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At least 19 recordsLinked to original sources

PubMed1992

EMSA: a SAAM service for the estimation of population parameters based on model fits to identically replicated experiments.

This paper presents a new technique for the aggregation of models to produce population parameter estimates based on a set of identically replicated experiments. After describing the theoretical basis for the technique we discuss tactical and strategic issues associated with its implementation in the SAAM software. Finally, we demonstrate its utility in the aggregation of models fitted to four simulated experiments.

Algorithms↗

PubMed1992

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.

Animals↗

PubMed2026

Azotobacter vinelandii AmrZ is a global regulator linking alginate production and c-di-GMP homeostasis.

Azotobacter vinelandii, a member of the Pseudomonadaceae, produces the exopolysaccharide alginate during vegetative growth; however, the circuitry linking alginate biosynthesis to lifestyle transitions remains poorly defined. Here, we show that the Ribbon-Helix-Helix transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels and motility. Deletion of amrZ abolished alginate synthesis, whereas chromosomal complementation restored it. A PalgD-gusA fusion and RT-qPCR demonstrated that algD, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of algD, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: PamrZ-gusA activity decreased in ΔamrZ, and purified AmrZ bound the amrZ promoter in EMSA. Moreover, PamrZ activity required the sigma factor AlgU, consistent with the presence of an AlgU promoter; this positive, AlgU-dependent feedback may stabilize AmrZ under alginate-inducing conditions. To probe AmrZ control of c-di-GMP, we implemented a riboswitch-based biosensor in A. vinelandii. The ΔamrZ strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant, whereas a phosphodiesterase mutant displayed elevated output, validating the assay. RNA-seq and RT-qPCR identified two DGC genes, AVAEIV_RS11610 and AVAEIV_RS18795, as AmrZ-activated targets; EMSA verified direct binding at the RS11610 regulatory region. By contrast, transcription of the principal vegetative DGC AvGReg was not AmrZ-regulated. Lower c-di-GMP in ΔamrZ correlated with larger swimming halos. Collectively, these genetic, biochemical and transcriptomic data support a model in which AmrZ directly activates algD and elevates c-di-GMP via selected DGCs, thereby promoting alginate synthesis while reducing motility. RNA-seq data also indicate that AmrZ influences broader cellular programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the Pseudomonas genus and reveals important differences in its targets and regulatory mechanisms.

Azotobacter vinelandii↗

PubMed1992

The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-kappa B-independent activation of the kappa B motif.

The kappa B transcriptional enhancer motif, present in many viruses, is broadly active in many cell types. It is recognized by c-Rel/HIVEN86A in DNA affinity precipitation (DNAP) assays and by the Rel-related p50 and p65 subunits of the nuclear factor NF-kappa B in electrophoretic mobility shift assays (EMSA). We have analyzed activities that bind the human immunodeficiency virus type 1 and simian virus 40 kappa B motifs in two human leukemia cell lines, Jurkat and H9. In both DNAP and EMSA analyses of Jurkat cell extracts, we detected multiple kappa B motif-binding activities in addition to c-Rel/HIVEN86A and p50-p65 NF-kappa B. In Jurkat cell nuclear extracts, EMSA analysis revealed at least six specific DNA-protein complexes, of which one comigrated with the p50-p65 NF-kappa B complex. Formation of all six complexes was enhanced by stimulation of the cells with phorbol 12-myristate-13-acetate and phytohemagglutinin but was differentially affected by the salt concentration in the binding reaction and by the conditions of Jurkat cell growth. Nuclear extracts from both unstimulated and stimulated H9 cells revealed similar levels of five kappa B motif-specific complexes, all of which displayed mobilities distinct from those of the Jurkat cell complexes. Indeed, a complex corresponding to p50-p65 NF-kappa B was not detectable in nuclear extracts from unstimulated H9 cells although such a complex was apparent in nuclear extracts from stimulated H9 cells. In contrast to the inducibility of a p50-p65 NF-kappa B-like complex, transcriptional enhancers composed of multimerized kappa B motifs displayed similar high levels of activity in both the unstimulated and stimulated H9 cells. Thus, the activity of the kappa B motif in H9 cells corresponded to the abundance of the H9 cell-specific kappa B motif complexes and not to the levels of p50-p65 NF-kappa B complex. These results suggest that the broad activity of the kappa B enhancer element is not only due to the broadly distributed NF-kappa B activator but also to cell type-specific kappa B motif-binding activities.

