Promoter Activity Research Articles

A type II toxin-antitoxin system, ECs3274-ECs3275, in enterohemorrhagic Escherichia coli O157.

The toxin-antitoxin (TA) genetic module controls various bacterial events. Novel toxins with different functions are still being discovered. This study aimed to determine whether the ECs3274-ECs3275 gene pair encoded by enterohemorrhagic Escherichia coli O157 functions as a TA system. To characterize this putative TA system, we analyzed the growth of E. coli expressing ECs3274, ECs3275, or both; the interaction between ECs3274 and ECs3275 using bacterial adenylate cyclase two-hybrid assays; and the DNA-binding ability of ECs3274 using gel-mobility shift assays. We observed that the ECs3274 antitoxin interacted with the ECs3275 toxin, was destabilized by Lon protease, and repressed its promoter activity via its helix-turn-helix (HTH) motif. These properties are consistent with those of typical type II TA antitoxins. Interestingly, ECs3275 has an HTH motif not observed in other TA toxins and is necessary for ECs3275 toxicity, suggesting that ECs3275 may exert its toxicity by regulating the expression of specific genes.

Transcriptional activity and variation analysis of genes critical for long-chain fatty acid (C≥16) elongation and desaturation in Pekin ducks

To deepen our understanding of long-chain fatty acid carbon chain elongation and desaturation in ducks, this study systematically analyzed the transcriptional activities of key gene promoters, including ELOVLs, FADSs, and SCDs. Predictive modeling coupled with statistical analysis revealed a prevalence of binding motifs for transcription factors, particularly those associated with Sp1, NF-1, and C/EBPalpha. Moreover, variation analysis of resequencing data from both wild and domestic ducks, specifically mallards and Pekin ducks, informed targeted mutagenesis within the core promoter regions of ELOVL2, ELOVL5, and ELOVL6. Notably, mutations at positions -56 G>C in ELOVL2, -52 T>C in ELOVL5, and -513 T>C in ELOVL6 significantly diminished transcriptional activity. These findings substantially enhance our understanding of the molecular mechanisms regulating the biosynthesis of long-chain fatty acids in ducks and support future genetic selection initiatives aimed at developing Pekin duck breeds with enhanced nutritional value.

A single dominant GLOBOSA allele accounts for repeated origins of hose-in-hose flowers in Sinningia (Gesneriaceae).

Plants bearing double flowers have long been cultivated as ornamental plants. Hose-in-hose flowers, bearing 2-whorled corolla tubes in whorls 1 and 2, are uncommon but recur in Sinningia (Gesnerioideae, Gesneriaceae). In this study, we selected 15 hose-in-hose cultivars as materials to explore the underlying molecular and genetic mechanisms of this floral architecture. We found that they originated from different hybridization events within the Dircaea clade. Three B-class MADS-box genes were globally expressed in all floral whorls, but only GLOBOSA1 (GLO1) has accumulated a dominant mutation, i.e., the insertion of a hAT-like miniature inverted-repeat transposable element (MITE) into its promoter, that co-segregated with the hose-in-hose phenotype. In addition, all 15 hose-in-hose cultivars contained the same dominant GLO1 allele. Transient gene expression assays confirmed the role of this MITE insertion in up-regulating the promoter activity of GLO1 by providing several cis-regulatory elements. Genetic transformation in heterologous Chirita pumila (Didymocarpoideae, Gesneriaceae) verified that this dominant GLO1 allele is sufficient to confer the hose-in-hose phenotype. We further demonstrated that both the GLO1 allele and the hAT-like MITE descended from wild S. cardinalis with single flowers. This study highlights the significance of wide hybridization in frequent gains of the dominant GLO1 allele and thereafter repeated occurrence of hose-in-hose flowers in Sinningia.

Molecular Mechanisms of Skatole-Induced Inflammatory Responses in Intestinal Epithelial Caco-2 Cells: Implications for Colorectal Cancer and Inflammatory Bowel Disease.

Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in intestinal epithelial cells significantly contribute to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Given our previous findings that TNF-α is upregulated in intestinal epithelial Caco-2 cells induced by skatole, a tryptophan-derived gut microbiota metabolite, the present study aimed to explore the relationship between skatole and IL-6, alongside TNF-α. Skatole elevated the promoter activity of IL-6 as well as TNF-α, and increased IL-6 mRNA expression and protein secretion. In addition to activating NF-κB, the NF-κB inhibitor BAY 11-7082 reduced skatole-induced cell survival and the mRNA expression of IL-6 and TNF-α. NF-κB activation was attenuated by the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 and the p38 inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. U126 and SB203580 also decreased the skatole-induced increase in IL-6 expression. When skatole-induced AhR activation was inhibited by CH223191, in addition to promoting NF-κB activation, IL-6 expression was enhanced in a manner similar to that previously reported for TNF-α. Taken together, these results suggest that skatole-elicited NF-κB activation induces IL-6 and TNF-α expression, although AhR activation partially suppresses this process. The ability of skatole to increase the expression of IL-6 and TNF-α may significantly affect the development and progression of these diseases. Moreover, the balance between NF-κB and AhR activation appears to govern the skatole-induced increases in IL-6 and TNF-α expression. Therefore, the present findings provide new insights into the mechanisms linking tryptophan-derived gut microbiota metabolites with colorectal disease.

The clock-associated LUX ARRHYTHMO regulates high-affinity nitrate transport in Arabidopsis roots.

The circadian clock organizes physiological processes in plants to occur at specific times of the day, optimizing efficient use of resources. Nitrate is a crucial inorganic nitrogen source for agricultural systems to sustain crop productivity. However, because nitrate fertilization has a negative impact on the environment, it is important to carefully manage nitrate levels. Understanding crop biological rhythms can lead to more ecologically friendly agricultural practices. Gating responses through the circadian clock could be a strategy to enhance root nitrate uptake and to limit nitrate runoff. In Arabidopsis, the NITRATE TRANSPORTER 2.1 (NRT2.1) gene encodes a key component of the high-affinity nitrate transporter system. Our study reveals that NRT2.1 exhibits a rhythmic expression pattern, with daytime increases and nighttime decreases. The NRT2.1 promoter activity remains rhythmic under constant light, indicating a circadian regulation. The clock-associated transcription factor LUX ARRHYTHMO (LUX) binds to the NRT2.1 promoter in vivo. Loss-of-function of LUX leads to increased NRT2.1 transcript levels and root nitrate uptake at dusk. This supports LUX acting as a transcriptional repressor and modulating NRT2.1 expression in a time-dependent manner. Furthermore, applying nitrate at different times of the day results in varying magnitudes of the transcriptional response in nitrate-regulated genes. We also demonstrate that a defect in the high-affinity nitrate transport system feeds back to the central oscillator by modifying the LUX promoter activity. In conclusion, this study uncovers a molecular pathway connecting the root nitrate uptake and circadian clock, with potential agro-chronobiological applications.

Prostaglandin E2 suppresses KCNH1 gene expression and inhibits the proliferation of CaSki cervical cells through its four prostanoid PTGER subtypes

The main risk factor for cervical cancer is the persistent infection of high-risk HPV subtypes, notably HPV16. Another contributing factor is proinflammatory prostaglandin E2 (PGE2), a lipid abundantly found in seminal fluid. PGE2, along with its receptors (PTGER1-4), contributes to cancer development; however, its specific role in the proliferation of cervical cancer models with high HPV16 copy numbers remains unclear. In this study, we investigated the effects of PGE2 on the proliferation of CaSki cells, a cell line with a high HPV16 viral load. Surprisingly, PGE2 inhibited CaSki cell proliferation, while it increased the proliferation of SiHa, HeLa, and C-33 A cervical cancer cells. The effect of PGE2 on CaSki cell proliferation was specific, as estradiol increased cell growth. Furthermore, PGE2 suppressed expression and promoter activity of the cervical tumoral marker KCNH1. To discern the specific role of each receptor in cell proliferation, we generated stable CaSki cell lines overexpressing each receptor alongside control cells with an empty vector. Notably, PGE2 significantly inhibited cell proliferation in all stable transfected CaSki cells, suppressing oncogenic KCNH1 expression and its promoter activity. In conclusion, our findings indicate that PGE2 inhibits the proliferation of CaSki cervical cancer cells with a high HPV16 load, at least in part, by suppressing the expression of the oncogenic KCNH1 gene.

