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The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Epstein-Barr virus (EBV) infects more than 90% of the human population worldwide and establishes lifelong infection in hosts by switching between latent and lytic infection. EBV latency can be reactivated under appropriate conditions, leading to expression of the viral lytic genes and production of infectious progeny viruses. EBV reactivation involves crosstalk between various factors and signaling pathways, and the subsequent complicated virus-host interplays determine whether EBV continues to propagate. However, the detailed mechanisms underlying these processes remain unclear. In this review, we summarize the critical factors regulating EBV reactivation and the associated mechanisms. This encompasses the transcription and post-transcriptional regulation of immediate-early (IE) genes, the functions of viral factors on viral DNA replication and progeny virus production, the mechanisms through which viral proteins disrupt and inhibit the host's innate immune response, and the host factors that modulate EBV reactivation. Finally, we explore the potential applications of novel technologies in studying EBV reactivation, providing novel insights into the investigation of mechanisms governing EBV reactivation and the development of anti-EBV therapeutic strategies. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) infects more than 90% of the human population worldwide and establishes lifelong infection in hosts by switching between latent and lytic infection. EBV latency can be reactivated under appropriate conditions, leading to expression of the viral lytic genes and production of infectious progeny viruses. EBV reactivation involves crosstalk between various factors and signaling pathways, and the subsequent complicated virus-host interplays determine whether EBV continues to propagate. However, the detailed mechanisms underlying these processes remain unclear. In this review, we summarize the critical factors regulating EBV reactivation and the associated mechanisms. This encompasses the transcription and post-transcriptional regulation of immediate-early (IE) genes, the functions of viral factors on viral DNA replication and progeny virus production, the mechanisms through which viral proteins disrupt and inhibit the host’s innate immune response, and the host factors that modulate EBV reactivation. Finally, we explore the potential applications of novel technologies in studying EBV reactivation, providing novel insights into the investigation of mechanisms governing EBV reactivation and the development of anti-EBV therapeutic strategies.

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

Objective To investigate the effect of human cytomegalovirus immediate-early protein 2 (HCMV-IE2) on fructose-induced hepatic steatosis in UL122 transgenic model mice. Methods The UL122 transgenic mice were established as experimental group, and age-matched wild-type mice were established as control group. The mice were fed with high-fructose (60%) diet for 8 weeks, and the changes in body weight and blood glucose were monitored. After the glucose tolerance test and the insulin tolerance test were performed, the mice were sacrificed, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, as well as the levels of triglyceride (TG) and total cholesterol (TC) in the liver. Liver tissue was collected, and HE staining and oil red O staining were used to observe histopathological changes; flow cytometry was used to observe macrophage polarization in the liver; RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of lipid synthesis-related genes in the liver. Results Compared with the control group, the experimental group had significant increases in body weight and fasting blood glucose level (F=12.78-100.05,P

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt's lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3'UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved among lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. IMPORTANCE Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

Pathogens use virulence factors to inhibit the immune system1. The guard hypothesis2,3 postulates that hosts monitor (or 'guard') critical innate immune pathways such that their disruption by virulence factors provokes a secondary immune response1. Here we describe a 'self-guarded' immune pathway in human monocytes, in which guarding and guarded functions are combined in one protein. We find that this pathway is triggered by ICP0, a key virulence factor of herpes simplex virus type 1, resulting in robust induction of anti-viral type I interferon (IFN). Notably, induction of IFN by ICP0 is independent of canonical immune pathways and the IRF3 and IRF7 transcription factors. A CRISPR screen identified the ICP0 target MORC34 as an essential negative regulator of IFN. Loss of MORC3 recapitulates the IRF3- and IRF7-independent IFN response induced by ICP0. Mechanistically, ICP0 degrades MORC3, which leads to de-repression of a MORC3-regulated DNA element (MRE) adjacent to the IFNB1 locus. The MRE is required in cis for IFNB1 induction by the MORC3 pathway, but is not required for canonical IFN-inducing pathways. As well as repressing the MRE to regulate IFNB1, MORC3 is also a direct restriction factor of HSV-15. Our results thus suggest a model in which the primary anti-viral function of MORC3 is self-guarded by its secondary IFN-repressing function-thus, a virus that degrades MORC3 to avoid its primary anti-viral function will unleash the secondary anti-viral IFN response.

Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre-mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage-priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis-induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization.

To address urgent need for strategies to limit mortality from coronavirus disease 2019 (COVID-19), this review describes experimental, clinical and epidemiological evidence that suggests that chronic sub-optimal hydration in the weeks before infection might increase risk of COVID-19 mortality in multiple ways. Sub-optimal hydration is associated with key risk factors for COVID-19 mortality, including older age, male sex, race-ethnicity and chronic disease. Chronic hypertonicity, total body water deficit and/or hypovolemia cause multiple intracellular and/or physiologic adaptations that preferentially retain body water and favor positive total body water balance when challenged by infection. Via effects on serum/glucocorticoid-regulated kinase 1 (SGK1) signaling, aldosterone, tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), aquaporin 5 (AQP5) and/or Na+/K+-ATPase, chronic sub-optimal hydration in the weeks before exposure to COVID-19 may conceivably result in: greater abundance of angiotensin converting enzyme 2 (ACE2) receptors in the lung, which increases likelihood of COVID-19 infection, lung epithelial cells which are pre-set for exaggerated immune response, increased capacity for capillary leakage of fluid into the airway space, and/or reduced capacity for both passive and active transport of fluid out of the airways. The hypothesized hydration effects suggest hypotheses regarding strategies for COVID-19 risk reduction, such as public health recommendations to increase intake of drinking water, hydration screening alongside COVID-19 testing, and treatment tailored to the pre-infection hydration condition. Hydration may link risk factors and pathways in a unified mechanism for COVID-19 mortality. Attention to hydration holds potential to reduce COVID-19 mortality and disparities via at least 5 pathways simultaneously.

ObjectiveThe present study examined the role of PPARβ/δ in hepatocellular carcinoma (HCC).MethodsThe effect of PPARβ/δ on HCC development was analyzed using PPARβ/δ-overexpressed liver cancer cells and PPARβ/δ-knockout mouse models.ResultsPPARβ/δ (-/-) mice were susceptible to diethylnitrosamine- (DEN-) induced HCC (87.5% vs. 37.5%, p < 0.05). In addition, PPARβ/δ-overexpressed HepG2 cells had reduced proliferation, migration, and invasion capabilities accompanied by increased apoptosis and cell cycle arrest at the G0/G1 phase. Moreover, differential gene expression profiling uncovered that the levels of serine/threonine-protein kinase (SGK-1) mRNA and its encoded protein were reduced in PPARβ/δ-overexpressed HepG2 cells. Consistently, elevated SGK-1 levels were found in PPARβ/δ (-/-) mouse livers as well as PPARβ/δ-knockdown human SMMC-7721 HCC cells. Chromatin immunoprecipitation (ChIP) assays followed by real-time quantitative polymerase chain reaction (qPCR) assays further revealed the binding of PPARβ/δ to the SGK-1 regulatory region in HepG2 cells.ConclusionsDue to the known tumor-promoting effect of SGK1, the present data suggest that PPARβ/δ-deactivated SGK1 is a novel pathway for inhibiting liver carcinogenesis.

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3 processing factor CPSF. It thereby induces the assembly of a dead-end 3 processing complex, blocking mRNA 3 cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3 processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer.

