RESUMEN

Cohesin is a protein complex that plays a key role in regulating chromosome structure and gene expression. While next-generation sequencing technologies have provided extensive information on various aspects of cohesin, integrating and exploring the vast datasets associated with cohesin are not straightforward. CohesinDB ( https://cohesindb.iqb.u-tokyo.ac.jp ) offers a web-based interface for browsing, searching, analyzing, visualizing, and downloading comprehensive multiomics cohesin information in human cells. In this protocol, we introduce how to utilize CohesinDB to facilitate research on transcriptional regulation and chromatin organization.

Asunto(s)

RESUMEN

The transcription factor WRKYs play pivotal roles in the adapting to adverse environments in plants. Prior research has demonstrated the involvement of OsWRKY70 in resistance against herbivores and its response to abiotic stress. Here, we reported the functional analysis of OsWRKY70 in immunity against fungal diseases and cold tolerance. The results revealed that OsWRKY70 was induced by various Magnaporthe oryzae strains. Knock out mutants of OsWRKY70, which were generated by the CRISPR/Cas9 system, exhibited enhanced resistance to M. oryzae. This was consistent with fortifying the reactive oxygen species (ROS) burst after inoculation in the mutants, elevated transcript levels of defense-responsive genes (OsPR1b, OsPBZ1, OsPOX8.1 and OsPOX22.3) and the observation of the sluggish growth of invasive hyphae under fluorescence microscope. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) validations demonstrated that differentially expressed genes were related to plant-pathogen interactions, hormone transduction and MAPK cascades. Notably, OsbHLH6, a key component of the JA signaling pathway, was down-regulated in the mutants compared to wild type plants. Further investigation confirmed that OsWRKY70 bound to the promoter of OsbHLH6 by semi-in vivo chromatin immunoprecipitation (ChIP). Additionally, the loss-function of OsWRKY70 impaired cold tolerance in rice. The enhanced susceptibility in the mutants characterized by excessive ROS production, elevated ion leakage rate and increased malondialdehyde content, as well as decreased activity of catalase (CAT) and peroxidase (POD) under low temperature stress was, which might be attributed to down-regulation of cold-responsive genes (OsLti6b and OsICE1). In conclusion, our findings indicate that OsWRKY70 negatively contributes to blast resistance but positively regulates cold tolerance in rice, providing a strategy for crop breeding with tolerance to stress.

RESUMEN

The yellow-fleshed loquat is abundant in carotenoids, which determine the fruit's color, provide vitamin A, and offer anti-inflammatory and anti-cancer health benefits. In this research, the impact of abscisic acid (ABA), a plant hormone, on carotenoid metabolism and flesh pigmentation in ripening loquat fruits was determined. Results revealed that ABA treatment enhanced the overall content of carotenoids in loquat fruit, including major components like ß-cryptoxanthin, lutein, and ß-carotene, linked to the upregulation of most genes in the carotenoid biosynthesis pathway. Furthermore, a transcription factor, EjWRKY6, whose expression was induced by ABA, was identified and was thought to play a role in ABA-induced carotenoid acceleration. Transient overexpression of EjWRKY6 in Nicotiana benthamiana and stable genetic transformation in Nicotiana tabacum with EjWRKY6 indicated that both carotenoid production and genes related to carotenoid biosynthesis could be upregulated in transgenic plants. A dual-luciferase assay proposed a probable transcriptional control between EjWRKY6 and promoters of genes associated with carotenoid production. To sum up, pre-harvest ABA application could lead to carotenoid biosynthesis in loquat fruit through the EjWRKY6-induced carotenoid biosynthesis pathway.

