Leucine Zipper Transcription Factor Research Articles

Role of the basic leucine zipper transcription factor BATF2 in modulating immune responses and inflammation in health and disease.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is known to exhibit tumor-suppressive activity in cancer cells. Within recent years, however, BATF2 has also emerged as an important transcriptional regulator of the immune system. Through its immunomodulatory function, BATF2 has been implicated in a variety of (patho)physiological processes, including host defense against infection, anti-tumor immunity, and maintenance of tissue inflammatory homeostasis. Below, we discuss recent literature that has provided insight into the role of BATF2 as a transcriptional regulator of immune responses in health and disease, including the cell types that express BATF2, the different diseases in which the immunomodulatory effects of BATF2 have been shown to play a role, and the molecular mechanisms through which BATF2 is thought to exert those effects. In doing so, we highlight that the immunological effects of BATF2 are highly context-dependent, and we point out overlap between the mechanisms of action of BATF2 in infectious disease and non-infectious disease. We also discuss areas of interest for future research, the clinical relevance of better understanding BATF2 function, and potential strategies for therapeutic modulation of BATF2.

GmTRAB1, a Basic Leucine Zipper Transcription Factor, Positively Regulates Drought Tolerance in Soybean (Glycine max. L).

The basic leucine zipper (bZIP) transcription factors play crucial roles in plant resistance to environmental challenges, but the biological functions of soybean bZIP members are still unclear. In this study, a drought-related soybean bZIP gene, GmTRAB1, was analyzed. The transcript of GmTRAB1 was upregulated under drought, ABA, and oxidative stresses. Overexpression of GmTRAB1 improved the osmotic stress tolerance of transgenic Arabidopsis and soybean hairy roots associated with increased proline content and activity of antioxidant enzymes and reduced accumulations of malonaldehyde and reactive oxide species. However, RNA interference silencing of GmTRAB1 in the soybean hairy roots improved drought sensitivity. Furthermore, GmTRAB1 increased the sensitivity of transgenic plants to ABA and participated in modulating ABA-regulated stomatal closure upon drought stress. In addition, GmTRAB1 stimulated the transcript accumulation of drought-, ABA-, and antioxidant-related genes to respond to drought. Collectively, this research will contribute to understanding the molecular mechanisms of bZIP transcription factors in soybean's resistance to drought.

Prokaryotic expression of wheat TaABI5-D3 gene and polyclonal antibody preparation

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

NFE2L2 and ferroptosis resistance in cancer therapy.

NFE2-like basic leucine zipper transcription factor 2 (NFE2L2, also known as NRF2), is a key transcription factor in the cellular defense against oxidative stress, playing a crucial role in cancer cell survival and resistance to therapies. This review outlines the current knowledge on the link between NFE2L2 and ferroptosis - a form of regulated cell death characterized by iron-dependent lipid peroxidation - within cancer cells. While NFE2L2 activation can protect normal cells from oxidative damage, its overexpression in cancer cells contributes to drug resistance by upregulating antioxidant defenses and inhibiting ferroptosis. We delve into the molecular pathways of ferroptosis, highlighting the involvement of NFE2L2 and its target genes, such as NQO1, HMOX1, FTH1, FTL, HERC2, SLC40A1, ABCB6, FECH, PIR, MT1G, SLC7A11, GCL, GSS, GSR, GPX4, AIFM2, MGST1, ALDH1A1, ALDH3A1, and G6PD, in ferroptosis resistance. Understanding the delicate balance between NFE2L2's protective and deleterious roles could pave the way for novel therapeutic strategies targeting NFE2L2 to enhance the efficacy of ferroptosis inducers in cancer therapy.

Evolutionary and Expression Analyses of the bZIP Family in Tea Plants (Camellia sinensis) and Functional Characterization of CsbZIP3/42/6 in Response to Environmental Stresses.