Base Sequence↗

PubMed2026

Binding analysis of the response regulator NarL protein to the promoter of the O6-methylguanine-DNA methyltransferase (ogt) gene in Salmonella Typhimurium.

BACKGROUND: Salmonella Typhimurium (STM) is a gram-negative bacterium that causes severe gastrointestinal disorders in both animals and humans. The regulation of DNA repair genes is critical for maintaining genomic stability of the bacteria. O6-methylguanine DNA methyltransferase (Ogt), plays a vital role in repairing alkylated DNA in STM; however, the transcriptional regulation of ogt gene remains poorly characterized. Furthermore, NarL is a transcriptional regulator, involved in the pathogenesis of STM under anaerobic condition. Therefore, this study investigated the interaction between NarL protein and the promoter region of the ogt gene. METHODS: In this study, narl gene was cloned in pET32a vector and NarL protein was expressed in Escherichia coli BL21 (DE3). Subsequently, the ogt gene promoter (pogt) was selected, amplified, cloned and its activity was evaluated. Electrophoretic mobility shift assay (EMSA), isothermal titration calorimetry (ITC), molecular docking were employed to elucidate the interaction between NarL protein and ogt promoter. Furthermore, the regulatory role of NarL in ogt gene expression was validated in vivo using RT-qPCR and β-galactosidase assay. RESULTS: This study resulted that NarL protein interacts specifically with the ogt promoter, as confirmed by EMSA and ITC, with ΔG of - 9.42 kcal mol⁻¹. Furthermore, RT-qPCR and β-galactosidase assays demonstrated that deletion of narl significantly (P ≤ 0.01) decreased ogt transcript levels and promoter activity than wild Salmonella Typhimurium, whereas exogenous supplementation of recombinant NarL protein restored the expression. These findings suggest that NarL plays a potential regulatory role in ogt gene expression in response to environmental signals. CONCLUSION: These findings highlight an interaction between NarL protein and the promoter region of ogt gene in Salmonella Typhimurium, linking nitrogen metabolism with the DNA repair pathway in STM, which may contribute to the bacterial survival under nitrosative stress.

Salmonella typhimurium↗

PubMed1992

Interferon gamma rapidly induces in human monocytes a DNA-binding factor that recognizes the gamma response region within the promoter of the gene for the high-affinity Fc gamma receptor.

Interferon gamma (IFN-gamma) transcriptionally activates several early-response genes in monocytes that are important for the ultimate phenotype of the activated macrophage. One of these genes is the high-affinity Fc receptor for IgG (Fc gamma RI). Recently, Pearse et al. [Pearse, R.N., Feinman, R. & Ravetch, J. V. (1991) Proc. Natl. Acad. Sci. USA 88, 11305-11309] defined within the promoter region of the Fc gamma RI gene an element, the gamma response region, which was necessary for IFN-gamma-induced enhancement of Fc gamma RI. In this report we describe the induction by IFN-gamma of a DNA-binding factor, FcRF gamma (Fc gamma RI DNA-binding factor, IFN-gamma induced), that specifically recognizes the gamma response region element. Electrophoretic mobility shift assays (EMSAs) demonstrated the presence of FcRF gamma in human monocytes within 1 min after exposure to IFN-gamma. On EMSA, FcRF gamma consisted of two complexes termed FcRF gamma 1 and FcRF gamma 2. The nuclear concentration of FcRF gamma rapidly increased, peaked at 15 min, and then fell after 1-2 hr. Dose-response studies revealed (i) as little as 0.05 ng of IFN-gamma per ml induced FcRF gamma, (ii) maximum activation occurred at 1 ng/ml, and (iii) steady-state levels of Fc gamma RI mRNA closely paralleled that of FcRF gamma. Since FcRF gamma was activated in cells normally not expressing Fc gamma RI RNA, other regulatory mechanisms must control Fc gamma RI-restricted tissue expression. Activation of FcRF gamma by IFN-gamma was inhibited by pretreatment with 500 nM staurosporin and 25 microM phenyl arsine oxide. These data suggest that a kinase and possibly a phosphatase activity are required for IFN-gamma-induced signaling of FcRF gamma in monocytes.