RESUMEN

More than a decade after the discovery of the classical cytoplasmic IκB proteins, IκBζ was identified as an additional member of the IκB family. Unlike cytoplasmic IκB proteins, IκBζ has distinct features, including its nuclear localization, preferential binding to NF-κB subunits, unique expression properties, and specialized role in NF-κB regulation. While the activation of NF-κB is primarily controlled by cytoplasmic IκB members at the level of nuclear entry, IκBζ provides an additional layer of NF-κB regulation in the nucleus, enabling selective gene activation. Human genome-wide association studies (GWAS) and gene knockout experiments in mice have elucidated the physiological and pathological roles of IκBζ. Despite the initial focus to its role in activated macrophages, IκBζ has since been recognized as a key player in the IL-17-triggered production of immune molecules in epithelial cells, which has garnered significant clinical interest. Recent research has also unveiled a novel molecular function of IκBζ, linking NF-κB and the POU transcription factors through its N-terminal region, whose role had remained elusive for many years.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays pivotal roles in both adaptive and innate immunities. GILT exhibits constitutive expression within antigen-presenting cells, whereas in other cell types, its expression is induced by interferon gamma (IFN-γ). Gaining insights into the precise molecular mechanism governing the induction of GILT protein by IFN-γ is of paramount importance for adaptive and innate immunities. In this study, we found that the 5' segment of GILT mRNA inhibited GILT protein expression regardless of the presence of IFN-γ. Conversely, the 3' segment of GILT mRNA suppressed GILT protein expression in the absence of IFN-γ, but it loses this inhibitory effect in its presence. Although the mTOR inhibitor rapamycin suppressed the induction of GILT protein expression by IFN-γ, the expression from luciferase sequence containing the 3' segment of GILT mRNA was resistant to rapamycin in the presence of IFN-γ, but not in its absence. Collectively, this study elucidates the mechanism behind GILT induction by IFN-γ: in the absence of IFN-γ, GILT mRNA is constitutively transcribed, but the translation process is hindered by both the 5' and 3' segments. Upon exposure to IFN-γ, a translation inhibitor bound to the 3' segment is liberated, and a translation activator interacts with the 3' segment to trigger the initiation of GILT translation.

Asunto(s)

RESUMEN

Cellular differentiation allows cells to transition between different functional states and adapt to various environmental cues. The diversity and plasticity of this process is beautifully exemplified by T cells responding to pathogens, which undergo highly specialized differentiation tailored to the ongoing infection. Such antigen-induced T cell differentiation is regulated at the transcriptional level by DNA-binding proteins and at the post-transcriptional level by RNA-binding proteins. Although traditionally defined as separate protein classes, a growing body of evidence indicates an overlap between these two groups of proteins, collectively coined DNA/RNA-binding proteins (DRBPs). In this review, we describe how DRBPs might bind both DNA and RNA, discuss the putative functional relevance of this dual binding, and provide an exploratory analysis into characteristics that are associated with DRBPs. To exemplify the significance of DRBPs in T cell biology, we detail the activity of several established and putative DRBPs during the T cell response. Finally, we highlight several methodologies that allow untangling of the distinct functionalities of DRBPs at the DNA and RNA level, including key considerations to take into account when applying such methods.

RESUMEN

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and end­stage renal disease, and is characterized by persistent proteinuria and decreased glomerular filtration rate. Despite extensive efforts, the increasing incidence highlights the urgent need for more effective treatments. Histone methylation is a crucial epigenetic modification, and its alteration can destabilize chromatin structure, thereby regulating the transcriptional activity of specific genes. Histone methylation serves a substantial role in the onset and progression of various diseases. In patients with DKD, changes in histone methylation are pivotal in mediating the interactions between genetic and environmental factors. Targeting these modifications shows promise in ameliorating renal histological manifestations, tissue fibrosis and proteinuria, and represents a novel therapeutic frontier with the potential to halt DKD progression. The present review focuses on the alterations in histone methylation during the development of DKD, systematically summarizes its impact on various renal parenchymal cells and underscores the potential of targeted histone methylation modifications in improving DKD outcomes.

Asunto(s)

RESUMEN

AIMS: Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable improvement in managing the progression of heart disease, developing effective therapies for heart failure (HF) remains a challenge. A better understanding of molecular mechanisms that maintain proper Ca2+ levels and contractility in the injured heart could be of therapeutic value. METHODS AND RESULTS: Here, we report that transcription factor zinc finger E-box-binding homeobox 2 (ZEB2) is induced by hypoxia-inducible factor 1-alpha (HIF1α) in hypoxic cardiomyocytes and regulates a network of genes involved in Ca2+ handling and contractility during ischaemic heart disease. Gain- and loss-of-function studies in genetic mouse models revealed that ZEB2 expression in cardiomyocytes is necessary and sufficient to protect the heart against ischaemia-induced diastolic dysfunction and structural remodelling. Moreover, RNA sequencing of ZEB2-overexpressing (Zeb2 cTg) hearts post-injury implicated ZEB2 in regulating numerous Ca2+-handling and contractility-related genes. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban at both serine-16 and threonine-17, implying enhanced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), thereby augmenting SR Ca2+ uptake and contractility. Furthermore, we observed a decrease in the activity of Ca2+-dependent calcineurin/NFAT signalling in Zeb2 cTg hearts, which is the main driver of pathological cardiac remodelling. On a post-transcriptional level, we showed that ZEB2 expression can be regulated by the cardiomyocyte-specific microRNA-208a (miR-208a). Blocking the function of miR-208a with anti-miR-208a increased ZEB2 expression in the heart and effectively protected from the development of pathological cardiac hypertrophy. CONCLUSION: Together, we present ZEB2 as a central regulator of contractility and Ca2+-handling components in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in regulating Ca2+ homeostasis in cardiomyocytes is an essential step towards the development of improved therapies for HF.

