Electrophoretic Mobility Shift Assay Research Articles

A bZIP type transcription factor from Picrorhiza kurrooa modulates the expression of terpenoid biosynthesis genes in Nicotiana benthamiana

bZIP family of transcription factors are DNA-binding proteins which bind ACGT motifs in the upstream promoter region of genes. bZIP proteins play significant roles in regulation of plant growth, secondary metabolism, development, defense and response to various abiotic and biotic stresses. Up to now, there is no report on molecular and functional characterization of bZIP transcription factors in Picrorhiza kurrooa, a medicinally important endangered herb of north western Himalayan region. The species is widely used in several commercially available drug formulations like livomap, livocare and livplus for treatment of liver disorders. The medicinal properties of P. kurrooa are due to bioactive picrosides, synthesized by isoprenoid pathway. The promoters of regulatory genes viz., Pkdxs and Pkhmgr of picroside biosynthesis pathway have been reported to contain ACGT cis-acting elements (bzip motifs) at different locations. In this study, the functionality of ACGT motifs was confirmed by electrophoretic mobility shift assay (EMSA). Full length PkbZIP gene was cloned through RACE method. In silico analysis revealed PkbZIP contained a conserved signature bZIP domain (N-x7-R/K-x9-L-x6-L-x6-L). Transcriptional activation and subcellular localization assay confirmed nuclear localization of PkbZIP. EMSA with purified PkbZIP confirmed in vitro binding of PkbZIP to ACGT elements in Pkdxs and Pkhmgr promoters. The expression pattern of PkbZIP positively correlated with the picroside (active medicinal component of the plant) content in different tissues, at 15 °C and under light conditions. Transient overexpression in Nicotiana benthamiana suggested a role of PkbZIP in terpenoid biosynthesis. PkbZIP might prove as potential candidate gene to be used in genetic engineering program for enhancing the picroside content in P. kurrooa.

Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

ZmABF4-ZmVIL2/ZmFIP37 module enhances drought tolerance in maize seedlings.

Drought, as a primary environmental factor, imposes significant constraints on developmental processes and productivity of plants. PHDs were identified as stress-responsive genes in a wide range of eukaryotes. However, the regulatory mechanisms governing PHD genes in maize under abiotic stress conditions are still largely unknown and require further investigation. Here, we identified a mutant, zmvil2, in the EMS mutant library with a C to T mutation in the exon of the Zm00001d053875 (VIN3-like protein 2, ZmVIL2), resulting in premature termination of protein coding. ZmVIL2 belongs to PHD protein family. Compared to WT, zmvil2 mutant exhibited increased sensitivity to drought stress. Consistently, overexpression of ZmVIL2 enhances drought resistance in maize. Y2H, BiFC, and Co-IP experiments revealed that ZmVIL2 directly interacts with ZmFIP37 (FKBP12-interacting protein of 37). zmfip37 knockout mutants also exhibit decreased drought tolerance. Interestingly, we demonstrated that ZmABF4 directly binds to the ZmVIL2 promoter to enhance its activity in yeast one hybrid (Y1H), electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assays. Therefore, we uncovered a novel model ZmABF4-ZmVIL2/ZmFIP37 that promotes drought tolerance in maize. Overall, these findings have enriched the knowledge of the functions of PHD genes in maize and provides genetic resources for breeding stress-tolerant maize varieties.

Evolutionary relationship of moso bamboo forms and a multihormone regulatory cascade involving culm shape variation.

Moso bamboo (Phyllostachys edulis) known as Mao Zhu (MZ) in Chinese exhibits various forms with distinct morphological characteristics. However, the evolutionary relationship among MZ forms and the mechanisms of culm shape variation are still lacking. Here, the main differences among MZ forms were identified as culm shape variation, which were confirmed by analysing MZ forms (799 bamboo culms) and MZ (458 bamboo culms) populations. To unravel the genetic basis underlying the morphological variations, 20 MZ forms were subjected to whole-genome resequencing. Further analysis yielded 3 230 107 high-quality SNPs and uncovered low genetic diversity and high genotype heterozygosity associated with MZ forms' formation. By integrating the SNP data of 427 MZ individuals representing 15 geographic regions, the origins of eight MZ forms were successfully traced using the phylogenetic tree and the identified common heterozygous loci. Meanwhile, transcriptomic analysis was performed using shoots from MZ and its two forms with culm shape variation. The results, combined with genomic analyses, demonstrated that hormone signalling related genes played crucial roles in culm variation. Co-expression network analysis uncovered genes associated with multiple plant hormone signal transduction, especially auxin and cytokinin were involved in culm shape variation. Furthermore, the regulatory relationships of a specific transcription factor and their target genes associated with auxin and ethylene signalling were validated by yeast one-hybrid, electrophoretic mobility shift assays, and dual-luciferase reporter. Overall, this study provides important insights into the culm shape variation formation in bamboo, which facilitates to breed new varieties with novel culms.