Base Sequence↗

PubMed2025

The toxin-antitoxin system SavRS contributes to vancomycin resistance in vancomycin-intermediate Staphylococcus aureus by mediating cell wall thickening.

BACKGROUND: The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has significantly challenged the treatment of S. aureus infection. Toxin-antitoxin (TA) systems have been reported to mediate bacterial stress adaptation and virulence, but their role in vancomycin resistance remains elusive. This study investigated the vancomycin resistance mechanism regulated by the TA system SavRS in VISA. METHODS: savRS mutants in Mu50 and XN108 were generated via homologous recombination. To investigate the regulatory mechanism of vancomycin resistance mediated by savRS in VISA, phenotypic analyses including MICs, growth kinetics and cell wall thickness measurements were performed. Expression of cell wall synthesis-related genes was analysed using quantitative RT-PCR (RT-qPCR) and promoter-lacZ reporter assay. Electrophoretic mobility shift assay (EMSA) was performed to assess the binding of SavRS to the promoters of the cell wall synthesis-related genes. Pull-down assay identified an upstream regulatory element of savRS associated with vancomycin resistance. Quantitative assessment of bacterial burden in murine organ systems following vancomycin administration revealed the critical regulatory role of savRS in mediating vancomycin resistance in vivo. RESULTS: Compared with the WT, the savRS mutant exhibited enhanced vancomycin sensitivity, accelerated growth and reduced cell wall thickness. Correspondingly, RT-qPCR revealed marked down-regulation of the cell wall synthesis-related genes (glyS, dltA, scdA, pbp2, ddl). EMSA and promoter-lacZ reporter assay confirmed direct binding of SavRS to a conserved promoter motif, MGHYYTCCTCA. Pull-down assay identified UspA as an upstream regulator of SavRS, demonstrating that UspA directly controls savRS transcription and modulates VISA resistance. Mouse infection experiments showed that savRS promotes VISA to vancomycin resistance in vivo. CONCLUSIONS: SavRS critically regulates vancomycin resistance in VISA.

Cell Wall↗

PubMed2026

The transcriptional regulator CasR controls mycobacterial antioxidant defense and biofilm formation via multiple direct targets.

AIMS: The antioxidant defense system of Mycobacterium tuberculosis is critical for pathogenicity and persistence within macrophages, yet the regulatory networks remain poorly understood. This study aims to elucidate the molecular mechanism by which the transcription factor CasR regulates antioxidant defense in mycobacteria through delineation of the regulatory axis linking CasR activity, target gene expression, and the antioxidant phenotype. METHODS AND RESULTS: Using Mycobacterium smegmatis as a model organism, we demonstrate that overexpression of CasR renders the bacteria significantly susceptible to hydrogen peroxide. Electrophoretic mobility shift assay (EMSA) and β-galactosidase reporter analyses reveal that CasR directly binds and represses the promoter of cyp144, an uncharacterized cytochrome P450-encoding gene. Deletion of casRMsmreduces biofilm formation, consistent with the expected derepression of cyp144Msm, a gene that negatively regulates both biofilm and oxidative stress tolerance. EMSA and β-galactosidase activity assays also demonstrate that CasR negatively regulates antioxidant gene katGI, suggesting that CasR exerts a broader, global regulatory role within the mycobacterial antioxidant defense network. Furthermore, we identify isoleucine 18 as a critical residue for the DNA-binding and regulatory function of CasR. CONCLUSION: This study establishes CasR as a pleiotropic transcriptional regulator that directly controls multiple antioxidant genes, including cyp144 and katGI, in mycobacteria. We report a previously unrecognized role for a cytochrome P450 family member in suppressing bacterial antioxidant capacity, as cyp144 overexpression reduces biofilm formation. These findings provide a valuable reference for further investigation into mycobacterial antioxidant mechanisms and identify CasR and Cyp144 as potential targets for the development of anti-tuberculosis drugs.