An activator-represssor complex of VvWRKYs regulate proanthocyanidins biosynthesis through co-targeting VvLAR in grape

Proanthocyanidins (PAs) are vital polyphenolic compounds in plants with various biological functions. Although WRKY transcription factors are known to play important roles, their specific involvement in regulating PAs metabolism in grapes remains underexplored. In this study, we identified six candidate WRKY genes potentially involved in PAs synthesis by transiently overexpressing them in Nicotiana tabacum leaves. Among these, VvWRKY57 was found to enhance PAs synthesis. Further functional analysis, achieved by overexpressing of VvWRKY57 in grape calli, confirmed its positive role in PAs biosynthesis. Using yeast one-hybrid (Y1H), dual-luciferase reporter (DLR) assays, and electrophoretic mobility shift assay (EMSA), we demonstrated that VvWRKY57 binds to the promoter of leucocyanidin reductase (VvLAR2) and stimulates its activity. Additionally, yeast two-hybrid (Y2H), bimolecular fluorescence complementary (BiFC), and pull-down assays revealed that VvWRKY57 forms heterodimers with VvWRKY20, while VvWTKY20 also forms homodimers. Interestingly, overexpression of VvWRKY20 was found to inhibit PAs synthesis. Y1H, DLR, and EMSA further showed that VvWRKY20 binds to the promoters of VvLAR1 and VvLAR2, repressing their transcription activity. When VvWRKY57 and VvWRKY20 were co-expressed, VvLAR2 promoter activity and PAs synthesis were suppressed. Moreover, we discovered that VvPUB26, an E3 ubiquitin ligase physically interacts with both VvWRKY57 and VvWRKY20. VvPUB26 mediated the degradation of VvWRKY20 but did not influence the degradation of VvWRKY57. In conclusion, this study highlights the regulatory interplay between WRKY transcription factors in PAs biosynthesis, offering insights into their distinct roles in modulating this important metabolic pathway in grapes.

PK205R targets the proximal element of IFN-I signaling pathway to assist African swine fever virus to escape host innate immunity at the early stage of infection.

African swine fever virus (ASFV) is a nuclear cytoplasmic large DNA virus (NCLDV) that causes devastating hemorrhagic diseases in domestic pigs and wild boars, seriously threatening the development of the global pig industry. IFN-I plays an important role in the body's antiviral response. Similar to other DNA viruses, ASFV has evolved a variety of immune escape strategies to antagonize IFN-I signaling and maintain its proliferation. In this study, we showed that the ASFV early protein pK205R strongly inhibited interferon-stimulated genes (ISGs) as well as the promoter activity of IFN-stimulated regulatory elements (ISREs). Mechanistically, pK205R interacted with the intracellular domains of IFNAR1 and IFNAR2, thereby inhibiting the interaction of IFNAR1/2 with JAK1 and TYK2 and hindering the phosphorylation and nuclear translocation of STATs. Subsequently, we generated a recombinant strain of the ASFV-pK205R point mutation, ASFV-pK205R7PM. Notably, we detected higher levels of ISGs in porcine alveolar macrophages (PAMs) than in the parental strain during the early stages of ASFV-pK205R7PM infection. Moreover, ASFV-pK205R7PM attenuated the inhibitory effect on IFN-I signaling. In conclusion, we identified a new ASFV immunosuppressive protein that increases our understanding of ASFV immune escape mechanisms.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host’s antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host’s innate antiviral immunity and thus provides insights into viral pathogenesis.

Bcwf regulates the white petal color in pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis]

Flower color is important in determining the ornamental value of Brassica species. However, our knowledge about the regulation of flower color in pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis] is limited. In this study, we investigated the molecular mechanism underlying white flower traits in pak choi by analyzing a genetic population with white and yellow flowers. Our genetic analysis revealed that the white trait is controlled by a single recessive gene called Bcwf. Through BSA-Seq and fine mapping, we identified a candidate gene, BraC02g039450.1, which is similar to Arabidopsis AtPES2 involved in carotenoid ester synthesis. Sequence analysis showed some mutations in the promoter region of Bcwf in white flowers. Tobacco transient assay confirmed that these mutations reduce the promoter's activity, leading to downregulation of Bcwf expression in white flowers. Furthermore, the silencing of Bcwf in pak choi resulted in lighter petal color and reduced carotenoid content. These findings provide new insights into the molecular regulation of white flower traits in pak choi and highlight the importance of Bcwf in petal coloring and carotenoid accumulation.

