Search Results (32)

The head rice rate, defined as the proportion of milled grains retaining at least three-quarters of their original length, has become a limiting factor that restricts the improvement of rice quality in China in recent years. Here, we characterized the role of ETHYLENE RESPONSIVE FACTOR34 (OsERF34), an APETALA2 (AP2/ERF) family TF, in the grain morphology, physiochemical properties, and processing quality of rice. Through CRISPR/Cas9-mediated knockout (Oserf34) and overexpression (OsERF34-OE) in the japonica cultivar ZH11, we demonstrate that OsERF34 exerts dose-dependent effects on grain morphology and processing traits. Oserf34 mutants exhibited significantly elevated chalkiness levels, with a 52.0% increase in percentage of grains with chalkiness(PGWC) and a 65.4% enhancement in chalkiness degree, with disordered and enlarged starch granules, reduced amylose content and skewed chain-length distribution (A/B1 chains increased but B2/B3 chains decreased), and displayed heightened starch solubility and swelling power but diminished milling resistance (shear hardness having fallen by 12.7–16.1% and compression hardness having fallen by 11.2–16.4%), culminating in doubled breakage rates and lower head rice rate (decreased by 6.7–9.0%) during processing. Strikingly, both mutants and OE lines showed analogous grain narrowing, yet the processing quality diverged. Mutants suffered structural fragility, while the OE lines enhanced mechanical robustness (compression hardness increased by 11.4–12.1%). The OsERF34-OE lines achieved 6.5–7.1% higher head rice rates. Our work positions OsERF34 as a dual-function regulator that governs grain morphology, regulating appearance and processing quality. These insights suggest that an overexpression of OsERF34 could improve processing efficiency, potentially laying a foundation for precision breeding. Full article

Genome organization is essential for precise spatial and temporal gene expression and relies on interactions between promoters and distal cis-regulatory elements (CREs), which constitute ~8% of the human genome. For the cystic fibrosis transmembrane conductance regulator (CFTR) gene, tissue-specific expression, especially in the pancreas, remains poorly understood. Unraveling its regulation could clarify the clinical heterogeneity observed in cystic fibrosis and CFTR-related disorders. To understand the role of 3D chromatin architecture in establishing tissue-specific expression of the CFTR gene, we mapped chromatin interactions and epigenomic regulation in Capan-1 pancreatic cells. Candidate CREs are validated by luciferase reporter assay and CRISPR knock-out. We identified active CREs not only around the CFTR gene but also outside the topologically associating domain (TAD). We demonstrate the involvement of multiple CREs upstream and downstream of the CFTR gene and reveal a cooperative effect of the −44 kb, −35 kb, +15.6 kb, and +37.7 kb regions, which share common predicted transcription factor (TF) motifs. We also extend our analysis to compare 3D chromatin conformation in intestinal and pancreatic cells, providing valuable insights into the tissue specificity of CREs in regulating CFTR gene expression. Full article

Fungal secondary metabolism (SM) is highly correlated with physiological processes that are typically regulated by pleiotropic regulators. In this study, we purposefully altered PratfA, a crucial regulator associated with oxidative stress in Penicillium raistrickii CGMCC 3.1066. After the knockout of PratfA, a novel polyketide (PK) raistrilide A (1) and the known nonribosomal peptide (NRP) tunicoidine (2) subsequently disappeared. Notably, compound 1 is a rare octaketone derivative and contains two unsubstituted cis-double bonds, demonstrating its unique biosynthetic mechanism. The knockout of PratfA resulted in the disappearance of 1–2 and greatly increased the susceptibility of ΔPratfA mutant strain to oxidative stress, rendering it nearly impossible to survive in such environments. At present, the OE⸬PratfA strain showed no phenotypic or oxidative stress sensitivity differences compared to the wild-type strain. Our findings highlight that the oxidative-stress-related transcription factor (TF) PratfA influences SM pathways in P. raistrickii. The manipulation of regulatory factors can guide the discovery of novel natural products (NPs). Full article