Biofilms↗

PubMed2026

An XRE-type regulator in Streptococcus mutans plays an important role in brpA expression and oxidative stress tolerance response.

This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the ∆xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response.IMPORTANCEStreptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.

Streptococcus mutans↗

PubMed2026

Transcription factor LbUBC positively regulates salt gland development and salt tolerance by directly binding to the LbTTG1 promoter and repressing its transcription.

KEY MESSAGE：: LbUBC enhances salt tolerance by promoting salt gland development via repressing LbTTG1, revealing a synergisticregulatory mechanism in Limonium bicolor. In the context of increasingly severe soil salinization, salt-tolerant genetic resources from halophytes show great application potential. In particular, the recretohalophyte Limonium bicolor, which possesses specialized salt gland structures, has become a key model for deciphering the molecular mechanisms underlying salt tolerance and salt gland development. In this study, using LbTTG1-overexpressing and -silenced lines, we demonstrate that LbTTG1 negatively regulates salt-gland development and salt tolerance. Through yeast one-hybrid, EMSA, and dual-luciferase assays, Lb7G33228 (LbUBC) was screened and verified as an upstream transcriptional regulator of LbTTG1. LbUBC enhances salt tolerance in L. bicolor by positively regulating salt-gland development, verified using LbUBC silence and overexpression strains. Interestingly, LbUBC represses the expression of its downstream target LbTTG1, thereby releasing the inhibitory effect of LbTTG1 on salt-gland development. In this manner, LbUBC positively regulates salt-gland development, achieving a dynamic balance in the regulation of salt-gland development and salt tolerance in L. bicolor. This study reveals a synergistic regulatory mechanism involving multiple genes, offering new insights for comprehensively dissecting the molecular regulatory network of salt-gland development.

Salt Tolerance↗

PubMed2026

CgMYC2 directly activates jasmonate-induced naringin biosynthesis in Citrus grandis 'Tomentosa'.

CgMYC2 links jasmonate signaling to naringin biosynthesis by binding G-box motifs and activating flavonoid-pathway promoters in Citrus grandis 'Tomentosa' Naringin, the predominant bitter-flavanone glycoside in Citrus grandis 'Tomentosa', has well-characterized biosynthetic enzymes, yet the transcriptional regulators coupling hormonal signals to pathway activation remain poorly understood. We demonstrate that CgMYC2, a jasmonate-responsive bHLH transcription factor, functions as a central activator of naringin biosynthesis. Exogenous methyl jasmonate (MeJA) treatment increased naringin content 3.45-fold in seedlings, coinciding with a rapid 6.6-fold induction of CgMYC2 that preceded the peak transcription of five core biosynthetic genes (CgPAL5, CgCHS, CgFNS, Cg7GlcT, and Cg1,2RhaT). Physical interaction between CgMYC2 and the JAZ protein CgJAZ3 was confirmed by pull-down and Co-IP assays, placing CgMYC2 within the canonical jasmonate signaling cascade. Y1H confirmed CgMYC2 binding to the Cg1,2RhaT promoter, EMSA demonstrated direct G-box-dependent binding to all five pathway promoters, and dual-luciferase assays showed transactivation of all five promoters, with the strongest activation for CgCHS. As complementary chromatin-level support, a single-sample CUT&Tag profile revealed G-box-enriched CgMYC2-associated chromatin regions across jasmonate-responsive and secondary-metabolic loci. Virus-induced gene silencing (VIGS) of CgMYC2 reduced naringin content by ~21% and suppressed biosynthetic gene expression, supporting its positive contribution. Furthermore, heterologous overexpression in tomato activated the flavonoid pathway and elevated 16 flavonoid compounds, consistent with evolutionary conservation of the MYC2-G-box regulatory logic. These findings establish CgMYC2 as a central, JA-responsive activator bridging jasmonate perception and naringin biosynthesis, providing a molecular framework for the targeted improvement of bitter-flavonoid traits in citrus.