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

An electrophoretic mobility shift assay using the protein isolated from host plants

BackgroundThe electrophoretic mobility shift assay (EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA.ResultsHere, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent EMSA method (PPF-EMSA). In PPF-EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent EMSA and super-shift fluorescent EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs.ConclusionAs transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.

A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D– Phenotypes Despite Normal RHD Exons

Introduction: With over 360 blood group antigens in systems recognized, there are antigens, such as RhD, which demonstrate a quantitative reduction in antigen expression due to nucleotide variants in the non-coding region of the gene that result in aberrant splicing or a regulatory mechanism. This study aimed to evaluate bioinformatically predicted GATA1-binding regulatory motifs in the RHD gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal RHD exons. Methods: Publicly available open chromatin region data were overlayed with GATA1 motif candidates in RHD. Genomic DNA from weak D, Del or D– samples with normal RHD exons (n = 13) was used to confirm RHD zygosity by quantitative PCR. Then, RHD promoter, intron 1, and intron 2 regions were amplified for Sanger sequencing to detect potential disruptions in the GATA1 motif candidates. Electrophoretic mobility shift assay (EMSA) was performed to assess GATA1-binding. Luciferase assays were used to assess transcriptional activity. Results: Bioinformatic analysis identified five of six GATA1 motif candidates in the promoter, intron 1 and intron 2 for investigation in the samples. Luciferase assays showed an enhancement in transcription for GATA1 motifs in intron 1 and for intron 2 only when the R2 haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel RHD c.1–110A>C variant disrupted the GATA1 motif in the promoter which was supported by a lack of a GATA1 supershift in the EMSA and 73% transcriptional activity in the luciferase assay. Two samples were D+/D– chimeras. Conclusion: The bioinformatic predictions enabled the identification of a novel DEL allele, RHD c.1–110A>C, which disrupted the GATA1 motif in the proximal promoter. Although the majority of the samples investigated here remain unexplained, we provide GATA1 targets which may benefit future RHD regulatory investigations.

The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa

Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.

Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Overexpression of a pearl millet WRKY transcription factor gene, PgWRKY74, in Arabidopsis retards shoot growth under dehydration and salinity-stressed conditions.

Pearl millet (Cenchrus americanus) is a cereal crop that can tolerate high temperatures, drought, and low-fertility conditions where other crops lose productivity. However, genes regulating this ability are largely unknown. Transcription factors (TFs) regulate transcription of their target genes, regulate downstream biological processes, and thus are candidates for regulators of such tolerance of pearl millet. PgWRKY74 encodes a group IIc WRKY TF in pearl millet and is downregulated by drought. PgWRKY74 may have a role in drought tolerance. The objective of this study was to gain insights into the physiological and biochemical functions of PgWRKY74. Yeast one-hybrid and gel shift assays were performed to examine transcriptional activation potential and deoxyribonucleic acid (DNA)-binding ability, respectively. Transgenic Arabidopsis thaliana plants overexpressing PgWRKY74-green fluorescent protein (GFP) fusion gene were generated and tested for growth and stress-responsive gene expression under mannitol and NaCl-stressed conditions. A construct with PgWRKY74 enabled yeast reporter cells to survive on test media in the yeast one-hybrid assays. The electrophoretic mobility of DNA with putative WRKY TF-binding motifs was lower in the presence of a recombinant PgWRKY74 protein than its absence. The PgWRKY74-GFP-overexpressing Arabidopsis plants exhibited smaller rosette areas than did wild-type plants under mannitol-stressed and NaCl-stressed conditions, and exhibited weaker expression of RD29B, which is induced by the stress-related phytohormone abscisic acid (ABA), under the mannitol-stressed condition. PgWRKY74 have transcriptional activation potential and DNA-binding ability, and can negatively regulate plant responses to mannitol and NaCl stresses, possibly by decreasing ABA levels or ABA sensitivity.

Targeted modulation of gene expression through receptor-specific delivery of small interfering RNA peptide conjugates.

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F7,P34]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects.

Cytochrome P450 SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11

Accumulating salinity in soil critically affected growth, development, and yield in plant. However, the mechanisms of plant against salt stress largely remain unknown. Herein, we identified a gene named SmCYP78A7a, which encoded a cytochrome P450 monooxygenase and belonged to the CYP78A sub-family, and its transcript level was significantly up-regulated by salt stress and down-regulated by dehydration stress. SmCYP78A7a located in the endoplasmic reticulum. Silencing of SmCYP78A7a enhanced susceptibility of eggplant to salt stress, and significantly down-regulated the transcript levels of salt stress defense related genes SmGSTU10 and SmWRKY11 as well as increased hydrogen peroxide (H2O2) content and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) enzyme activities. In addition, SmCYP78A7a transient expression enhanced eggplant tolerance to salt stress. By chromatin immunoprecipitation PCR (ChIP-PCR), luciferase reporter assay, and electrophoretic mobility shift assay (EMSA), SmWRKY11 activated SmCYP78A7a expression by directly binding to the W-box 6–8 (W-box 6, W-box 7, and W-box 8) within SmCYP78A7a promoter to confer eggplant tolerance to salt stress. In summary, our finds reveal that SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11, and provide a new insight into regulatory mechanisms of eggplant to salt stress.