Basic leucine zipper (bZIP) transcription factors play crucial roles in various biological processes and responses to environmental stresses. However, the functions of the bZIP family in tea plants remain largely unexplored. Here, we identified 74 bZIP genes in tea plants (Camellia sinensis) and classified them into 12 phylogenetic groups, supported by analyses of conserved motifs and gene structures. Cis-element analysis provided insights into the potential roles of CsbZIP genes in phytohormone signaling and stress responses. Tissue-specific expression analysis demonstrated differential expression profiles of CsbZIP genes, suggesting their tissue- and stage-specific functions. Additionally, varying expression levels under different abiotic stresses indicated functional divergence of the CsbZIP family during the long-term evolution. Notably, CsbZIP3/42/6 were identified as positive regulators of drought and salt stress responses but negative regulators in response to pathogen infection, and CsbZIP42 could interact with CsbZIP3 and CsbZIP6 in regulating these environmental stresses. This study provides valuable information on potential applications for improving stress tolerance and overall plant health of tea plants.

OsPUB75-OsHDA716 mediates deactivation and degradation of OsbZIP46 to negatively regulate drought tolerance in rice.

Histone deacetylases (HDACs) play crucial roles in plant stress responses via modification of histone as well as non-histone proteins; however, how HDAC-mediated deacetylation of non-histone substrates affects protein functions remains elusive. Here, we report that the Reduced Potassium Dependency3/Histone Deacetylase1 (RPD3/HDA1)-type histone deacetylase OsHDA716 and plant U-box (PUB) E3 ubiquitin ligase OsPUB75 form a complex to regulate rice drought response via deactivation and degradation of basic leucine zipper (bZIP) transcription factor OsbZIP46 in rice (Oryza sativa). OsHDA716 decreases ABA-induced drought tolerance, and mechanistic investigations showed that OsHDA716 interacts with and deacetylates OsbZIP46, a key regulator in ABA signaling and drought response, thus inhibiting its transcriptional activity. Furthermore, OsHDA716 recruits OsPUB75 to facilitate ubiquitination and degradation of deacetylated OsbZIP46. Therefore, the OsPUB75-OsHDA716 complex exerts double restrictions on the transcriptional activity and protein stability of OsbZIP46, leading to repression of downstream drought-responsive gene expression and consequently resulting in reduced drought tolerance. Conversely, OsbZIP46 acts as an upstream repressor to repress OsHDA716 expression, and therefore OsHDA716 and OsbZIP46 form an antagonistic pair to reciprocally inhibit each other. Genetic evidence showed that OsHDA716 works with OsbZIP46 in a common pathway to antagonistically regulate rice drought response, revealing that plants can fine-tune stress responses by the complex interplay between chromatin regulators and transcription factors. Our findings unveil an acetylation-dependent regulatory mechanism governing protein functions and shed light on the precise coordination of activity and stability of key transcription factors through a combination of different post-translational modifications.

The genetic architecture of protein interaction affinity and specificity

The encoding and evolution of specificity and affinity in protein-protein interactions is poorly understood. Here, we address this question by quantifying how all mutations in one protein, JUN, alter binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. We fit a global thermodynamic model to the data to reveal that most affinity changing mutations equally affect JUN’s affinity to all its interaction partners. Mutations that alter binding specificity are relatively rare but distributed throughout the interaction interface. Specificity is determined both by features that promote on-target interactions and by those that prevent off-target interactions. Approximately half of the specificity-defining residues in JUN contribute both to promoting on-target binding and preventing off-target binding. Nearly all specificity-altering mutations in the interaction interface are pleiotropic, also altering affinity to all partners. In contrast, mutations outside the interface can tune global affinity without affecting specificity. Our results reveal the distributed encoding of specificity and affinity in an interaction interface and how coiled-coils provide an elegant solution to the challenge of optimizing both specificity and affinity in a large protein family.

Comparative transcriptome analysis and identification of candidate bZIP transcription factors involved in anthraquinone biosynthesis in Rheum officinale Baill