Citrus↗

PubMed1992

Interferon-alpha-induced gene expression: evidence for a selective effect of ouabain on activation of the ISGF3 transcription complex.

Binding of interferons (IFNs) to their cell surface receptors stimulates rapid translocation of cytoplasmic proteins to the nucleus and the expression of a variety of cellular genes within minutes. Translocated proteins subsequently bind to the interferon-stimulated response element (ISRE) located in the promoters of all IFN-activated cellular genes. We report here that ouabain, a specific inhibitor of the Na/K ATPase, selectively inhibited transcription of several IFN-alpha-induced cellular RNAs under conditions in which some other well-described signal transduction pathways remained intact. The latter included induction of human metallothionein 2A (HMT2A) by phorbol ester and induction of IP-10 RNA by IFN-gamma. Ouabain itself induced RNA of the protooncogene c-fos which conversely was inhibited by IFN-alpha. Specificity of the ouabain effects on IFN alpha-induced RNAs with respect to a direct action on the Na/K ATPase was shown with a transfected monkey CV-1 cell line which expresses the ouabain-insensitive rat alpha 1 subunit. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from ouabain-treated cells demonstrated that ouabain decreased IFN alpha-induced binding of the ISGF3 complex to the ISRE. Reconstitution experiments showed that this effect of ouabain is not due to the inhibition of IFN alpha activation of the ISGF3 alpha subcomponent, which occurs in the cytoplasm, but a selective depletion of the ISGF3 gamma factor which in concert with activated ISGF3 alpha induces interferon-stimulated gene (54 kDa) transcription. These findings imply that intracellular ion balance can selectively regulate the half-life of the ISGF3 gamma protein or the ability of this protein to complex with ISGF3 alpha to activate IFN alpha-regulated cellular genes.

Base Sequence↗

PubMed2026

Inhibitory mechanism of phloretin on the AgrA LytTR domain-agr operon complex formation and its application in beef.

Staphylococcus aureus (S. aureus) represents a major foodborne pathogen whose enterotoxin production poses significant challenges to food safety due to its high environmental resistance and limited efficacy of conventional sterilization. Since the expression of enterotoxins is predominantly governed by the agr quorum sensing system, targeting this regulatory pathway has become a strategic choice for virulence control. This study elucidated the mechanism by which phloretin, a potential quorum sensing inhibitor, interferes with the agr system to attenuate virulence. To achieve this, the recombinant AgrA LytTR domain was expressed and purified, and its interaction with phloretin was characterized using thermal shift assays (TSA), electrophoretic mobility shift assays (EMSA), and molecular dynamics (MD) simulations. The results showed that phloretin specifically binds to the AgrA LytTR domain, enhancing its thermal stability and disrupting AgrA LytTR-agr operon binding by reducing the free energy of interaction between them, without causing significant structural rearrangement. Mechanistic analysis indicated that phloretin sterically hinders key β-sheet turn residues (HIS169, ASN201, ARG233), thereby impairing DNA recognition, downregulating RNAIII transcription, and inhibiting agr signaling. In cooked beef, phloretin significantly inhibited the secretion of enterotoxins and α-hemolysin, while delaying lipid oxidation and protein degradation, and maintaining the meat texture. These findings suggested that phloretin is a multifunctional substance with anti-virulence, antioxidant, and preservative properties, demonstrating its potential as a natural food preservative.