RESUMEN

Background: Colorectal cancer (CRC) remains a significant global health challenge, often characterized by late-stage metastasis and poor prognosis. The Runt-related transcription factor 1 (RUNX1) plays a dual role as both an oncogene and a tumor suppressor in various cancers, including CRC. However, the specific regulatory mechanisms of RUNX1 in CRC, particularly its direct roles, are not fully understood. Objective: This study aimed to investigate the role of RUNX1 in CRC progression and its interaction with Mucin 13 (MUC13) as a potential regulatory target. Methods: RUNX1 expression was analyzed in CRC tissues and cell lines compared to controls. In vitro and in vivo assays were conducted to assess the effects of RUNX1 overexpression and knockdown on cell behavior. ChIP-seq and RNA-seq analyses were performed to identify RUNX1 targets, with a focus on MUC13. Results: RUNX1 expression was significantly upregulated in CRC tissues and cells, correlating with advanced pathological characteristics and poor patient outcomes. RUNX1 overexpression enhanced CRC cell proliferation, migration, invasion, and G2/M phase arrest, while its knockdown had the opposite effects. MUC13 was identified as a direct transcriptional target of RUNX1, with its expression contributing to the activation of the Wnt/ß-catenin signaling pathway. Disruption of MUC13 partially reversed the malignant phenotypes induced by RUNX1. Conclusion: RUNX1 promotes CRC progression by upregulating MUC13 and activating the Wnt/ß-catenin pathway. This RUNX1-MUC13 axis represents a potential therapeutic target for managing CRC.

Asunto(s)

RESUMEN

B-Myb, also known as MYB proto-oncogene like 2 (MYBL2), is an important transcription factor implicated in transcription regulation, cell cycle and tumorigenesis. However, the molecular mechanism underlying B-Myb-controlled transactivation in different cell contexts as well as its functional implication in cancers remains elusive. In this study, we have conducted a comprehensive genome-wide analysis of B-Myb binding sites in multiple immortalized or cancer cell lines and identified its critical target genes. The results revealed that B-Myb regulates a common set of core cell cycle genes and cell type-specific genes through collaboration with other important transcription factors (e.g. NFY and MuvB complex) and binding to cell type-invariant promoters and cell type-specific enhancers and super-enhancers. KIF2C, UBE2C and MYC were further validated as B-Myb target genes. Loss-of-function analysis demonstrated that KIF2C knockdown inhibited tumor cell growth both in vitro and in vivo, suppressed cell motility and cell cycle progression, accompanied with defects in microtubule organization and mitosis, strongly suggesting that KIF2C is a critical regulator of cancer cell growth and mitosis, and maintains high cancer cell motility ability and microtubule dynamics. Pan-cancer transcriptomic analysis revealed that the overexpression of both B-Myb and KIF2C presents as independent prognostic markers in various types of cancer. Notably, B-Myb associates with NFYB, binds to target gene promoters, enhancers and super-enhancers, and provokes a cascade of oncogenic gene expression profiles in cancers. Overall, our results highly suggest the critical implication of B-Myb-mediated gene regulation in cancers, and the promising therapeutic and prognostic potentials of B-Myb and KIF2C for cancer diagnosis and treatment.

Asunto(s)

RESUMEN

Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response SPX DOMAIN GENE3, nitrate reductase and two nitrate transporters. Altered nutrient sensing, acquisition or distribution are likely a consequence of adaption to growth on marginal, low-nutrient input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments.

RESUMEN

Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, and GAL10) for galactose catabolism through the Leloir pathway, and a "GALLAC" cluster containing the transcriptional activator gene LAC9, second copies of GAL1 and GAL10, and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences.IMPORTANCEThis study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia. Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products.