Herpes Simplex Virus Type 2 Blocks IFN-β Production through the Viral UL24 N-Terminal Domain-Mediated Inhibition of IRF-3 Phosphorylation.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus, the cause of genital herpes, and its infection can increase the risk of HIV-1 infection. After initial infection, HSV-2 can establish lifelong latency within the nervous system, which is likely associated with the virus-mediated immune evasion. In this study, we found that HSV-2 UL24 significantly inhibited the activation of the IFN-β promoter and the production of IFN-β at both mRNA and protein levels. Of importance, the inhibitory effect of HSV-2 on IFN-β production was significantly impaired in the context of HSV-2 infection when UL24 was knocked down. Additional studies revealed that, although the full-length HSV-2 UL24 affected cell cycle and viability to some extent, its N-terminal 1-202AA domain showed no obvious cytotoxicity while its C-terminal 201-281 AA domain had a minimal impact on cell viability. Further studies showed that the N-terminal 1-202 AA domain of HSV-2 UL24 (HSV-2 UL24-N) was the main functional region responsible for the inhibition of IFN-β production mediated by HSV-2 UL24. This domain significantly suppressed the activity of RIG-IN, MAVS, TBK-1, IKK-ε, or the IRF-3/5D-activated IFN-β promoter. Mechanistically, HSV-2 UL24-N suppressed IRF-3 phosphorylation, resulting in the inhibition of IFN-β production. The findings of this study highlight the significance of HSV-2 UL24 in inhibiting IFN-β production, revealing two potential roles of UL24 during HSV-2 infection: facilitating immune evasion and inducing cell cycle arrest.

γ-Glutamylcyclotransferase is transcriptionally regulated by c-Jun and controls proliferation of glioblastoma stem cells through Notch1 levels.

Glioblastoma stem cells (GSCs) have been reported to cause poor prognosis of glioblastoma by contributing to therapy resistance. γ-Glutamylcyclotransferase (GGCT) is highly expressed in various cancer types, including glioblastoma, and its inhibition suppresses cancer cell growth. However, the mechanism of GGCT overexpression and its function in GSCs are unknown. In this study, we show that GGCT is highly expressed in GSCs established from a mouse glioblastoma model and its knockdown suppresses their proliferation. Effects of NRas and its downstream transcription factor c-Jun on GGCT expression were analyzed; NRas knockdown reduced c-Jun and GGCT expression. Knockdown of c-Jun also reduced expression levels of GGCT and inhibited cell proliferation. Consistent with this, pharmacological inhibition of c-Jun with SP600125 reduced GGCT and inhibited GSC proliferation. Furthermore, the GGCT promoter-reporter assay with mutagenesis demonstrated that c-Jun regulates the activity of the GGCT promoter via AP-1 consensus sequence. Gene expression analysis revealed that GGCT knockdown showed a repressive effect on the Delta-Notch pathway and decreased Notch1 expression. Notch1 knockdown alone inhibited the GSC proliferation, confirming that Notch1 is functional in this model. Forced expression of the Notch1 intracellular domain restored the growth inhibitory effect of GGCT knockdown. Moreover, GGCT knockdown inhibited GSC tumorigenic potential in vivo. These results indicate that GGCT, whose expression is promoted by c-Jun, plays an important role in the proliferation and tumorigenic potential of GSCs, and that the phenotype caused by its knockdown is contributed by a decrease in Notch1. Thus, GGCT may represent a novel therapeutic target for attacking GSCs.

Singapore grouper iridovirus VP12 evades the host antiviral immune response by targeting the cGAS-STING signalling pathway.

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus. Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

The essential function of cathepsin X of the orange-spotted grouper, Epinephelus coioides during SGIV infection

Cathepsin X, a class of cysteine proteases in the lysosome, involved in intracellular protein degradation processes. Numerous reports revealed that many kinds of cysteine proteases played a crucial role in pathogen invasion. To investigate the relationship between cathepsin X of teleost fish and virus infection, EcCX was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcCX included 909 nucleotides and encoded a protein consisting of 302 amino acids, which shared 75% and 56% identity with zebrafish and humans, respectively. The protein EcCX mainly consisted of a signal peptide (1–19 aa), a pro-pre-peptide region (20–55 aa), and a mature cysteine protease region (56–302 aa). Subcellular localization analysis showed that EcCX was mainly distributed in the cytoplasm, but EcCX ectoped to the vicinity of apoptotic vesicles in FHM cells during SGIV infection. Following stimulation with SGIV or Poly (dA:dT), there was a notable rise in the expression levels of EcCX. EcCX overexpression facilitated virus infection, upregulated the production of inflammatory factors, and induced the activation of the NF-κB promoter. Furthermore, the overexpression of EcCX also accelerated the process of SGIV-induced apoptosis, potentially by enhancing the promoter activity of P53 and AP-1. Overall, our findings demonstrated a correlation between the function of EcCX and SGIV infection, providing a new understanding of the mechanisms involved in fish virus infection.