Phloretin↗

PubMed2025

Multi-omics reveals an ecdysone-activated Eip75B-FABP signaling axis coordinating nutrient metabolism for development in Hermetia illucens.

INTRODUCTION: Efficient nutrient storage is essential for insect development and energy homeostasis; however, the mechanisms coordinating nutrient allocation during ontogeny are not well understood. Elucidating these systems may yield valuable insights to insect metabolic adaptation. OBJECTIVES: This study aimed to identify regulatory modules governing nutrient metabolism in insects, focusing on hormonal and metabolic interplay. METHODS: Multi-omics profiling (proteomics, phosphoproteomics, and transcriptomics) was conducted throughout the life cycle, from egg to adult, to identify metabolic regulators. RNAi was utilized for gene knockdown, followed by qRT-PCR and mitochondrial DNA quantification to evaluate knockdown efficiency and its metabolic implications. Assessments of nutrient metabolism were performed using assays for triglycerides, crude protein, and fatty acid synthase. EMSA and BODIPY staining examined transcriptional regulation and lipid droplet dynamics. RESULTS: Utilizing an integrative multi-omics approach, this study elucidates the temporal metabolic regulators in insects. A conserved regulatory module was identified in which the PPAR homolog, ecdysone-induced protein 75B (Eip75B), functions as a transcriptional activator of fatty acid binding protein (FABP), sustaining lipid metabolic homeostasis during the larval stage. PPARγ modulators (rosiglitazone and GW9662) alter lipid accumulation, along with the expression of Eip75B and FABP, which was measured by qRT-PCR. Furthermore, the deficiency of FABP may reprogram metabolic pathways by inhibiting lipid storage and promoting mitochondrial β-oxidation, as supported by increased mitochondrial DNA copy number, as well as enhancing protein synthesis. This metabolic change could be modulated by ecdysone signaling, as hormonal supplementation effectively rescued the lipid loss phenotype. Our results establish the ecdysone-Eip75B-FABP signaling axis as a central regulatory module that integrates hormonal and nutrient-sensing signals to control insect nutritional metabolism. CONCLUSION: The ecdysone-Eip75B-FABP axis integrates hormonal and nutrient signals to regulate metabolic plasticity, underscoring a universal strategy for developmental energy allocation. The data also offer potential implications for research on metabolic disorders and bioenergy applications.

Animals↗

PubMed2026

The transcription factor SbWRKY6 confers cadmium tolerance via activating SbPLAC8-17 expression in sorghum.

Cadmium (Cd) is a widespread environmental pollutant that severely threatens crop productivity and food safety. However, the regulatory mechanisms underlying Cd detoxification and tolerance in sorghum remain largely elusive. Herein, we functionally characterized SbWRKY6, a Cd‑induced WRKY transcription factor that localizes to the nucleus and functions as a transcriptional activator. Stable overexpression of SbWRKY6 significantly enhanced Cd tolerance in sorghum, as evidenced by improved growth performance, mitigated oxidative damage, and decreased Cd concentration in plant tissues, whereas silencing of SbWRKY6 resulted in a Cd-hypersensitive phenotype with exacerbated toxicity symptoms. Mechanistically, we identified SbPLAC8-17, a member of the Plant Cadmium Resistance (PCR)/PLAC8 family, as a critical downstream target of SbWRKY6. Heterologous expression of SbPLAC8-17 functionally complemented the Cd‑sensitive phenotype of the yeast mutant ∆ycf1 and reduced intracellular Cd accumulation. Further yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter (Dual-LUC) assays confirmed that SbWRKY6 directly binds to the SbPLAC8-17 promoter and transcriptionally activates its expression. In vivo silencing of SbPLAC8-17 significantly impaired cellular Cd²⁺ efflux and aggravated Cd toxicity in sorghum. Additionally, the rapid Cd-induced transcriptional upregulation of SbMPK3 and its direct physical interaction with SbWRKY6 suggest a potential upstream regulatory module that remains to be functionally validated. Collectively, this study elucidates a novel SbWRKY6‑SbPLAC8-17 transcriptional cascade that positively regulates Cd tolerance by facilitating Cd²⁺ efflux, providing promising genetic targets for phytoremediation and molecular breeding of safe sorghum cultivars for Cd-contaminated fields.