Salvia miltiorrhiza, the valuable traditional Chinese medicinal plant, has been used in clinics for thousands of years. The water-soluble salvianolic acid compounds are bioactive substances used in treating many diseases. Gibberellins (GAs) are growth-promoting phytohormones that regulate plant growth and development. Previous studies have demonstrated that GAs can promote salvianolic acid accumulation in S. miltiorrhiza; however, the underlying mechanism requires further investigation. Here, we identified a GA-induced R2R3MYB transcription factor (TF), SmMYB71, from a transcriptome library of GA-treated S. miltiorrhiza. SmMYB71 was highly expressed in the root of S. miltiorrhiza and localized to the nucleus. SmMYB71-knockout hairy roots showed higher salvianolic acid accumulation compared to wild lines. Overexpressing SmMYB71 in S. miltiorrhiza hairy roots significantly decreased the content of salvianolic acid by downregulating key salvianolic acid biosynthesis enzymes such as SmRAS and SmCYP98A14. The GCC box in the promoter of SmMYB71 can bind with SmERF115, suggesting that SmMYB71 is regulated by SmERF115 in salvianolic acid biosynthesis. These findings demonstrate a novel regulatory role of SmMYB71 in GA-mediated phenolic acid biosynthesis. With the development of CRISPR/Cas9-based genome editing technology, the SmMYB71 regulation mechanism of salvianolic acid biosynthesis provides a potential target gene for metabolic engineering to increase the quality of S. miltiorrhiza. Full article

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars. Full article

Transcriptional regulatory networks (TRNs) associated with recombinant protein (rProt) synthesis in Yarrowia lipolytica are still under-described. Yet, it is foreseen that skillful manipulation with TRNs would enable global fine-tuning of the host strain’s metabolism towards a high-level-producing phenotype. Our previous studies investigated the transcriptomes of Y. lipolytica strains overproducing biochemically different rProts and the functional impact of transcription factors (TFs) overexpression (OE) on rProt synthesis capacity in this species. Hence, much knowledge has been accumulated and deposited in public repositories. In this study, we combined both biological datasets and enriched them with further experimental data to investigate an interplay between TFs and rProts synthesis in Y. lipolytica at transcriptional and functional levels. Technically, the RNAseq datasets were extracted and re-analyzed for the TFs’ expression profiles. Of the 140 TFs in Y. lipolytica, 87 TF-encoding genes were significantly deregulated in at least one of the strains. The expression profiles were juxtaposed against the rProt amounts from 125 strains co-overexpressing TF and rProt. In addition, several strains bearing knock-outs (KOs) in the TF loci were analyzed to get more insight into their actual involvement in rProt synthesis. Different profiles of the TFs’ transcriptional deregulation and the impact of their OE or KO on rProts synthesis were observed, and new engineering targets were pointed. Full article

Iron is a vital element involved in a plethora of metabolic activities. Mammalian systemic iron homeostasis is mainly modulated by hepcidin, the synthesis of which is regulated by a number of proteins, including the hemochromatosis-associated proteins Hfe and Transferrin Receptor 2 (TfR2). Macrophages play versatile functions in iron homeostasis by storing iron derived from the catabolism of erythrocytes and supplying iron required for erythropoiesis. The absence of Hfe in macrophages causes a mild iron deficiency in aged mice and leads to an overproduction of the iron exporter Ferroportin 1 (Fpn1). Conversely, TfR2 gene silencing in macrophages does not influence systemic iron metabolism but decreases transcription of the macrophage Fpn1 in adult mice and modulates their immune response. This study investigated cellular and systemic iron metabolism in adult and aged male mice with macrophage-specific Hfe and TfR2 silencing (double knock-out, DKO). Serum iron parameters were significantly modified in aged animals, and significant differences were found in hepatic hepcidin transcription at both ages. Interestingly, splenic iron content was low in adult DKOs and splenic Fpn1 transcription was significantly increased in DKO animals at both ages, while the protein amount does not reflect the transcriptional trend. Additionally, DKO macrophages were isolated from mice bone marrow (BMDMs) and showed significant variations in the transcription of iron genes and protein amounts in targeted mice compared to controls. Specifically, Tranferrin Receptor 1 (TfR1) increased in DKO adult mice BMDMs, while the opposite is observed in the cells of aged DKO mice. Fpn1 transcript was significantly decreased in the BMDMs of adult DKO mice, while the protein was reduced at both ages. Lastly, a significant increase in Erythropoietin production was evidenced in aged DKO mice. Overall, our study reveals that Hfe and TfR2 in macrophages regulate hepatic Hepc production and affect iron homeostasis in the spleen and BMDMs, leading to an iron deficiency in aged animals that impairs their erythropoiesis. Full article

In this study, we investigated the function of a gene associated with iron metabolism using CRISPR-Cas9 and RNA sequencing in SHK-1 salmon cells. Our objective was to understand how different guide RNA (gRNA) sequences against the transferrin gene tf could influence gene expression and cellular processes related to iron uptake. RNA-Seq analysis was performed to evaluate the transcriptomic effects of two distinct gRNA targets with high knock-out (KO) efficiencies for the targeted tf gene in the SHK-1 genome. Our results showed no significant differential expression in transferrin-related transcripts between wild-type and CRISPR-edited cells; however, there were major differences between their transcriptomes, indicating complex transcriptional regulation changes. Enrichment analysis highlighted specific processes and molecular functions, including those related to the nucleus, cytoplasm, and protein binding. Notably, different sgRNAs targeting tf might result in different mutations at DNA levels in SHK-1 salmon cells. Full article