Homeobox B9 promotes the invasion and metastasis of hepatocellular carcinoma cells via the EZH2–MIR203A–SNAI2 axis

BackgroundResearch has elucidated that homeobox B9 (HOXB9), an important transcriptional activator, plays a pivotal role in promoting the invasion and metastasis of hepatocellular carcinoma (HCC) cells. However, the mechanism by which HOXB9 promotes the invasion and metastasis of HCC cells is incompletely understood and needs further exploration.MethodsHOXB9 and snail family transcriptional repressor 2 (SNAI2) expression were analyzed using qRT-PCR and western blotting. The invasion and metastasis of hepatocellular carcinoma (HCC) cells were investigated using in vitro and in vivo assays. The H3K27me3 enrichment and HOXB9 interaction with microRNA 203a (MIR203A) or SNAI2 were detected using ChIP-qPCR. Transcriptional activities of SNAI2 and MIR203A promoter were detected using dual-luciferase reporter assays. Co-IP and GST pull-down assays were performed to confirm the binding between HOXB9 and EZH2.ResultsHOXB9 and SNAI2 were highly expressed in HCC tissues and their expression was positively intercorrelated and associated with poor prognosis in patients with HCC. In vitro and in vivo experiments confirmed that HOXB9 can upregulate the expression of SNAI2 to promote the invasion and metastasis of HCC cells. Furthermore, HOXB9 elevated SNAI2 expression by inhibiting MIR203A expression, a tumor suppressor gene, in HCC cells. Mechanistically, HOXB9 recruited enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) through interaction with its WD-binding domain, which increased EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) at the MIR203A promoter region, in turn repressing the transcriptional activity and expression of MIR203A and consequently increasing the SNAI2 level in HCC cells. Finally, empirical evidence from in vitro and in vivo studies confirmed that mitigation of the HOXB9-mediated enhancement of epigenetic silencing of MIR203A inhibited SNAI2 expression, impeding the invasion and metastasis of HCC cells.ConclusionsOur study reveals a novel mechanism by which HOXB9 promotes the invasion and metastasis of HCC cells and expands the understanding of the function of HOXB9 in tumor progression and provides a novel therapeutic strategy for curtailing HCC invasion and metastasis.

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.

Histone H4K12 lactylation promotes malignancy progression in triple-negative breast cancer through SLFN5 downregulation

Lactylation, a newly identified post-translational modification, is uncertain in its implication in triple-negative breast cancer (TNBC). In this study, we analyzed 60 TNBC samples using immunohistochemical staining and revealed elevated levels of pan-lactylated proteins and specific histone H4K12 lactylation in tumor tissues, correlating with TNBC progression. Lactate exposure in TNBC cell lines significantly induced lysine lactylation at the H4K12 site, leading to alterations in gene profiles and reduced apoptosis. These effects were attenuated by DCA or sodium Oxamate, inhibitors of endogenous lactate production. Gene sequencing showed an increase in Schlafen 5 (SLFN5) expression in TNBC cells treated with Oxamate, contrasting with the effects of lactate exposure. Analysis of TNBC tissues showed a negative correlation between H4K12 lactylation and SLFN5 protein levels. Overexpression of SLFN5 countered the effects of lactate on apoptosis and tumor growth, highlighting its pivotal role in TNBC malignancy. CUT&Tag sequencing indicated that lactylated H4K12 potentially binds to the SLFN5 promoter region. Luciferase reporter assays further verified that lactate-induced suppression of SLFN5 promoter activity is mediated by wild-type H4K12, but not by its R or A mutants, verified by both in vitro and in vivo apoptosis detection in response to lactate and Oxamate stimulation. These results establish that H4K12 lactylation, induced by lactate in TNBC cells, specifically suppresses SLFN5 expression, contributing to TNBC malignancy. Our findings illuminate a critical histone lactylation-dependent carcinogenic pathway in TNBC.

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

Rs2564978(T) allele associated with severe influenza a disrupts binding site for myeloid differentiation factor PU.1 and reduces <i>CD55/DAF</i> gene promoter activity in macrophages

An inhibitor of the complement system CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity during influenza A infection, as well as with vascular complications in pathologies involving excessive activation of the complement system. Using a luciferase reporter system, we performed functional analysis of the single nucleotide polymorphism rs2564978 located in the promoter of the CD55 gene in human pro-monocytic cell line U937. We have shown a decreased activity in activated U937 cells of the CD55 gene promoter carrying minor rs2564978(T) allele associated with the severe course of influenza A(H1N1)pdm09. Using bioinformatic resources, we determined that transcription factor PU.1 can potentially bind to the CD55 promoter region containing rs2564978 in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was determined by genetic knockdown of PU.1 using small interfering RNAs under specific monocyte activation conditions.

CodY controls the SaeR/S two-component system by modulating branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus.IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.