The CD8+ T cell response to the intracellular parasite Toxoplasma gondii varies dramatically between mouse strains, resulting in stark differences in control of the parasite. Protection in BALB/c mice can be attributed to an unusually strong and protective MHC-1 Ld-restricted CD8+ T cell response directed against a peptide derived from the parasite antigen GRA6. The MHC-1 Ld molecule has limited peptide binding compared to conventional MHC molecules such as Kb or Db, which correlates with polymorphisms associated with elite control of HIV in humans. To investigate the link between the unusual MHC-1 molecule Ld and the generation of elite controller CD8+ T cell responses, we compared the GRA6-Ld specific T cell response to the well-studied OVA-Kb specific response, and demonstrated that GRA6-Ld specific T cells are significantly more protective and resistant to exhaustion in chronic T. gondii infection. To further investigate the connection between limited peptide presentation and robust T cell responses, we used CRISPR/Cas9 to generate mice with a point mutation (W97R) in the peptide-binding groove of Ld that results in broader peptide binding. We investigated the effect of this Ld W97R mutation on another robust Ld-restricted response against the IE1 peptide during Murine Cytomegalovirus (MCMV) infection. This mutation leads to an increase in exhaustion markers in the IE1-Ld specific CD8+ T cell response. Our results indicate that limited peptide binding by MHC-1 Ld correlates with the development of robust and protective CD8+ T cell responses that may avoid exhaustion during chronic infection.

In cancer, aberrant growth factor receptor signaling reprograms cellular metabolism and global gene transcription to drive aggressive growth, but the underlying mechanisms are not well-understood. Here we show that in the highly lethal brain tumor glioblastoma (GBM), mTOR complex 2 (mTORC2), a critical core component of the growth factor signaling system, couples acetyl-CoA production with nuclear translocation of histone-modifying enzymes including pyruvate dehydrogenase and class IIa histone deacetylases to globally alter histone acetylation. Integrated analyses in orthotopic mouse models and in clinical GBM samples reveal that mTORC2 controls iron metabolisms via histone H3 acetylation of the iron-related gene promoter, promoting tumor cell survival. These results nominate mTORC2 as a critical epigenetic regulator of iron metabolism in cancer.

Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity.IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

Potassium (K+) secretion by kidney tubule cells is central to electrolyte homeostasis in mammals. In the K+ secretory "principal" cells of the distal nephron, electrogenic Na+ transport by the epithelial sodium channel (ENaC) generates the electrical driving force for K+ transport across the apical membrane. Regulation of this process is attributable in part to aldosterone, which stimulates the gene transcription of the ENaC-regulatory kinase, SGK1. However, a wide range of evidence supports the conclusion that an unidentified aldosterone-independent pathway exists. We show here that in principal cells, K+ itself acts through the type 2 mTOR complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion. The effect depends on changes in K+ concentration on the blood side of the cells, and requires basolateral membrane K+-channel activity. However, it does not depend on changes in aldosterone, or on enhanced distal delivery of Na+ from upstream nephron segments. These data strongly support the idea that K+ is sensed directly by principal cells to stimulate its own secretion by activating the mTORC2-SGK1 signaling module, and stimulate ENaC. We propose that this local effect acts in concert with aldosterone and increased Na+ delivery from upstream nephron segments to sustain K+ homeostasis.

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.

The TREX complex mediates the passage of bulk cellular mRNA export to the nuclear export factor TAP/NXF1 via the export adaptors ALYREF or UIF, which appear to act in a redundant manner. TREX complex recruitment to nascent RNA is coupled with 5' capping, splicing and polyadenylation. Therefore to facilitate expression from their intronless genes, herpes viruses have evolved a mechanism to circumvent these cellular controls. Central to this process is a protein from the conserved ICP27 family, which binds viral transcripts and cellular TREX complex components including ALYREF. Here we have identified a novel interaction between HSV-1 ICP27 and an N-terminal domain of UIF in vivo, and used NMR spectroscopy to locate the UIF binding site within an intrinsically disordered region of ICP27. We also characterized the interaction sites of the ICP27 homolog ORF57 from KSHV with UIF and ALYREF using NMR, revealing previously unidentified binding motifs. In both ORF57 and ICP27 the interaction sites for ALYREF and UIF partially overlap, suggestive of mutually exclusive binding. The data provide a map of the binding sites responsible for promoting herpes virus mRNA export, enabling future studies to accurately probe these interactions and reveal the functional consequences for UIF and ALYREF redundancy.