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the “host phase” and the viral TF Tat during the “viral phase”, which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure. Full article

Sclerotinia sclerotiorum is a fungal pathogen with a broad range of hosts, which can cause diseases and pose a great threat to many crops. Fungal-specific Zn₂Cys₆ transcription factors (TFs) constitute a large family prevalent among plant pathogens. However, the function of Zn₂Cys₆ TFs remains largely unknown. In this study, we identified and characterized SsZNC1, a Zn₂Cys₆ TF in S. sclerotiorum, which is involved in virulence, sclerotial development, and osmotic stress response. The expression of SsZNC1 was significantly up-regulated in the early stages of S. sclerotiorum infection on Arabidopsis leaves. The target deletion of SsZNC1 resulted in reduced virulence on Arabidopsis and oilseed rape. In addition, sclerotial development ability and growth ability under hyperosmotic conditions of SsZNC1 knockout transformants were reduced. A transcriptomic analysis unveiled its regulatory role in key cellular functions, including cellulose catabolic process, methyltransferase activity, and virulence, etc. Together, our results indicated that SsZNC1, a core regulatory gene involved in virulence, sclerotial development and stress response, provides new insight into the transcription regulation and pathogenesis of S. sclerotiorum. Full article

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits. Full article

The increasing prevalence of antimicrobial resistance and the limited availability of new antimicrobial agents have created an urgent need for new approaches to combat these issues. One such approach involves reevaluating the use of old antibiotics to ensure their appropriate usage and maximize their effectiveness, as older antibiotics could help alleviate the burden on newer agents. An example of such an antibiotic is chloramphenicol (CHL), which is rarely used due to its hematological toxicity. In the current study, we employed a previously published transposon mutant library in MG1655/pTF2::bla_CTX-M-1, containing over 315,000 unique transposon insertions, to identify the genetic factors that play an important role during growth in the presence of CHL. The list of conditionally essential genes, collectively referred to as the secondary resistome (SR), included 67 genes. To validate our findings, we conducted gene knockout experiments on six genes: arcA, hfq, acrZ, cls, mdfA, and nlpI. Deleting these genes resulted in increased susceptibility to CHL as demonstrated by MIC estimations and growth experiments, suggesting that targeting the products encoded from these genes may reduce the dose of CHL needed for treatment and hence reduce the toxicity associated with CHL treatment. Thus, the gene products are indicated as targets for antibiotic adjuvants to favor the use of CHL in modern medicine. Full article

Rice (Oryza sativa) exhibits tremendous aluminum (Al)-tolerance. The C2H2-transcription factor (TF) ART1 critically regulates rice Al tolerance via modulation of specific gene expression. However, little is known about the posttranscriptional ART1 regulation. Here, we identified an ART1-interacted gene OsNAC016 via a yeast two-hybrid (Y2H) assay. OsNAC016 was primarily expressed in roots and weakly induced by Al. Immunostaining showed that OsNAC016 was a nuclear protein and localized in all root cells. Knockout of OsNAC016 did not alter Al sensitivity. Overexpression of OsNAC016 resulted in less Al aggregation within roots and enhanced Al tolerance in rice. Based on transcriptomic and qRT-PCR evaluations, certain cell-wall-related or ART-regulated gene expressions such as OsMYB30 and OsFRDL4 were altered in OsNAC016-overexpressing plants. These results indicated that OsNAC016 interacts with ART1 to cooperatively regulate some Al-tolerance genes and is a critical regulatory factor in rice Al tolerance. Full article

Understanding condition-specific biological mechanisms from RNA-seq data requires comprehensive analysis of gene expression data, from the gene to the network level. However, this requires computational expertise, which limits the accessibility of data analysis for understanding biological mechanisms. Therefore, the development of an easy-to-use and comprehensive analysis system is essential. In response to this issue, we present TFNetPropX, a user-friendly web-based platform designed to perform gene-level, gene-set-level, and network-level analysis of RNA-seq data under two different conditions. TFNetPropX performs comprehensive analysis, from DEG analysis to network propagation, to predict TF-affected genes with a single request, and provides users with an interactive web-based visualization of the results. To demonstrate the utility of our system, we performed analysis on two TF knockout RNA-seq datasets and effectively reproduced biologically significant findings. We believe that our system will make it easier for biological researchers to gain insights from different perspectives, allowing them to develop diverse hypotheses and analyses. Full article

It is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5–E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches. Full article