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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.

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., (© 2024. The Author(s).)

Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The bZIP transcription factors play crucial roles in various aspects of plant biology, including development, defence mechanisms, senescence, and responses to both biotic and abiotic environmental stresses. Myristica fragrans Houtt. transcriptome analysis has identified 15 bZIP transcription factors, each exhibiting major conserved domains and motifs such as BRLZ, MFMR, and DOG1. Functional characterisation of these identified MfbZIP factors indicates their predominant localisation within the nucleus. Phylogenetic analysis reveals that MfbZIP factors cluster into three subgroups alongside annotated bZIP sequences from Magnolia sinica and Arabidopsis thaliana. Moreover, gene ontology (GO) analysis highlights several key functions of MfbZIP, including involvement in defence responses, abscisic acid-induced signalling pathways, and DNA-binding transcription factor activity. Further investigation through KEGG pathway analysis reveals that the amino acid sequences of MfbZIP contain binding motifs for proteins such as TGA, implicated in plant hormone signal transduction pathways associated with disease resistance. To confirm the disease-defence-related activity of the TGA binding protein within MfbZIP, we employed amino acid sequences for 3-D ab initio modelling. Subsequently, we analysed TGA-NPR1 interactions using docking and molecular dynamics simulation analysis. These analyses shed light on the functional and structural aspects of TGA, demonstrating its stable association with NPR1 protein and its significance in the expression of PR1 protein, thus playing a pivotal role in defence responses against pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The basic leucine zipper (bZIP) is a widely found transcription factor family that plays regulatory roles in a variety of cellular processes including cell growth and development and various stress responses. However, the bZIP gene family has not been well studied at a genome-wide scale in Fusarium graminearum (Fg) , a potent pathogen of cereal grains. In the present study, we conducted a genome-wide identification, characterization, and expression profiling of 22 F. graminearum bZIP ( FgbZIP ) genes at different developmental stages and under various abiotic stresses. All identified FgbZIPs were categorized into nine groups based on their sequence similarity and phylogenetic tree analysis. Furthermore, the gene structure analysis, conserved motif analysis, chromosomal localization, protein network studies, and synteny analysis were performed. The symmetry of the exon and intron varied with the phylogenetic groups. The post-translational modifications (PTMs) analysis also predicted several phosphorylation sites in FgbZIPs, indicating their functional diversity in cellular processes. The evolutionary study identified many orthogroups among eight species and also predicted several gene duplication events in F. graminearum . The protein modeling indicated the presence of a higher number of α-helices and random coils in their structures. The expression patterns of FgbZIP genes showed that 5 FgbZIP genes, including FgbZIP_1.1 , FgbZIP_1.3 , FgbZIP_2.6 FgbZIP_3.1 and FgbZIP_4.3 , had high expression at different growth and conidiogenesis stages. Similarly, eight genes including FgbZIP_1.1 , FgbZIP_1.6 , FgbZIP_2.3 , FgbZIP_2.4 , FgbZIP_4.1 , FgbZIP_4.2 , FgbZIP_4.3 and FgbZIP_4.6 demonstrated their putative role in response to various abiotic stresses. In summary, these results provided basic information regarding FgbZIPs which are helpful for further functional analysis.

The occurrence of frequent, extreme high temperatures affects agriculture and causes irreversible damage during the ripening period of grapes. Breeding high-temperature-tolerant varieties of grapes is the main way to deal with this challenge, thus necessitating research on the regulatory mechanism of high-temperature tolerance. Extreme high temperature causes the mismatch of proteins in the endoplasmic reticulum in plant cells and initiates the unfolded protein response (UPR). The transcription factor bZIP60 participates in the UPR process. In the present study, VvbZIP60 and VvbZIP60s (unconventional splicing of VvbZIP60) were cloned and expressed in a transgenic system to verify heat tolerance. VvbZIP60s was found to be a key gene in adapting to heat stress. VvbZIP60s/60u interacted with VvHSP83 as observed in two yeast hybrids, with bimolecular fluorescence complementation and pull-down assays. VvHSP83 is also a key gene for plants to adapt to heat stress by participating in the renaturation and degradation of denatured proteins under adversity, causing plants to resist high temperatures. This study provides a basis for analyzing the mechanism of high-temperature tolerance in grapes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