RESUMEN

In plant-pathogen interactions, numerous pathogens secrete polygalacturonase (PG) to degrade plants cell walls, whereas plants produce PG-inhibiting protein (PGIP) that specifically binds to pathogen-derived PG to inhibit its activity and resist pathogen infection. In the present study, we dshowed that PpPGIP1 was significantly upregulated in peaches after Monilinia fructicola infection, and the prokaryotic expression of the PpPGIP1 protein inhibited M. fructicola by mitigating its PG activity. Transient overexpression of PpPGIP1 in peaches significantly enhanced their resistance to M. fructicola. PpPGIP1 promoter had several W-box the defense elements that can bind to WRKY transcription factors. Transcriptome analysis identified 20 differentially expressed WRKY genes, including the classic disease resistance gene WRKY33. PpWRKY33 is significantly upregulated in M. fructicola infected peaches. PpWRKY33 is localized in the nucleus and can bind to the W-box in the PpPGIP1 promoter to transcriptional activate the expression of PpPGIP1. Transient overexpression PpWRKY33 upregulated PpPGIP1 expression in peaches, and silencing PpWRKY33 decreased the PpPGIP1 expression. These results indicated that PpPGIP1 positively regulates fungal disease resistance in peaches and is transcriptionally activated by PpWRKY33. These findings reveal the disease resistant role of PpPGIP1 in peaches, and provide new insights into its transcriptional regulation.

RESUMEN

INTRODUCTION: Lignin is a principal constituent of the secondary cell wall, which plays a role in both plant growth and defensing against stress, such as low temperature and pest infestation. Additionally, it also accumulates in fleshy fruits and negatively affects fruit quality. Red-fleshed loquat is temperature sensitive and exhibits cold-induced lignification. A number of technologies have been developed, for example, Low Temperature Conditioning (LTC) treatment, which has been applied in order to relieve the symptom of cold injury. OBJECTIVES: The present study seeks to elucidate the regulatory mechanism underlying cold-induced lignification in loquat fruit. METHODS: The target genes were isolated through the analysis of transcriptome. The gene function was analyzed by transient transgenic method in tobacco leaves and loquat fruit, respectively, as well as stable overexpression in liverwort. The regulatory mechanism study was achieved by in vitro protein-protein interaction assays, dual-luciferase assay, and EMSA. RESULTS: In the present study, the Xylem NAC Domain transcription factor EjXND1 was identified as a repressor of loquat fruit lignification. It was demonstrated that EjXND1 could interact with the characterized lignin activator EjHB1, resulting in a diminution of the activation of EjHB1 on EjPRX12 promoter. Furthermore, two highly methylated regions were identified in the promoter of EjXDN1. One of these regions exhibited a negative correlation between methylation level and EjXND1 expression. Additionally, it was shown that hypermethylation of this region weaken the binding affinity of EjXND1 activators to its promoter. CONCLUSION: The EjXND1 plays a role in modified Low Temperature Conditioning (mLTC) treatment that alleviates cold-induced lignification in red-fleshed loquat fruit by targeting the EjHB1-EjPRX12 module and EjXND1 is regulated by the dynamic of DNA methylation level in the promoter.

RESUMEN

The fate and stability of messenger RNA (mRNA), from transcription to degradation is regulated by a dynamic shuttle of epigenetic modifications and RNA binding proteins in maintaining healthy cellular homeostasis and disease development. While Transforming Growth Factor Beta 1 (TGFß1) has been implicated as a key regulator for diabetic retinopathy, a microvascular complication of diabetes, the RNA binding proteins post-transcriptionally regulating its expression remain unreported in the ocular context. Further, dysfunction of TGFß1 signalling is also strongly associated with angiogenesis, inflammatory responses and tissue fibrosis in many eye conditions leading to vision loss. In this study, computational and molecular simulations were initially carried out to identify Human Antigen R (HuR) binding sites in TGFß1 mRNA and predict the structural stability of these RNA-protein interactions. These findings were further validated through in vitro experiments utilizing Cobalt Chloride (CoCl2) as a hypoxia mimetic agent in human retinal microvascular endothelial cells (HRMVEC). In silico analysis revealed that HuR preferentially binds to the 5'-UTR of TGFß1 and displayed more stable interaction than the 3'UTR. Consistent with in silico analysis, RNA immunoprecipitation demonstrated a robust association between HuR and TGFß1 mRNA specifically under hypoxic conditions. Further, silencing of HuR significantly reduced TGFß1 protein expression upon CoCl2 treatment. Thus, for the first time in ocular pathological milieu, direct evidence of HuR- TGFß1 mRNA interaction under conditions of hypoxia has been reported in this study providing valuable insights into RNA binding proteins as therapeutic targets for ocular diseases associated with TGFß1 dysregulation.

