training. The subcortical networks centered around the limbic areas that emerged in the early stage have been implicated in rewards, pleasures, and fears. The connectivities between the motor cortex, somatosensory cortex, and striatum that consolidated in the late stage have been implicated in motor learning. Our multimodal longitudinal study successfully revealed temporal shifts in brain regions involved in behavioral learning together with the underlying cellular-level plasticity between these regions. Our study represents a first step towards establishing a new experimental paradigm that combines rsfMRI and immunohistochemistry to link macroscopic and microscopic mechanisms involved in learning.

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

EGR1 modulates EPHB4-induced trophoblast dysfunction in recurrent spontaneous abortion†.

Recurrent spontaneous abortion, defined as at least three unexplained abortions occurring before the 20-24 week of pregnancy, has a great impact on women's quality of life. Ephrin receptor B4 has been associated with trophoblast function in preeclampsia. The present study aimed to verify the hypothesis that ephrin receptor B4 regulates the biological functions of trophoblasts in recurrent spontaneous abortion and to explore the upstream mechanism. Ephrin receptor B4 was overexpressed in mice with recurrent spontaneous abortion. Moreover, ephrin receptor B4 inhibited trophoblast proliferation, migration, and invasion while promoting apoptosis. Downregulation of early growth response protein 1 expression in mice with recurrent spontaneous abortion led to ephrin receptor B4 overexpression. Poor expression of WT1-associated protein in mice with recurrent spontaneous abortion reduced the modification of early growth response protein 1 mRNA methylation, resulting in decreased early growth response protein 1 mRNA stability and expression. Overexpression of WT1-associated protein reduced the incidence of recurrent spontaneous abortion in mice by controlling the phenotype of trophoblasts, which was reversed by early growth response protein 1 knockdown. All in all, our findings demonstrate that dysregulation of WT1-associated protein contributes to the instability of early growth response protein 1, thereby activating ephrin receptor B4-induced trophoblast dysfunction in recurrent spontaneous abortion. Our study provides novel insights into understanding the molecular pathogenesis of recurrent spontaneous abortion.

EGR1 functions as a new host restriction factor for SARS-CoV-2 to inhibit virus replication through the E3 ubiquitin ligase MARCH8.

IMPORTANCE: Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Caffeine upregulates SIRT3 expression to ameliorate astrocytes-mediated HIV-1 Tat neurotoxicity via suppression of EGR1 signaling pathway.

Caffeine is one of the most popular consumed psychostimulants that mitigates several neurodegenerative diseases. Nevertheless, the roles and molecular mechanisms of caffeine in HIV-associated neurocognitive disorders (HAND) remain largely unclear. Transactivator of transcription (Tat) is a major contributor to the neuropathogenesis of HAND in the central nervous system. In the present study, we determined that caffeine (100 µM) treatment significantly ameliorated Tat-induced decreased astrocytic viability, oxidative stress, inflammatory response and excessive glutamate and ATP release, thereby protecting neurons from apoptosis. Subsequently, SIRT3 was demonstrated to display neuroprotective effects against Tat during caffeine treatment. In addition, Tat downregulated SIRT3 expression via activation of EGR1 signaling, which was reversed by caffeine treatment in astrocytes. Overexpression of EGR1 entirely abolished the neuroprotective effects of caffeine against Tat. Furthermore, counteracting Tat or caffeine-induced differential expression of SIRT3 abrogated the neuroprotection of caffeine against Tat-triggered astrocytic dysfunction and neuronal apoptosis. Taken together, our study establishes that caffeine ameliorates astrocytes-mediated Tat neurotoxicity by targeting EGR1/SIRT3 signaling pathway. Our findings highlight the beneficial effects of caffeine on Tat-induced astrocytic dysfunction and neuronal death and propose that caffeine might be a novel therapeutic drug for relief of HAND.

EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer.

BACKGROUND: Gemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. METHODS: The following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry. FINDINGS: We revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group. CONCLUSIONS: Our study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.