AFP1 interacts with ABI5 and negatively regulates the abscisic acid (ABA) signaling by accelerating ABI5's degradation during the seed germination phase in Arabidopsis, but the underlying mechanism remains unclear. Moreover, the molecular basis of the interaction between AFP1 homologs and ABI5 has yet to be elucidated. In this study, the patterns of their interactions with ABI5 were investigated in detail. We found that AFP2/3/4 can bind two regions of ABI5, one is ABI5 1aa to 135aa and another is ABI5 202aa to 213aa . However, AFP1 only interacts with the second region of ABI5, i.e. ABI5 202aa to 213aa . Prior research has shown that ABI5 1aa to 135aa is related to the transcriptional activity of ABI5. Thus, our results suggest that AFPs may also modulate ABI5, by directly binding to its transcriptional activation domain, thereby influencing its transcriptional activity. Further, interactions between AFPs and ABI5 were not affected if the Ser 42th in the ABI5-SnRK2 motif were mutated respectively to Glu or Ala. Nevertheless, interactions between AFPs and ABI5 were eliminated if the Thr 47th and Thr 206th of ABI5 were mutated respectively to Glu or Ala. Since the two residues of Thr 47th and Thr 206th were located in the phosphorylation motifs of CKII, AFPs might regulate the activities of ABI5 transcription factor through a CKII-dependent pathway., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2021. Published by Elsevier Inc.)

Transcription factors are regulatory proteins known to modulate gene expression. These are the critical component of signaling pathways and help in mitigating various developmental and stress responses. Among them, bZIP, BBR, and BZR transcription factor families are well known to play a crucial role in regulating growth, development, and defense responses. However, limited data is available on these transcription factors in Triticum aestivum. In this study, bZIP, BBR, and BZR sequences from Brachypodium distachyon, Oryza sativa, Oryza barthii, Oryza brachyantha, T. aestivum, Triticum urartu, Sorghum bicolor, Zea mays were retrieved, and dendrograms were constructed to analyze the evolutionary relatedness among them. The sequences clustered into one group indicated a degree of evolutionary correlation highlighting the common lineage of cereal grains. This analysis also exhibited that these genes were highly conserved among studied monocots emphasizing their common ancestry. Furthermore, these transcription factor genes were evaluated for envisaging conserved motifs, gene structure, and subcellular localization in T. aestivum. This comprehensive computational analysis has provided an insight into transcription factor evolution that can also be useful in developing approaches for future functional characterization of these genes in T. aestivum. Furthermore, the data generated can be beneficial in future for genetic manipulation of economically important plants., Competing Interests: The authors have declared that no competing interests exist.

Proteins are folded to avoid exposure of the nonpolar groups to water because water-mediated interactions between nonpolar groups are a promising factor in the thermodynamic stabilities of proteins-which is a well-accepted view as one of the unique effects of hydrophobic interactions. This article poses a critical question for this classical view by conducting an accurate solvation free-energy calculation for a thermodynamic cycle of a protein folding using a liquid-state density functional theory. Here, the solvation-free energy for a leucine zipper formation was examined in the coiled-coil protein GCN4-p1, a typical model for hydrophobic interactions, which demonstrated that water-mediated interactions were unfavorable for the association of nonpolar groups in the native state, while the dispersion forces between them were, instead, responsible for the association. Furthermore, the present analysis well predicted the isolated helical state stabilized by pressure, which was previously observed in an experiment. We reviewed the problems in the classical concept and semiempirical presumption that the energetic cost of the hydration of nonpolar groups is a driving force of folding., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate β-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed β-propeller fold containing three unusual loops. A model of the Rho1-Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The progression of cancer involves not only the gradual evolution of cells by mutations in DNA but also alterations in the gene expression induced by those mutations and input from the surrounding microenvironment. Such alterations contribute to cancer cells' abilities to reprogram metabolic pathways and undergo epithelial-to-mesenchymal transition (EMT), which facilitate the survival of cancer cells and their metastasis to other organs. Recently, BTB and CNC homology 1 (BACH1), a heme-regulated transcription factor that represses genes involved in iron and heme metabolism in normal cells, was shown to shape the metabolism and metastatic potential of cancer cells. The growing list of BACH1 target genes in cancer cells reveals that BACH1 promotes metastasis by regulating various sets of genes beyond iron metabolism. BACH1 represses the expression of genes that mediate cell-cell adhesion and oxidative phosphorylation but activates the expression of genes required for glycolysis, cell motility, and matrix protein degradation. Furthermore, BACH1 represses FOXA1 gene encoding an activator of epithelial genes and activates SNAI2 encoding a repressor of epithelial genes, forming a feedforward loop of EMT. By synthesizing these observations, we propose a "two-faced BACH1 model", which accounts for the dynamic switching between metastasis and stress resistance along with cancer progression. We discuss here the possibility that BACH1-mediated promotion of cancer also brings increased sensitivity to iron-dependent cell death (ferroptosis) through crosstalk of BACH1 target genes, imposing programmed vulnerability upon cancer cells. We also discuss the future directions of this field, including the dynamics and plasticity of EMT., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