RESUMEN

BACKGROUND: Alternative splicing is a pivotal mechanism of post-transcriptional modification that contributes to the transcriptome plasticity and proteome diversity in metazoan cells. Although many splicing regulations around the exon/intron regions are known, the relationship between promoter-bound transcription factors and the downstream alternative splicing largely remains unexplored. RESULTS: In this study, we present computational approaches to unravel the regulatory relationship between promoter-bound transcription factor binding sites (TFBSs) and the splicing patterns. We curated a fine dataset that includes DNase I hypersensitive site sequencing and transcriptomes across fifteen human tissues from ENCODE. Specifically, we proposed different representations of TF binding context and splicing patterns to examine the associations between the promoter and downstream splicing events. While machine learning models demonstrated potential in predicting splicing patterns based on TFBS occupancies, the limitations in the generalization of predicting the splicing forms of singleton genes across diverse tissues was observed with carefully examination using different cross-validation methods. We further investigated the association between alterations in individual TFBS at promoters and shifts in exon splicing efficiency. Our results demonstrate that the convolutional neural network (CNN) models, trained on TF binding changes in the promoters, can predict the changes in splicing patterns. Furthermore, a systemic in silico substitutions analysis on the CNN models highlighted several potential splicing regulators. Notably, using empirical validation using K562 CTCFL shRNA knock-down data, we showed the significant role of CTCFL in splicing regulation. CONCLUSION: In conclusion, our finding highlights the potential role of promoter-bound TFBSs in influencing the regulation of downstream splicing patterns and provides insights for discovering alternative splicing regulations.

Asunto(s)

RESUMEN

Three types of highly promising small RNA therapeutics, namely, small interfering RNAs (siRNAs), microRNAs (miRNAs) and the RNA subtype of antisense oligonucleotides (ASOs), offer advantages over small-molecule drugs. These small RNAs can target any gene product, opening up new avenues of effective and safe therapeutic approaches for a wide range of diseases. In preclinical research, synthetic small RNAs play an essential role in the investigation of physiological and pathological pathways as silencers of specific genes, facilitating discovery and validation of drug targets in different conditions. Off-target effects of small RNAs, however, could make it difficult to interpret experimental results in the preclinical phase and may contribute to adverse events of small RNA therapeutics. Out of the two major types of off-target effects we focused on the hybridization-dependent, especially on the miRNA-like off-target effects. Our main aim was to discuss several approaches, including sequence design, chemical modifications and target prediction, to reduce hybridization-dependent off-target effects that should be considered even at the early development phase of small RNA therapy. Because there is no standard way of predicting hybridization-dependent off-target effects, this review provides an overview of all major state-of-the-art computational methods and proposes new approaches, such as the possible inclusion of network theory and artificial intelligence (AI) in the prediction workflows. Case studies and a concise survey of experimental methods for validating in silico predictions are also presented. These methods could contribute to interpret experimental results, to minimize off-target effects and hopefully to avoid off-target-related adverse events of small RNA therapeutics.

RESUMEN

Cotton fiber length is basically determined by well-coordinated gene expression and phosphatidylinositol phosphates (PIPs) accumulation during fiber elongation but the regulatory mechanism governing PIPs transport remains unknown. Here, we report a MYB transcription factor GhMYB30D04 in Gossypium hirsutum that promotes fiber elongation through modulating the expression of PIP transporter gene GhLTPG1. Knockout of GhMYB30D04 gene in cotton (KO) results in a reduction of GhLTPG1 transcripts with lower accumulation of PIPs, leading to shorter fibers and lower fiber yield. Conversely, GhMYB30D04 overexpression (GhMYB30D04-OE) causes richer PIPs and longer cotton fibers, mimicking the effects of exogenously applying PIPs on the ovules of GhMYB30D04-KO and wild type. Furthermore, GhMYB30D04 interacts with GhHD1, the crucial transcription factor of fiber initiation, to form an activation complex stabilized by PIPs, both of which upregulate GhLTPG1 expression. Comparative omics-analysis revealed that higher and extended expressions of LTPG1 in fiber elongation mainly correlate with the variations of the GhMYB30D04 gene between two cotton allotetraploids, contributing to longer fiber in G. babardense. Our work clarifies a mechanism by which GhHD1-GhMYB30D04 form a regulatory module of fiber elongation to tightly control PIP accumulation. Our work still has an implication that GhMYB30D04-GhHD1 associates with development transition from fiber initiation to elongation.

RESUMEN

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.