Transcription factor EgGRP2A regulates EgFATA expression and promotes oleic acid accumulation in oil palm (Elaeis guineensis)

The oil palm (Elaeis guineensis) is emerging as the world's most important and prolific oilseed crop, celebrated for its impressive oil yield. However, the molecular intricacies that govern lipid metabolism and fatty acid accumulation in oil palm fruits remain relatively underexplored. This study reveals a significant correlation between the expression of EgGRP2A, a transcription factor, and the expression of EgFATA in the oil palm. Yeast one-hybrid analysis and electrophoretic mobility shift assays (EMSA) reveal and confirm the binding interactions between EgGRP2A and the promoter region of EgFATA. Subsequent experiments in oil palm protoplasts show that transient overexpression of EgGRP2A leads to a marked upregulation of EgFATA expression. Conversely, downregulation of EgGRP2A in transgenic oil palm embryoids leads to a significant reduction in EgFATA expression. Metabolite profiling in the transgenic embryoids reveals a significant reduction in unsaturated fatty acids, particularly oleic acid. These findings promise profound insights into the regulatory orchestration of EgFATA and the synthesis of fatty acids, particularly oleic acid, in the oil palm. Furthermore, the results lay the foundation for future breeding and genetic improvement efforts aimed at increasing oleic acid content in oil palm varieties.

OsBBP1, a newly identified protein containing DUF630 and DUF632 domains confers drought tolerance in rice

Domain of unknown function (DUF) protein families, which are uncharacterized and numerous within the Pfam database. Recently, studies have demonstrated that DUFs played crucial roles in plant development, but whether, or how, they function in drought resistance remain unclear. In this study, we identified the Os03g0321500 gene, encoding OsbZIP72 binding protein 1 (OsBBP1), as a target of OsbZIP72 using chromatin immunoprecipitation sequencing in rice. OsBBP1 is a novel member of DUFs, which localize both in the nuclei and cytoplasm of rice protoplasts. Furthermore, yeast one-hybrid and electrophoretic mobility shift assays confirmed the specific binding between OsbZIP72 and OsBBP1. Additionally, a luciferase reporter analysis illustrated that OsbZIP72 activated the expression of OsBBP1. Drought tolerance experiments demonstrate that the OsBBP1 CRISPER-CAS9 transgenic mutants were sensitive to drought stress, but the transgenic OsBBP1 over-expressing rice plants showed enhanced drought resistance. Moreover, drought tolerance experiments in a paddy field suggested that OsBBP1 contributed to less yield or yield-related losses under drought conditions. Mechanistically, OsBBP1 might confer drought resistance by inducing more efficient reactive oxygen species (ROS) scavenging. Several ROS scavenging-related genes showed increased expression levels in OsBBP1 overexpression lines and decreased expression levels in OsBBP1 CRISPER-CAS9 mutants under drought conditions. Thus, OsBBP1, acting downstream of OsbZIP72, contributes to drought resistance and causes less yield or yield-related losses under drought conditions.

A novel IKZF1 variant in a family with autosomal dominant CVID: A case for expanding exon coverage in inborn errors of immunity

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20–30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high.

Targeting G4 motifs of various stem cell makers with designed peptide for therapeutic applications

Noncanonical secondary structures formed by Guanine-rich DNA sequences fold into four-stranded structures called the G-quadruplexes (G4s). Targeting G-quadruplexes is considered an attractive approach toward drug intervention. Here, we have studied the targeting of G4s of stem cell markers with designed short peptide (named as QW10) using biophysical and biochemical techniques. Our CD studies showed that G4 sequences of stem cell markers formed mixed G-quadruplexes in 100 mM Na+, 100 mM K+ and 100 mM K+ +40 wt% PEG 200. On titrating these structures with an increasing concentration of QW10 peptide, we observed a significant decrease in CD intensity followed by the complete disappearance of G4 CD signatures confirming their destabilization not only in dilute conditions but also under cell-mimicking molecular crowding conditions. Our electrophoretic mobility shift assay and significant decrease in the Tm values confirmed the significant destabilization of G4 structures Fluorescence results showed the formation of high-affinity G4 complex-peptide complex with binding affinities in the micromolar (µM) range of 2–8 µM in different ionic conditions. First time, this study may give insight into the use of peptides as leads for the development of more potent and selective ligands to regulate the potential therapeutic applications of cancer stem cell markers.