TGA transcription factor is a member of the D subfamily of the basic region-leucine zippers (bZIP) family. It is a type of transcription factor that was first identified in plants and is the main regulator in plant development and physiological processes, including morphogenesis and seed formation in response to abiotic and biotic stress and maintaining plant growth. The present study examined the sequence of the MaTGA8 transcription factor, the sequence of which belonged to subfamily D of the bZIP and had multiple cis-acting elements such as the G-box, TCA-element, TGACG-element, and P-box. Quantitative real time polymerase chain reaction (qRT-PCR) analyses showed that MaTGA8 was significantly down-regulated by the soil-borne fungus Fusarium oxysporum f. sp. cubense race 4 (Foc TR4). Under the induction of salicylic acid (SA), MaTGA8 was down-regulated, while different members of the MaNPR1 family responded significantly differently. Among them, MaNPR11 and MaNPR3 showed an overall upward trend, and the expression level of MaNPR4 , MaNPR8, and MaNPR13 was higher than other members. MaTGA8 is a nuclear-localized transcription factor through strong interaction with MaNPR11 or weaker interaction with MaNPR4 , and it is implied that the MaPR gene can be activated. In addition, the MaTGA8 transgenic Arabidopsis has obvious disease resistance and higher chlorophyll content than the wild-type Arabidopsis with the infection of Foc TR4. These results indicate that MaTGA8 may enhance the resistance of bananas to Foc TR4 by interacting with MaNPR11 or MaNPR4 . This study provides a basis for further research on the application of banana TGA transcription factors in Foc TR4 stress and disease resistance and molecular breeding programs.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.

Background: The bZIP gene family, which is widely present in plants, participates in varied biological processes including growth and development and stress responses. How do the genes regulate such biological processes? Systems biology is powerful for mechanistic understanding of gene functions. However, such studies have not yet been reported in poplar., Results: In this study, we identified 86 poplar bZIP transcription factors and described their conserved domains. According to the results of phylogenetic tree, we divided these members into 12 groups with specific gene structures and motif compositions. The corresponding genes that harbor a large number of segmental duplication events are unevenly distributed on the 17 poplar chromosomes. In addition, we further examined collinearity between these genes and the related genes from six other species. Evidence from transcriptomic data indicated that the bZIP genes in poplar displayed different expression patterns in roots, stems, and leaves. Furthermore, we identified 45 bZIP genes that respond to salt stress in the three tissues. We performed co-expression analysis on the representative genes, followed by gene set enrichment analysis. The results demonstrated that tissue differentially expressed genes, especially the co-expressing genes, are mainly involved in secondary metabolic and secondary metabolite biosynthetic processes. However, salt stress responsive genes and their co-expressing genes mainly participate in the regulation of metal ion transport, and methionine biosynthetic., Conclusions: Using comparative genomics and systems biology approaches, we, for the first time, systematically explore the structures and functions of the bZIP gene family in poplar. It appears that the bZIP gene family plays significant roles in regulation of poplar development and growth and salt stress responses through differential gene networks or biological processes. These findings provide the foundation for genetic breeding by engineering target regulators and corresponding gene networks into poplar lines.

Maintenance of stem-cell identity requires proper regulation of enhancer activity. Both transcription factors OCT4/SOX2/NANOG and histone methyltransferase complexes MLL/SET1 were shown to regulate enhancer activity, but how they are regulated in embryonic stem cells (ESCs) remains further studies. Here, we report a transcription factor BACH1, which directly interacts with OCT4/SOX2/NANOG (OSN) and MLL/SET1 methyltransferase complexes and maintains pluripotency in mouse ESCs (mESCs). BTB domain and bZIP domain of BACH1 are required for these interactions and pluripotency maintenance. Loss of BACH1 reduced the interaction between NANOG and MLL1/SET1 complexes, and decreased their occupancy on chromatin, and further decreased H3 lysine 4 trimethylation (H3K4me3) level on gene promoters and (super-) enhancers, leading to decreased enhancer activity and transcription activity, especially on stemness-related genes. Moreover, BACH1 recruited NANOG through chromatin looping and regulated remote NANOG binding, fine-tuning enhancer-promoter activity and gene expression. Collectively, these observations suggest that BACH1 maintains pluripotency in ESCs by recruiting NANOG and MLL/SET1 complexes to chromatin and maintaining the trimethylated state of H3K4 and enhancer-promoter activity, especially on stemness-related genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Torsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodelling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total twist of a DNA fragment, we address the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 0.5° per dinucleotide step, starting from the relaxed state to a maximum of 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorbing most of the torsional stress become more torsionally rigid, as they are involved in protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavourable. Our data suggest that specifically bound BZIP factors could act as torsional stress insulators, modulating the propagation of torsional stress along the chromatin fibre, which might promote cooperative binding of collaborative DNA-binding factors.