A fundamental signal-processing problem is how biological systems maintain phenotypic states (i.e., canalization) long after degradation of initial catalyst signals. For example, to efficiently replicate, herpesviruses (e.g., human cytomegalovirus, HCMV) rapidly counteract cell-mediated silencing using transactivators packaged in the tegument of the infecting virion particle. However, the activity of these tegument transactivators is inherently transient-they undergo immediate proteolysis but delayed synthesis-and how transient activation sustains lytic viral gene expression despite cell-mediated silencing is unclear. By constructing a two-color, conditional-feedback HCMV mutant, we find that positive feedback in HCMV's immediate-early 1 (IE1) protein is of sufficient strength to sustain HCMV lytic expression. Single-cell time-lapse imaging and mathematical modeling show that IE1 positive feedback converts transient transactivation signals from tegument pp71 proteins into sustained lytic expression, which is obligate for efficient viral replication, whereas attenuating feedback decreases fitness by promoting a reversible silenced state. Together, these results identify a regulatory mechanism enabling herpesviruses to sustain expression despite transient activation signals-akin to early electronic transistors-and expose a potential target for therapeutic intervention.

In order to determine the impact of the epigenetic response to traumatic stress on post-traumatic stress disorder (PTSD), this study examined longitudinal changes of genome-wide blood DNA methylation profiles in relation to the development of PTSD symptoms in two prospective military cohorts (one discovery and one replication data set). In the first cohort consisting of male Dutch military servicemen (n=93), the emergence of PTSD symptoms over a deployment period to a combat zone was significantly associated with alterations in DNA methylation levels at 17 genomic positions and 12 genomic regions. Evidence for mediation of the relation between combat trauma and PTSD symptoms by longitudinal changes in DNA methylation was observed at several positions and regions. Bioinformatic analyses of the reported associations identified significant enrichment in several pathways relevant for symptoms of PTSD. Targeted analyses of the significant findings from the discovery sample in an independent prospective cohort of male US marines (n=98) replicated the observed relation between decreases in DNA methylation levels and PTSD symptoms at genomic regions in ZFP57, RNF39 and HIST1H2APS2. Together, our study pinpoints three novel genomic regions where longitudinal decreases in DNA methylation across the period of exposure to combat trauma marks susceptibility for PTSD.

Kaposi's sarcoma-associated herpesvirus (KSHV) employs diverse mechanisms to subvert host immune responses, contributing to its infection and pathogenicity. As an immune evasion strategy, KSHV encodes the Membrane-Associated RING-CH (MARCH)-family E3 ligases, K3, and K5, which target and remove several immune regulators from the cell surface. In this study, we investigate the impact of K3 and K5 on lymphotoxin receptor (LTβR) ligands, LTβ and LIGHT, which are type II transmembrane proteins and function as pivotal immune mediators during virus infection. Upon co-expression of viral MARCH proteins with LTβR ligands, we showed that K3 and K5 selectively targeted LTβ, but not LIGHT, for the downregulation of surface expression. Specifically, K3 and K5 E3 ligases interacted with the transmembrane domain of LTβ. Intriguingly, K3 interacted with an immature form of LTβ, whereas K5 targeted the fully mature form. Subsequent biochemical analyses revealed that K3 disrupted the initial steps of N-glycosylation maturation of LTβ. This interference resulted in the sequestration of LTβ within the endoplasmic reticulum, impeding its trafficking to the plasma membrane. Consequently, the K3-mediated downregulation of LTβ surface expression suppressed the LTβR downstream signaling pathway. These findings uncover a novel mechanism by which KSHV K3 E3 ligase inhibits the membrane trafficking pathway of the LTβ inflammatory ligand through glycosylation interference, potentially evading LTβR-mediated antiviral immunity., (© 2025 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFβ signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFβ signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFβ-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.

Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days after in utero electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2+ cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed nonradially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2+ callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be responsible for the increased neural stem cell exit from the VZ and cortical migrational defects, respectively. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings, such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in the context of the embryonic brain using an in utero gene transfer tool. Surprisingly, IE2, but not HCMV IE1 or murine CMV ie3, interferes pleiotropically with key neurodevelopmental processes, including neural stem cell regulation, proper positioning of migrating neurons, and the callosal axon projections important for communication between the hemispheres. Our data suggest that the wide spectrum of clinical outcomes, ranging from mental retardation to microcephaly, caused by congenital HCMV infection can be sufficiently explained in terms of IE2 action alone.