Rhubarb is a traditional medicinal plant in China, whose pharmacological effects derive mainly from its anthraquinones. However, the regulatory mechanism affecting anthraquinone biosynthesis in R. officinale remains poorly understood. We assembled a high-quality, full-length transcriptome using single-molecule real-time (SMRT) sequencing. 274 unigenes potentially involved in the biosynthesis of anthraquinones, including those in the shikimate, polyketide, MVA and MEP pathways, were identified based on full-length transcriptome. Differentially expressed genes (DEGs) induced by MeJA treatment and DEGs between different tissues were identified through next-generation sequencing (NGS), revealing the genes that may be involved in the biosynthesis of anthraquinones. The basic leucine zipper (bZIP) transcription factors of R. officinale were systematically identified. Key genes such as RobZIP50 and RobZIP53 were systematically identified and found to be associated with anthraquinone biosynthesis in R. officinale through differential expression, co-expression and protein interaction analyses. RobZIP50 and RobZIP53 were highly expressed in roots and rhizomes, and significantly increased after 12 h of MeJA treatment. Additionally, both RobZIP50 and RobZIP53 were localized exclusively in the nucleus, with RobZIP53 showing significant transcriptional activity. Taken together, our results suggest that RobZIP53 may play a role in regulating anthraquinone biosynthesis in R. officinale.

Identification of transcription factor BnHDG4-A08 as a novel candidate associated with the accumulation of oleic, linoleic, linolenic, and erucic acid in Brassica napus.

We screened 47 significantly associated haplotype blocks for oleic, linoleic, linolenic, and erucic acid, with 17 blocks influencing multiple traits. A novel candidate of transcription factor BnHDG4 A08 influencing oleic, linoleic, linolenic, and erucic acid was identified, by a joint strategy of haplotype-based genome-wide association study, genomic resequencing, gene cloning, and co-expression network Fatty acid (FA) composition determines the quality and economic value of rapeseed oil (Brassica napus). However, the molecular network of FAs is unclear. In the current study, multi-strategies of haplotype-based genome-wide association study (GWAS), genomic resequencing, gene cloning, and co-expression network were joint to reveal novel genetic factors influencing FA accumulation in rapeseed. We identified 47 significantly associated haplotype blocks for oleic, linoleic, linolenic, and erucic acid, with 17 blocks influencing multiple traits, using a haplotype-based GWAS with phenotype data from 203 Chinese semi-winter accessions. A total of 61 rapeseed orthologs involved in acyl-lipid metabolism, carbohydrate metabolism, or photosynthesis were identified in these 17 blocks. Among these genes, BnHDG4-A08, encoding a class IV homeodomain leucine-zipper transcription factor, exhibited two single-nucleotide polymorphisms (SNPs) in the exon and intron, with significant associations with oleic, linoleic, linolenic, and erucic acid. Gene cloning further validated two SNPs in the exon of BnHDG4-A08 in a population with 75 accessions, leading to two amino acid changes (T372A and P366L) and significant variation of oleic, linoleic, linolenic, and erucic acid. A competitive allele-specific PCR (KASP) marker based on the SNPs was successfully developed and validated. Moreover, 98 genes exhibiting direct interconnections and high weight values with BnHDG4-A08 were identified through co-expression network analysis using transcriptome data from 13 accessions. Our study identified a novel FA candidate of transcription factor BnHDG4-A08 influencing oleic, linoleic, linolenic, and erucic acid. This gene provides a potential promising gene resource for the novel mechanistic understanding of transcription factors regulating FA accumulation.

BATF is involved in the malignant phenotype and epithelial-mesenchymal transition of colon cancer cells via ERK/PD-L1 signaling.

Transcription factors have emerged as primary regulators in colon cancer. Basic Leucine Zipper Transcription Factor (BATF) was found to be differentially expressed in colon cancer. This study aimed to explore the impact of BATF on the malignant phenotype and epithelial-mesenchymal transition (EMT) process. Based on The Cancer Genome Atlas (TCGA) data, the correlation between BATF and patients' overall prognosis was analyzed. BATF expression in epithelial and colon cancer cells was evaluated. By knocking down its levels in colon cancer cells, its effects on the malignant phenotype, apoptosis, EMT progression, and ERK/PD-L1 were evaluated. Cells were treated with ERK/PD-L1 agonists, and the BATF cell regulation was re-examined. BATF levels were negatively correlated with patients' overall survival. BATF is upregulated in colon cancer cell lines, and BATF knockdown in HCT116 cells suppressed the malignant cellular phenotypes (proliferation, migration, and invasion) and increased apoptosis. BATF knockdown inhibited EMT and ERK/PD-L1 signaling activation, whereas upon agonist treatment, BATF potency was disrupted. This study revealed that BATF is involved in the malignant phenotype and EMT of colon cancer cells, and this process may be mediated by ERK/PD-L1 signaling.

BZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry.

Basic leucine zipper (bZIP) transcription factors (TFs) play a crucial role in anthocyanin accumulation in plants. In addition to bZIP TFs, abscisic acid (ABA) increases anthocyanin biosynthesis. Therefore, this study aimed to investigate whether bZIP TFs are involved in ABA-induced anthocyanin accumulation in sweet cherry and elucidate the underlying molecular mechanisms. Specifically, the BLAST method was used to identify bZIP genes in sweet cherry. Additionally, we examined the expression of ABA- and anthocyanin-related genes in sweet cherry following the overexpression or knockdown of a bZIP candidate gene. In total, we identified 54 bZIP-encoding genes in the sweet cherry genome. Basic leucine zipper 6 (bZIP6) showed significantly increased expression, along with increased anthocyanin accumulation in sweet cherry. Additionally, yeast one-hybrid and dual-luciferase assays indicated that PavbZIP6 enhanced the expression of anthocyanin biosynthetic genes (PavDFR, PavANS, and PavUFGT), thereby increasing anthocyanin accumulation. Moreover, PavbZIP6 interacted directly with the PavBBX6 promoter, thereby regulating PavNCED1 to promote abscisic acid (ABA) synthesis and enhance anthocyanin accumulation in sweet cherry fruit. Conclusively, this study reveals a novel mechanism by which PavbZIP6 mediates anthocyanin biosynthesis in response to ABA and contributes to our understanding of the mechanism of bZIP genes in the regulation of anthocyanin biosynthesis in sweet cherry.

Analysis of bZIP transcription factors in Rhododendron simsii and functional study of RsbZIP6 in regulating anthocyanin biosynthesis

The basic leucine zipper (bZIP) transcription factors play a critical role in various plant biological processes, including anthocyanin biosynthesis. This study focuses on Rhododendron simsii, a notable ornamental species with insufficiently explored bZIP transcription factors. We identified 66 bZIP transcription factors in the R. simsii genome and conducted comprehensive bioinformatics analyses to determine their gene localization, phylogenetic relationships, grouping, gene/protein structure, duplication events, synteny, and expression profiles. Our analysis identified RsbZIP6, a homolog of HY5 known to influence anthocyanin biosynthesis in many plants, as a potential regulator of this pathway. We cloned the complete coding sequence of RsbZIP6, which encodes a 170-amino acid protein spanning 510 bp. Subcellular localization analysis verified the nuclear presence of the RsbZIP6 protein. RT-qPCR analysis revealed the highest expression of RsbZIP6 in petals, which correlated with anthocyanin accumulation. Transgenic experiments indicated that overexpressing RsbZIP6 in Arabidopsis enhanced anthocyanin accumulation by upregulating genes involved in anthocyanin biosynthesis (4CL, CHS, CHI, DFR, F3H, F3’H, ANS and UF3GT). Our findings enhance understanding of the bZIP transcription factor family in R. simsii and underscore the vital role of RsbZIP6 in anthocyanin biosynthesis, providing insights for future genetic enhancement strategies.

GWAS and polygenic risk score of severe COVID-19 in Eastern Europe.

COVID-19 disease has infected more than 772 million people, leading to 7 million deaths. Although the severe course of COVID-19 can be prevented using appropriate treatments, effective interventions require a thorough research of the genetic factors involved in its pathogenesis. We conducted a genome-wide association study (GWAS) on 7,124 individuals (comprising 6,400 controls who had mild to moderate COVID-19 and 724 cases with severe COVID-19). The inclusion criteria were acute respiratory distress syndrome (ARDS), acute respiratory failure (ARF) requiring respiratory support, or CT scans indicative of severe COVID-19 infection without any competing diseases. We also developed a polygenic risk score (PRS) model to identify individuals at high risk. We identified two genome-wide significant loci (P-value <5 × 10-8) and one locus with approximately genome-wide significance (P-value = 5.92 × 10-8-6.15 × 10-8). The most genome-wide significant variants were located in the leucine zipper transcription factor like 1 (LZTFL1) gene, which has been highlighted in several previous GWAS studies. Our PRS model results indicated that individuals in the top 10% group of the PRS had twice the risk of severe course of the disease compared to those at median risk [odds ratio = 2.18 (1.66, 2.86), P-value = 8.9 × 10-9]. We conducted one of the largest studies to date on the genetics of severe COVID-19 in an Eastern European cohort. Our results are consistent with previous research and will guide further epidemiologic studies on host genetics, as well as for the development of targeted treatments.