In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.

Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.

Activator protein 1 (AP-1) is one of the largest families of basic leucine zipper (bZIP) transcription factors in eukaryotic cells. How AP-1 proteins achieve target DNA binding specificity remains elusive. In Saccharomyces cerevisiae, the AP-1-like protein (Yap) family comprises eight members (Yap1 to Yap8) that display distinct genomic target sites despite high sequence homology of their DNA binding bZIP domains. In contrast to the other members of the Yap family, which preferentially bind to short (7-8 bp) DNA motifs, Yap8 binds to an unusually long DNA motif (13 bp). It has been unclear what determines this unique specificity of Yap8. In this work, we use molecular and biochemical analyses combined with computer-based structural design and molecular dynamics simulations of Yap8-DNA interactions to better understand the structural basis of DNA binding specificity determinants. We identify specific residues in the N-terminal tail preceding the basic region, which define stable association of Yap8 with its target promoter. We propose that the N-terminal tail directly interacts with DNA and stabilizes Yap8 binding to the 13 bp motif. Thus, beside the core basic region, the adjacent N-terminal region contributes to alternative DNA binding selectivity within the AP-1 family., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The generation of catalytically active metalloproteins inside living mammalian cells is a major research challenge at the interface between catalysis and cell biology. Herein we demonstrate that basic domains of bZIP transcription factors, mutated to include two histidine residues at i and i+4 positions, react with palladium(II) sources to generate catalytically active, stapled pallado-miniproteins. The resulting constrained peptides are efficiently internalized into living mammalian cells, where they perform palladium-promoted depropargylation reactions without cellular fixation. Control experiments confirm the requirement of the peptide scaffolding and the palladium staple for attaining the intracellular reactivity., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Olive ( Olea europaea .L) is an economically important oleaginous crop and its fruit cold-pressed oil is used for edible oil all over the world. The basic region-leucine zipper ( bZIP ) family is one of the largest transcription factors families among eukaryotic organisms; its members play vital roles in environmental signaling, stress response, plant growth, seed maturation, and fruit development. However, a comprehensive report on the bZIP gene family in olive is lacking. In this study, 103 OebZIP genes from the olive genome were identified and divided into 12 subfamilies according to their genetic relationship with 78 bZIPs of A. thaliana . Most OebZIP genes are clustered in the subgroup that has a similar gene structure and conserved motif distribution. According to the characteristics of the leucine zipper region, the dimerization characteristics of 103 OebZIP proteins were predicted. Gene duplication analyses revealed that 22 OebZIP genes were involved in the expansion of the bZIP family. To evaluate the expression patterns of OebZIP genes, RNA-seq data available in public databases were analyzed. The highly expressed OebZIP genes and several lipid synthesis genes ( LPGs ) in fruits of two varieties with different oil contents during the fast oil accumulation stage were examined via qRT-PCR. By comparing the dynamic changes of oil accumulation, OebZIP1 , OebZIP7 , OebZIP22 , and OebZIP99 were shown to have a close relationship with fruit development and lipid synthesis. Additionally, some OebZIP had a significant positive correlation with various LPG genes. This study gives insights into the structural features, evolutionary patterns, and expression analysis, laying a foundation to further reveal the function of the 103 OebZIP genes in olive.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Basic leucine zipper (bZIP) transcription factor plays an important role in various biological processes, such as response to biotic and abiotic stresses. In this study we performed a systematic investigation and analysis of bZIP gene family in Gossypium hirsutum to predict their functions in response to different abiotic stresses. A total of 207 bZIP genes were identified from Gossypium hirsutum genome and classified into 13 subfamilies through phylogenetic analysis, which was testified by the analysis of conserved motifs and exon-intron structures. Annotation of GHbZIPs was performed based on well-studied Arabidopsis bZIPs to speculate the gene function. RNA-seq analysis was conducted to identify the co-expressed and differentially expressed bZIPs under cold, heat, salt and PEG treatments. Promoter analysis and interaction network of GHbZIP proteins demonstrated that ABA-activated signaling pathway was pivotal in the regulation of GHbZIPs, and GHbZIPs involved in ER stress were supposed to function through interaction with other GHbZIPs and ABA pathway. Cis-elements in the upstream and downstream of GHbZIPs interaction network were also discussed. These findings provided us with clues about functions of bZIP in Gossypium hirsutum., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico / in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.