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain.

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.ImportanceGammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Abstract: Despite its high prevalence, effective treatment for degenerative mitral regurgitation (MR) remains elusive. Although the mineralocorticoid-receptor antagonist spironolactone, in conjunction with renin-angiotensin-aldosterone system inhibitors, has been shown to reduce mortality in patients with heart failure with reduced ejection fraction, its efficacy in managing degenerative MR is uncertain. In this study, we aimed to compare the effectiveness of valsartan (a renin-angiotensin system inhibitor), spironolactone, and combination therapy in mitigating MR-induced myocardial dysfunction. Using a mini-invasive model of degenerative MR, we administered valsartan (31 mg/kg/d), spironolactone (80 mg/kg/d), or a combination of both to rats over a 4-week period. Serial echocardiography and pressure-volume loops were utilized to assess cardiac function and hemodynamics. Rats with degenerative MR treated with valsartan or spironolactone alone did not show significant improvement in myocardial dysfunction. In contrast, combination therapy resulted in significant improvement. Similarly, the pressure-volume relationship was significantly improved in rats treated with the combination therapy compared with that in rats treated with a single therapy. Mechanistically, combination therapy effectively suppressed circulating and cardiac expression of aldosterone- and apoptosis-associated proteins. Overall, combination treatment with valsartan and spironolactone significantly attenuated the degenerative MR-induced myocardial stress and dysfunction, suggesting a potential therapeutic avenue for managing degenerative MR-induced heart failure., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The epithelial Na(+) channel (ENaC) is decisive for sodium reabsorption by the aldosterone-sensitive distal nephron (ASDN) of the kidney. ENaC is regulated by the serum- and glucocorticoid-inducible kinase 1 (SGK1), a kinase genomically upregulated by several hormones including glucocorticoids and mineralocorticoids. SGK1 is activated by the serine/threonine kinase mammalian target of rapamycin (mTOR) isoform mTORC2. SGK1 knockout (sgk1(-/-) mice) impairs renal Na(+) retention during salt depletion. The mTOR catalytic site inhibitor, PP242, but not mTORC1 inhibitor rapamycin, inhibits ENaC, decreases Na(+) flux in isolated perfused tubules and induces natriuresis in wild-type mice. PP242 does not lead to further impairment of Na(+) reabsorption in sgk1(-/-) mice. The mTORC2/SGK1 sensitive renal Na(+) retention leads to extracellular volume expansion with increase of blood pressure. A SGK1 gene variant (prevalence ∼ 3-5% in Caucasians, ∼ 10% in Africans) predisposes to hypertension, stroke, obesity and type 2 diabetes. Future studies will be required to define the role of mTORC2 in the regulation of further SGK1 sensitive transport proteins, such as further ion channels, carriers and the Na(+)/K(+)-ATPase. Moreover, studies are required disclosing the impact of mTORC2 on SGK1 sensitive disorders, such as hypertension, obesity, diabetes, thrombosis, stroke, inflammation, autoimmune disease, fibrosis and tumour growth.

Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate-early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with oncogenic properties. Here, we show how HCMV IEs are preferentially expressed in glioma stem-like cells (GSC), where they colocalize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSCs that are endogenously infected with HCMV, attenuating IE expression by an RNAi-based strategy was sufficient to inhibit tumorsphere formation, Sox2 expression, cell-cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSCs elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSCs, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and proinflammatory cytokines, resembling the therapeutically resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miR-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSCs infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared with mock-infected human GSCs. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in GBM cells.