MAFB-mediated CEBPA regulated human urothelium growth through Wnt/β-catenin signaling pathway

MAFB is essential for regulating male-type urethral differentiation, and especially, its variation can contribute to hypospadias in mice. However, the potential mechanism is still unclear. Here we observed that the basic leucine zipper (bZIP) transcription factor MAFB and CCAAT/enhancer-binding protein alpha (CEBPA) could promote human urothelium SV-HUC-1 growth. Moreover, MAFB and CEBPA expression were reduced in the prepuce tissues of hypospadias patients. Based on transcriptome sequencing analysis and Western blot, MAFB knockdown was found to suppress CEBPA protein expression and repress Wnt/β-catenin signaling in urothelium cells. Meanwhile, we observed blocked cell-cycle progression from the G1 to the S phase, inhibited cell proliferation, and activated apoptosis. Furthermore, MAFB could facilitate CEBPA transcription and regulate the proliferation of urothelium. The above results indicated that MAFB-mediated inhibition of urothelial SV-HUC-1 growth resulted from inhibiting the Wnt/β-catenin signaling pathway by down-regulating CEBPA. Our findings provide new insight into the understanding of genes associated with hypospadias and the pathogenic mechanism of this disorder.

Insights into the bZIP gene family in Osmanthus fragrans and the role of OfbZIP98 in the callus and flower

Osmanthus fragrans has important ornamental and economic value. As the callus re-differentiation process is difficult, a genetic transformation system has not been successfully established in O. fragrans. The basic leucine zipper (bZIP) transcription factors play an important role in plant growth and development. A total of 116 OfbZIP genes were identified in O. fragrans using bioinformatics methods. According to the evolutionary relationships, these genes were divided into 12 subfamilies and were found to be unevenly distributed on 23 chromosomes of O. fragrans. The quantitative real-time PCR results showed that the expression trend of OfbZIP98 was consistent with the proliferation of O. fragrans callus. The subcellular localization and yeast self-activation results showed that OfbZIP98 was localized in the nucleus and had self-activation activity. Further, phenotypic observation of over-expressed tobacco showed that compared with the wild type (WT), the transgenic strain formed callus 4 d earlier, and callus re-differentiation began on day 15. Analysis of the callus differentiation rate showed that after 20 d of culture, the differentiation rate of the leaf callus of the transgenic strain was about twice that of the WT plants. In addition, measurements of corolla diameter, corolla circumference, corolla area, and corolla tube diameter indicated that the petals of the transgenic strain were significantly larger than the WT plants. Our results showed that OfbZIP98 has the important function of promoting callus growth, re-differentiation, and flower organ enlargement. These findings provide new insights into the potential role of OfbZIP98 in the growth and development of O. fragrans.

β-cell dedifferentiation in HOMA-βlow and HOMA-βhigh subjects.

β-cell dedifferentiation ratio is increased in type 2 diabetes; but its direct link to in vivo β-cell function in human remains unclear. The present study was designed to investigate whether β-cell dedifferentiation in situ was closely associated with β-cell function in vivo and to identify targets crucial for β-cell dedifferentiation/function in human. We acquired HOMA-β values, calculated the number of hormone-negative endocrine cells and evaluated important markers and novel candidates for β-cell dedifferentiation/function on paraneoplastic pancreatic tissues from 13 patients with benign pancreatic cystic neoplasm (PCN) or intrapancreatic accessory spleen. Both β-cell dedifferentiation ratio and dedifferentiation marker (Aldh1a3) were inversely related with in vivo β-cell function (HOMA-β) and in situ β-cell functional markers Glut2 and Ucn3 in human. Moreover, the islets from HOMA-βlow subjects were manifested as 1) increased β-cell dedifferentiation ratio, 2) enriched dedifferentiation maker Aldh1a3, and 3) lower expression of Glut2 and Ucn3, compared to those from HOMA-βhigh subjects. We found that basic leucine zipper transcription factor 2 (Bach2) expression was significantly induced in islets from HOMA-βlow patients and was positively correlated with the ratio of β-cell dedifferentiation in human. Our findings emphasize the contribution of β-cell dedifferentiation to β-cell dysfunction in human. The Bach2 induction in β-cells with higher frequency of dedifferentiation observed in HOMA-βlow subjects reinforce its distinctive role as a pharmaceutical target of β-cell dedifferentiation for the treatment of human diabetes.