The transcription factor Bach1 impairs angiogenesis after ischemic injury by suppressing Wnt/β-catenin signaling; however, the specific domains responsible for the anti-angiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients and is the early induction gene after ischemia. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were significantly lower and ROS levels were higher in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human endothelial cells was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF and HO-1 expression in endothelial cells. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal domain of TCF4. Bach1, but not Bach1-ΔBTB, also co-precipitated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy., Competing Interests: Declaration of competing interest None., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H 2 O 2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein-protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically disordered proteins and their targets. We illustrate the method with reference to interactions between the activation domain of the human T-cell leukemia virus type-I (HTLV-1) basic leucine zipper protein (HBZ) and its cellular binding partner, the KIX domain of the transcriptional coactivator CBP.

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The human voltage-gated proton channel [Hv1 (1) or VSDO (2) ] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn 2+ -induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H + concentration is required for the protonation of side chains in the Zn 2+ -induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

It is well known that the DNA-binding specificity of transcription factors (TFs) is influenced by protein-protein interactions (PPIs). However, the underlying molecular mechanisms remain largely unknown. In this work, we adopted the cAMP-response element-binding protein (CREB) of the basic leucine zipper (bZIP) TF family as a model system, and a workflow of combined bioinformatics and molecular modeling analysis of protein-DNA interaction was tested. First, the multiple sequence alignment and SDPsite method were used to find potential bZIP family binding specificity determining positions (SDPs) within the protein-protein interaction region. Second, the mutation system was analyzed using molecular dynamics simulation. Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy calculations confirmed the enhancement of the binding affinity of the mutation, which was in agreement with experimental results. The root mean square fluctuation (RMSF) and hydrogen bonding changes suggested an open and close protein dimerization process after the system was mutated, which resulted in the change of the hydrogen bonding of the protein-DNA interface and a slight conformational change. We believe that this work will contribute to understanding the protein-protein interaction-regulated binding specificity of bZIP transcription factors.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR., Competing Interests: JP, PW No competing interests declared, (© 2019, Peschek and Walter.)

Hac1p is a key transcription factor regulating the unfolded protein response (UPR) induced by abnormal accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) in Saccharomyces cerevisiae . The accumulation of unfolded/misfolded proteins is sensed by protein Ire1p, which then undergoes trans-autophosphorylation and oligomerization into discrete foci on the ER membrane. HAC1 pre-mRNA, which is exported to the cytoplasm but is blocked from translation by its intron sequence looping back to its 5'UTR to form base-pair interaction, is transported to the Ire1p foci to be spliced, guided by a cis-acting bipartite element at its 3'UTR (3'BE). Spliced HAC1 mRNA can be efficiently translated. The resulting Hac1p enters the nucleus and activates, together with coactivators, a large number of genes encoding proteins such as protein chaperones to restore and maintain ER homeostasis and secretary protein quality control. This review details the translation regulation of Hac1p production, mediated by the nonconventional splicing, in the broad context of translation control and summarizes the evolution and diversification of the UPR signaling pathway among fungal, metazoan and plant lineages.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Vrille (Vri), a basic leucine zipper transcription factor, plays important roles in insect circadian clock regulation, tracheal development, proliferation, flight and metamorphosis. Here, Helicoverpa armigera was used as a model to investigate the role of Vri in larval moulting and metamorphosis. Sequence analysis results revealed that H. armigera Vri (HaVri) shares a high amino acid identity with other Lepidoptera Vri homologues. Spatial-temporal expression pattern data showed that HaVri expression was highly abundant in larval moulting and metamorphosis stages and was mainly expressed in the midgut and Malpighian tubule during metamorphosis. HaVri knockdown by RNA interference in the fourth-instar larvae prevented larval moulting, and HaVri knockdown in the fifth-instar larvae suppressed midgut remodelling and delayed or blocked metamorphosis. Further studies confirmed that 20-hydroxyecdysone (20E) activated HaVri expression via its heterodimer receptors, ecdysone receptor (EcRB1) and ultraspiracle protein (USP1), whereas methoprene [juvenile hormone analogue (JHA)] promoted HaVri expression via its intracellular receptor methoprene-tolerant (Met1). However, 20E and JHA can counteract each other in the activation of HaVri expression. Together, the present results suggested that HaVri was involved in larval moulting and metamorphosis and was regulated by 20E and JHA in H. armigera., (© 2018 The Royal Entomological Society.)