Herpesviruses cause life-long infections by evading the host immune system and establishing latent infections. All mammalian herpesviruses express an essential multifunctional protein that is typified by ICP27 encoded by Herpes Simplex Virus 1. The only region that is conserved among the diverse members of the ICP27 family is a predicted globular domain that has been termed the ICP27 homology domain. Here we present the first crystal structure of the ICP27 homology domain, solved to 1.9 Å resolution. The protein is a homo-dimer, adopting a novel intertwined fold with one CHCC zinc-binding site per monomer. The dimerization, which was independently confirmed by SEC-MALS and AUC, is stabilized by an extensive network of intermolecular contacts, and a domain-swap involving the two N-terminal helices and C-terminal tails. Each monomer contains a lid motif that can clamp the C-terminal tail of its dimeric binding partner against its globular core, without forming any distinct secondary structure elements. The binding interface was probed with point mutations, none of which had a noticeable effect on dimer formation; however deletion of the C-terminal tail region prevented dimer formation in vivo. The structure provides a template for future biochemical studies and modelling of ICP27 homologs from other herpesviruses.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Diabetic nephropathy (DN) is a common clinical syndrome in diabetic patients. Functional characterization of non-coding (ncRNAs) involved in the progression of DN can provide insights into the diagnosis and therapeutic management of DN. Human kidney proximal tubular epithelial cells (HK-2) were challenged by high glucose (HG, 50 mM) as a cell model of DN. The expression level of long non-coding RNA (lncRNA) ZFAS1 was quantified by qRT-PCR. The proteins and cytokines related to fibrosis and scortosis in DN (NLRP3, GSDMD-N, IL-1β and Caspase 1, fibronectin, collagen I, collagen III, IL-1β, and IL-18) were examined by western blot or ELISA. RNA precipitation and luciferase reporter activity experiments were conducted to assess the molecular associations. ZFAS1 and SGK1 were highly induced in HK-2 cells challenged with HG, while miR-525-5p downregulated upon HG treatment. ZFAS1 knockdown attenuated HG-induced fibrosis and scortosis in HK-2 cells by reducing the levels of NLRP3, GSDMD-N, Caspase 1, fibronectin, collagen I/III, IL-1β, and IL-18. Mechanically, ZFAS1 knockdown protected HK-2 cells from HG-induced injury by upregulating miR-525-5p and repressing SGK1 expression. Overall, our results suggest that knocking down ZFAS1 may be formulated as a protective strategy in ameliorating DN progression through regulating miR-525-5p/SGK1 pathway. Targeting ZFAS1 could be further explored as a potential approach for the management of DN., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Long-term memory formation requires de novo RNA and protein synthesis. Using differential display PCR, we found that the NCoR1 cDNA fragment is differentially expressed between fast learners and slow learners, with fast learners showing a lower expression level than slow learners in the water maze learning task. Fast learners also show lower NCoR1 mRNA and protein expression levels. In addition, spatial training decreases both NCoR1 mRNA and protein expression, whereas NCoR1 conditional knockout (cKO) mice show enhanced spatial memory. In studying the molecular mechanism, we found that spatial training decreases the association between NCoR1 and DEC2. Both NCoR1 and DEC2 suppress the expression of BDNF, integrin α3 and SGK1 through C/EBPα binding to their DNA promoters, but overexpression of DEC2 in NCoR1 cKO mice rescues the decreased expression of these proteins compared with NCoR1 loxP mice overexpressing DEC2. Further, spatial training decreases DEC2 expression. Spatial training also enhances C/EBPα binding to Bdnf, Itga3 and Sgk1 promoters, an effect also observed in fast learners, and both NCoR1 and DEC2 control C/EBPα activity. Whereas knockdown of BDNF, integrin α3 or SGK1 expression impairs spatial learning and memory, it does not affect Y-maze performance, suggesting that BDNF, integrin α3 and SGK1 are involved in long-term memory formation, but not short-term memory formation. Moreover, NCoR1 expression is regulated by the JNK/c-Jun signaling pathway. Collectively, our findings identify DEC2 as a novel interacting protein of NCoR1 and elucidate the novel roles and mechanisms of NCoR1 and DEC2 in negative regulation of spatial memory formation., (© 2024. The Author(s).)