Decreased RNA-binding protein heterogeneous nuclear ribonucleoprotein U improves multiple myeloma sensitivity to lenalidomide.

Multiple myeloma (MM) is an incurable plasma cell cancer in the bone marrow. Immunomodulatory drugs, such as lenalidomide (LEN) and pomalidomide, are backbone agents in MM treatment, and LEN resistance is commonly seen in the MM clinic. In this study, we presented that heterogeneous nuclear ribonucleoprotein U (hnRNPU) affected MM resistance to LEN via the regulation of target mRNA translation. hnRNPULow MM cells exhibited upregulated CRBN and IKZF1 proteins, stringent IKZF1/3 protein degradation upon LEN addition and increased sensitivity to LEN. RNA pulldown assays and RNA electrophoretic mobility shift assays revealed that hnRNPU bound to the 3'-untranslated region of CRBN and IKZF1 mRNA. A sucrose gradient assay suggested that hnRNPU specifically regulated CRBN and IKZF1 mRNA translation. The competition of hnRNPU binding to its target mRNAs by small RNAs with hnRNPU-binding sites restored MM sensitivity to LEN. hnRNPU function invivo was confirmed in an immunocompetent MM mouse model constructed by the inoculation of Crbn-humanized murine 5TGM1 cells into CrbnI391V/+ mice. Overall, this study suggests a novel mechanism of LEN sensitivity in which hnRNPU represses CRBN and IKZF1 mRNA translation.

Cold-shock Domain Protein A (CSDA) Influences Hepatocyte Growth Factor-medicated Cell Proliferation and Metastasis in Gastric Cancer Cells.

A role for cold-shock domain (CSD) proteins in abnormal cell proliferation has been suggested in the literature. The aim of this study was to investigate the effect of hepatocyte growth factor (HGF)-induced up-regulation of CSD protein A (CSDA) expression on vascular endothelial growth factor (VEGF) expression and its role in gastric cancer cell invasion and proliferation. We assessed effects on two gastric cancer cell lines using reverse transcription-polymerase chain reaction, western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and CSDA knockdown with short hairpin RNA. Hepatocyte growth factor (HGF) elevates CSDA levels in gastric cancer cell lines. To elucidate the mechanism by which HGF prompts CSDA expression and its impact on vascular endothelial growth factor (VEGF), we applied the Mitogen Activated Protein Kinase (MAPK) inhibitor PD098059 and conducted analyses using western blot. Following the administration of PD098059, a reduction in the protein levels of HGF-stimulated VEGF was observed. Additionally, silencing of CSDA resulted in diminished levels of both VEGF and phosphorylated extracellular signal-regulated kinase (ERK). The suppression of CSDA also led to reduced HGF-induced cell proliferation and diminished invasive capabilities in vitro. Furthermore, our research pinpointed a potential activator protein-1 (AP-1) binding site within the VEGF promoter zone, validating its activity via chromatin immunoprecipitation assays. Electrophoretic mobility shift assays further disclosed that HGF-induced CSDA augmentation correlates with an increase in AP-1 binding to VEGF. CSDA is crucial for the proliferation of gastric cancer cells, and the inhibition of this protein could impede the advancement of gastric cancer.

Unraveling the role of PlARF2 in regulating deed formancy in Paeonia lactiflora.

PlARF2 can positively regulate the seed dormancy in Paeonia lactiflora Pall. and bind the RY cis-element. Auxin, a significant phytohormone influencing seed dormancy, has been demonstrated to be regulated by auxin response factors (ARFs), key transcriptional modulators in the auxin signaling pathway. However, the role of this class of transcription factors (TFs) in perennials with complex seed dormancy mechanisms remains largely unexplored. Here, we cloned and characterized an ARF gene from Paeonia lactiflora, named PlARF2, which exhibited differential expression levels in the seeds during the process of seed dormancy release. The deduced amino acid sequence of PlARF2 had high homology with those of other plants and contained typical conserved Auxin_resp domain of the ARF family. Phylogenetic analysis revealed that PlARF2 was closely related to VvARF3 in Vitis vinifera. The subcellular localization and transcriptional activation assay showed that PlARF2 is a nuclear protein possessing transcriptional activation activity. The expression levels of dormancy-related genes in transgenic callus indicated that PlARF2 was positively correlated with the contents of PlABI3 and PlDOG1. The germination assay showed that PlARF2 promoted seed dormancy. Moreover, TF Centered Yeast one-hybrid assay (TF-Centered Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay analysis (Dual-Luciferase) provided evidence that PlARF2 can bind to the 'CATGCATG' motif. Collectively, our findings suggest that PlARF2, as TF, could be involved in the regulation of seed dormancy and may act as a repressor of germination.