The UV Resistance Locus 8 (UVR8) photoreceptor controls UV-B mediated photomorphogenesis in Arabidopsis. The aim of this work is to collect and characterize different molecular reporters of photomorphogenic UV-B responses. Browsing available transcriptome databases, we identified sets of genes responding specifically to this radiation and are controlled by pathways initiated from the UVR8 photoreceptor. We tested the transcriptional changes of several reporters and found that they are regulated differently in different parts of the plant. Our experimental system led us to conclude that the examined genes are not controlled by light piping of UV-B from the shoot to the root or signalling molecules which may travel between different parts of the plant body but by local UVR8 signalling. The initiation of these universal signalling steps can be the induction of Elongated Hypocotyl 5 (HY5) and its homologue, HYH transcription factors. We found that their transcript and protein accumulation strictly depends on UVR8 and happens in a tissue autonomous manner. Whereas HY5 accumulation correlates well with the UVR8 signal across cell layers, the induction of flavonoids depends on both UVR8 signal and a yet to be identified tissue-dependent or developmental determinant.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the largest gene families, and play crucial roles in many processes, including stress responses, hormone effects. The TF family also participates in plant growth and development. However, limited information is available for these genes in moso bamboo ( Phyllostachys edulis ), one of the most important non-timber forest products in the world. In the present study, 154 putative PhebZIP genes were identified in the moso bamboo genome. The phylogenetic analyses indicate that the PhebZIP gene proteins classify into 9 subfamilies and the gene structures and conserved motifs that analyses identified among all PhebZIP proteins suggested a high group-specificity. Microsynteny and evolutionary patterns analyses of the non-synonymous (Ka) and synonymous (Ks) substitution rates and their ratios indicated that paralogous pairs of PhebZIP genes in moso bamboo underwent a large-scale genome duplication event that occurred 7-15 million years ago (MYA). According to promoter sequence analysis, we further selected 18 genes which contain the higher number of cis -regulatory elements for expression analysis. The result showed that these genes are extensively involved in GA-, ABA- and MeJA-responses, with possibly different mechanisms. The tissue-specific expression profiles of PhebZIP genes in five plant tissues/organs/developmental stages suggested that these genes are involved in moso bamboo organ development, especially seed development. Subcellular localization and transactivation activity analysis showed that PhebZIP47 and PhebZIP126 were localized in the nucleus and PhebZIP47 with no transcriptional activation in yeast. Our research provides a comprehensive understanding of PhebZIP genes and may aid in the selection of appropriate candidate genes for further cloning and functional analysis in moso bamboo growth and development, and improve their resistance to stress during their life.

Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.

Background: The basic leucine zipper (bZIP) transcription factor is one of the most abundant and conserved transcription factor families. In addition to being involved in growth and development, bZIP transcription factors also play an important role in plant adaption to abiotic stresses., Results: A total of 41 bZIP genes that encode 66 proteins were identified in Ipomoea trifida. They were distributed on 14 chromosomes of Ipomoea trifida. Segmental and tandem duplication analysis showed that segmental duplication played an important role in the ItfbZIP gene amplification. ItfbZIPs were divided into ten groups (A, B, C, D, E, F, G, H, I and S groups) according to their phylogenetic relationships with Solanum lycopersicum and Arabidopsis thaliana. The regularity of the exon/intron numbers and distributions is consistent with the group classification in evolutionary tree. Prediction of the cis-acting elements found that promoter regions of ItfbZIPs harbored several stress responsive cis-acting elements. Protein three-dimensional structural analysis indicated that ItfbZIP proteins mainly consisted of α-helices and random coils. The gene expression pattern from transcriptome data and qRT-PCR analysis showed that ItfbZIP genes expressed with a tissue-specific manner and differently expressed under various abiotic stresses, suggesting that the ItfbZIPs were involved in stress response and adaption in Ipomoea trifida., Conclusions: Genome-wide identification, gene structure, phylogeny and expression analysis of bZIP gene in Ipomoea trifida supplied a solid theoretical foundation for the functional study of bZIP gene family and further facilitated the molecular breeding of sweet potato.

Heme is one of the key factors involved in the oxidative stress response of cells. The transcriptional repressor Bach1 plays an important role in this response through its heme-binding activity. Heme inhibits the transcriptional-repressor activity of Bach1, and can occur in two binding modes: 5- and 6-coordinated binding. The Cys-Pro (CP) motif has been determined to be the heme-binding motif of Bach family proteins. The sequence of Bach1 includes six CP motifs, and four CP motifs are functional. With the aim of elucidating the molecular mechanism of heme-Bach1 regulation, we conducted biophysical analyses focusing on the C-terminal region of mouse Bach1 (residues 631-739) which is located after the bZip domain and includes one functional CP motif. UV-Vis spectroscopy indicated that the CP motif binds heme via 5-coordinated bond. A mutant, which included a cysteine to alanine substitution at the CP motif, did not show 5-coordination, suggesting that this binding mode is specific to the CP motif. Surface plasmon resonance revealed that the binding affinity and stoichiometry of heme with the Bach1 C-terminal region were K D = 1.37 × 10 -5 M and 2.3, respectively. The circular dichroism spectrum in the near-UV region exhibited peaks for heme binding to the CP motif. No significant spectral shifts were observed in the far-UV region when samples with and without heme were compared. Therefore, disordered-ordered transition such as "coupled folding and binding" is not involved in the Bach1-heme system. Consequently, the heme response of this C-terminal region is accomplished by disorder-disorder conformational alteration.