Transcriptional regulation and functional validation analysis of the McbZIP1 in Mentha canadensis L.

Monoterpenoids are the main components of Mentha canadensis essential oil. Monoterpene biosynthetic pathways have been explored, but the regulatory mechanisms remain unclarified. We identified an abscisic acid (ABA)-inducible A-type basic leucine zipper (bZIP) transcription factor McbZIP1 that was localized in the nucleus and positively regulates monoterpene synthesis. McbZIP1 was expressed in most M. canadensis tissues and was induced under ABA, mannitol, and NaCl treatments. McbZIP1 had transcriptional activity in yeast and the N terminus (amino acids 75–117) was sufficient for transactivation. Yeast one-hybrid and Dual-Luciferase assays showed that McbZIP1 binds to ABA-responsive elements in the promoter region of limonene synthase gene. Yeast two-hybrid and biomolecular fluorescence complementation assays revealed that McbZIP1 interacts with McSnRK2.4. Overexpression of McbZIP1 in peppermint resulted in dramatically up-regulated monoterpene biosynthesis gene levels and increased menthol contents. The results support a transcriptional regulation mechanism in which McbZIP1 serves as a positive regulator of menthol biogenesis. These findings contribute to the molecular mechanism of monoterpenoid biogenesis, which may have uses in genetic engineering and menthol production.

Two high-quality Prototheca zopfii genomes provide new insights into their evolution as obligate algal heterotrophs and their pathogenicity.

The genus Prototheca, characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii. The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.

Nrf2/ASPM axis regulated vasculogenic mimicry formation in hepatocellular carcinoma under hypoxia.

Hypoxic microenvironment is a common feature of most solid tumors including hepatocellular carcinoma (HCC). Vasculogenic mimicry (VM) formation by tumor cells could provide blood supply to tumor cells under hypoxia. NFE2 like basic leucine zipper (bZIP) transcription factor 2 (Nrf2), a regulator of cellular homeostasis, may promote tumor progression in the hypoxic conditions. However, the role and regulatory mechanisms of Nrf2 in HCC are not fully elucidated. Nrf2 and assembly factor for spindle microtubules (ASPM) expression modulations were conducted by lentiviral transfections. Western blot, immunofluorescence, ChIP-qPCR, dual-luciferase reporter gene assay, flow cytometry, RNA sequencing, multiple bioinformatics databases analysis, cell function assays in vitro, mouse model in vivo and human HCC tissues were employed to assess the effect of Nrf2/ASPM axis on HCC progression under hypoxia. Nrf2 and ASPM expression facilitated epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) feature, and VM formation of HCC cells under hypoxia. Furthermore, Nrf2-regulated ASPM expression, via binding directly to the promoter region of ASPM and transcriptionally promoting ASPM expression. ASPM re-expression in Nrf2 knockdown cells or ASPM knockdown in Nrf2 overexpression cells reversed the cellular function caused by Nrf2. Meantime, retinol metabolism pathway was disrupted following abnormal ASPM expression. Nrf2/ASPM axis in murine models accelerated tumor growth and VM, corroborating in vitro findings. All-trans retinoic acid treatment reversed stemness and VM of HCC cells in vitro and in vivo. Clinically, Nrf2 and ASPM expressions were related to poor prognosis of HCC patients. Nrf2 drives EMT, CSCs characteristics and VM in HCC under hypoxia through the modulation of ASPM. Retinol metabolism pathway was dysregulated in HCC cells with ASPM overexpression. Nrf2/ASPM axis and related pathway provided potential therapeutic target for HCC.

Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA2 is driven by importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to enhanced yellow fluorescent protein (EYFP) show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent with tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell type differentiation of the epidermis.