Sorghum↗

PubMed2026

GmMYB29 activates Gm4CL3 to enhance soybean resistance to Heterodera glycines.

Soybean cyst nematode is a devastating soil-borne pathogen that severely limits soybean yield worldwide. To uncover downstream target genes of the resistance-associated transcription factor GmMYB29, we combined ChIP-seq and RNA-seq data from T3-generation GmMYB29-overexpressing soybean plants, alongside Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, to screen candidate genes carrying transcription factor binding peaks within the 2000 bp region upstream of transcription start sites (TSS). Four orthogonal molecular assays-yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), dual-luciferase reporter (LUC) system, and GUS histochemical staining-collectively confirmed the specific physical interaction between GmMYB29 and the promoter of Gm4CL3. We generated transgenic soybean hairy roots overexpressing Gm4CL3 (OX-Gm4CL3) and CRISPR-Cas9-mediated Gm4CL3 knockout lines (KO-Gm4CL3), with wild-type (WT) plants serving as controls. Inoculation assays using SCN 3 demonstrated that OX-Gm4CL3 roots displayed substantially improved SCN resistance, while KO-Gm4CL3 roots were hypersusceptible to nematode infection. Mechanistic investigations revealed that Gm4CL3 promotes lignin deposition in root tissues to block SCN penetration. Furthermore, GmMYB29 and Gm4CL3 act synergistically to activate lignin biosynthetic pathways and strengthen soybean resistance against SCN 3 (SCN Race 3, the dominant physiological race in Northeast China). In summary, this study functionally characterizes Gm4CL3 and defines a previously unreported GmMYB29-Gm4CL3 regulatory cascade that mediates plant defense against SCN. This module functions independent of classic SCN resistance loci rhg1/Rhg4, providing new genetic resources for SCN-resistant soybean molecular breeding.

Glycine max↗

PubMed1992

Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism.

Transcription from the human immunodeficiency virus type 1 (HIV-1) provirus is activated by a cellular factor, NF kappa B, recognizing the tandemly repeated 10-base-pair sequences, termed the kappa B sequence, present in the enhancer region within the viral long terminal repeat (LTR). Using electrophoretic mobility shift assay (EMSA), which demonstrates specific DNA-protein interaction in vitro, we could demonstrate that reducto-oxidative modulation of NF kappa B dramatically changes its DNA binding activity and that a cellular physiological reducing catalyst, thioredoxin (TRX) also known as adult T cell leukemia derived factor (ADF), fully restored the DNA-binding activity of the oxidized NF kappa B. We also observed that purified TRX/ADF protein could augment gene expression from HIV LTR as demonstrated by transient chloramphenicol acetyltransferase (CAT) assay. These observations confirmed the previous notion that ADF might be an inducing factor of cellular interleukin-2 receptor alpha subunit (IL-2R alpha) through the kappa B sequence that is a common central cis-regulatory element in both IL-2R alpha and HIV gene expression. These observations indicate that reducto-oxidative regulation (or redox regulation) of a cysteine residue(s) on the NF kappa B molecule might play an important role in its specific DNA interaction and that it might provide a clue to the understanding of a pathway of cellular signal transduction to NF kappa B that is independent from the known pathways involving protein phosphorylation.

Amino Acid Sequence↗

PubMed2025

Phosphoregulation of RAD51AP1 function in homology-directed repair.

Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-Associated-Protein 1 (RAD51AP1) which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types. Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.

CDK1/2↗