Sequence-specific transcription factors (TFs) represent one of the largest groups of proteins that is targeted for SUMO post-translational modification, in both yeast and humans. SUMO modification can have diverse effects, but recent studies showed that sumoylation reduces the interaction of multiple TFs with DNA in living cells. Whether this relates to a general role for sumoylation in TF binding site selection, however, has not been fully explored because few genome-wide studies aimed at studying such a role have been reported. To address this, we used genome-wide analysis to examine how sumoylation regulates Sko1, a yeast bZIP TF with hundreds of known binding sites. We find that Sko1 is sumoylated at Lys 567 and, although many of its targets are osmoresponse genes, the level of Sko1 sumoylation is not stress-regulated and the modification does not depend or impinge on its phosphorylation by the osmostress kinase Hog1. We show that Sko1 mutants that cannot bind DNA are not sumoylated, but attaching a heterologous DNA binding domain restores the modification, implicating DNA binding as a major determinant for Sko1 sumoylation. Genome-wide chromatin immunoprecipitation (ChIP-seq) analysis shows that a sumoylation-deficient Sko1 mutant displays increased occupancy levels at its numerous binding sites, which inhibits the recruitment of the Hog1 kinase to some induced osmostress genes. This strongly supports a general role for sumoylation in reducing the association of TFs with chromatin. Extending this result, remarkably, sumoylation-deficient Sko1 binds numerous additional promoters that are not normally regulated by Sko1 but contain sequences that resemble the Sko1 binding motif. Our study points to an important role for sumoylation in modulating the interaction of a DNA-bound TF with chromatin to increase the specificity of TF-DNA interactions., Competing Interests: The authors have declared that no competing interests exist.

Spin relaxation in solution-state NMR spectroscopy is a powerful approach to explore the conformational dynamics of biological macromolecules. Probability distribution functions for overall or internal correlation times have been used previously to model spectral density functions central to spin-relaxation theory. Applications to biological macromolecules rely on transverse relaxation rate constants, and when studying nanosecond timescale motions, sampling at ultralow frequencies is often necessary. Consequently, appropriate distribution functions necessitate spectral density functions that are accurate and convergent as frequencies approach zero. In this work, the inverse Gaussian probability distribution function is derived from general properties of spectral density functions at low and high frequencies for macromolecules in solution, using the principle of maximal entropy. This normalized distribution function is first used to calculate the correlation function, followed by the spectral density function. The resulting model-free spectral density functions are finite at a frequency of zero and can be used to describe distributions of either overall or internal correlation times using the model-free ansatz. To validate the approach, 15 N spin-relaxation data for the bZip transcription factor domain of the Saccharomyces cerevisiae protein GCN4, in the absence of cognate DNA, were analyzed using the inverse Gaussian probability distribution for intramolecular correlation times. The results extend previous models for the conformational dynamics of the intrinsically disordered, DNA-binding region of the bZip transcription factor domain., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Spatial and temporal expression of genes is essential for maintaining phenotype integrity. Transcription factors (TFs) modulate expression patterns by binding to specific DNA sequences in the genome. Along with the core binding motif, the flanking sequence context can play a role in DNA-TF recognition. Here, we employ high-throughput in vitro and in silico analyses to understand the influence of sequences flanking the cognate sites in binding of three most prevalent eukaryotic TF families (zinc finger, homeodomain and bZIP). In vitro binding preferences of each TF toward the entire DNA sequence space were correlated with a wide range of DNA structural parameters, including DNA flexibility. Results demonstrate that conformational plasticity of flanking regions modulates binding affinity of certain TF families. DNA duplex stability and minor groove width also play an important role in DNA-TF recognition but differ in how exactly they influence the binding in each specific case. Our analyses further reveal that the structural features of preferred flanking sequences are not universal, as similar DNA-binding folds can employ distinct DNA recognition modes.

Photozipper (PZ) is a light-activatable basic leucine zipper (bZIP) protein composed of a bZIP domain and a light-oxygen-voltage-sensing domain of aureochrome-1. Blue light induces dimerization and subsequently increases the affinity of PZ for the target DNA sequence. We prepared site-directed PZ mutants in which Asn131 (N131) in the basic region was substituted with Ala and Gln. N131 mutants showed spectroscopic and dimerization properties almost identical to those of wild-type PZ and an increase in helical content in the presence of the target sequence. Quantitative analyses by an electrophoretic mobility shift assay and quartz crystal microbalance (QCM) measurements demonstrated that the half-maximal effective concentrations of N131 mutants to bind to the target sequence were significantly higher than those of PZ. QCM data also revealed that N131 substitutions accelerated the dissociation without affecting the association, suggesting that a base-specific interaction of N131 occurred after the association between PZ and DNA. Activation of PZ by illumination decreased both the standard errors and the unstable period of QCM data. Optical control of transcription factors will provide new knowledge of the recognition of the target sequence.

Appropriate subcellular localization of regulatory factors is critical for cellular function. Pap1, a nucleocytoplasmic shuttling transcription factor of Schizosaccharomyces pombe , is redox regulated for localization and antistress function. In this study, we find that overproduction of a peptide conjugate containing the nuclear export signal of Oxs1, a conserved eukaryotic protein that, along with Pap1, regulates certain diamide responsive genes, can retain Pap1 in the nucleus before stress by competing for nuclear export. The nuclear retention of Pap1 upregulates several drug resistance genes to prime the cells for higher tolerance to disulfide stress. Overproduction of Oxs1 also upregulates these same genes, not by competing for export but by binding directly to the drug resistance gene promoters for Pap1-mediated activation. Of medical relevance is that this may suggest a gene therapy approach of using nuclear export signal conjugates to suppress the nuclear export of biomolecules., (Copyright © 2018 by the Genetics Society of America.)

The transcription factor BTB and CNC homology 1 (Bach1) is widely expressed in most mammalian tissues and functions primarily as a transcriptional suppressor by heterodimerizing with small Maf proteins and binding to Maf recognition elements in the promoters of targeted genes. It has a key regulatory role in the production of reactive oxygen species, cell cycle, heme homeostasis, hematopoiesis, and immunity and has been shown to suppress ischemic angiogenesis and promote breast cancer metastasis. This review summarizes how Bach1 controls these and other cellular and physiological and pathological processes. Bach1 expression and function differ between different cell types. Thus, therapies designed to manipulate Bach1 expression will need to be tightly controlled and tailored for each specific disease state or cell type.

The human T cell leukemia virus I basic leucine zipper protein (HTLV-1 HBZ) maintains chronic viral infection and promotes leukemogenesis through poorly understood mechanisms involving interactions with the KIX domain of the transcriptional coactivator CBP and its paralog p300. The KIX domain binds regulatory proteins at the distinct MLL and c-Myb/pKID sites to form binary or ternary complexes. The intrinsically disordered N-terminal activation domain of HBZ (HBZ AD) deregulates cellular signaling pathways by competing directly with cellular and viral transcription factors for binding to the MLL site and by allosterically perturbing binding of the transactivation domain of the hematopoietic transcription factor c-Myb. Crystal structures of the ternary KIX:c-Myb:HBZ complex show that the HBZ AD recruits two KIX:c-Myb entities through tandem amphipathic motifs (L/V)(V/L)DGLL and folds into a long α-helix upon binding. Isothermal titration calorimetry reveals strong cooperativity in binding of the c-Myb activation domain to the KIX:HBZ complex and in binding of HBZ to the KIX:c-Myb complex. In addition, binding of KIX to the two HBZ (V/L)DGLL motifs is cooperative; the structures suggest that this cooperativity is achieved through propagation of the HBZ α-helix beyond the first binding motif. Our study suggests that the unique structural flexibility and the multiple interaction motifs of the intrinsically disordered HBZ AD are responsible for its potency in hijacking KIX-mediated transcription pathways. The KIX:c-Myb:HBZ complex provides an example of cooperative stabilization in a transcription factor:coactivator network and gives insights into potential mechanisms through which HBZ dysregulates hematopoietic transcriptional programs and promotes T cell proliferation., Competing Interests: The authors declare no conflict of interest.

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1's C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood.

Sequence-specific DNA binding recruits transcription factors (TFs) to the genome to regulate gene expression. Here, we perform high resolution mapping of CEBP proteins to determine how sequence dictates genomic occupancy. We demonstrate a fundamental difference between the sequence repertoire utilized by CEBPs in vivo versus the palindromic sequence preference reported by classical in vitro models, by identifying a palindromic motif at <1% of the genomic binding sites. On the native genome, CEBPs bind a diversity of related 10 bp sequences resulting from the fusion of degenerate and canonical half-sites. Altered DNA specificity of CEBPs in cells occurs through heterodimerization with other bZip TFs, and approximately 40% of CEBP-binding sites in primary human cells harbor motifs characteristic of CEBP heterodimers. In addition, we uncover an important role for sequence bias at core-motif-flanking bases for CEBPs and demonstrate that flanking bases regulate motif function across mammalian bZip TFs. Favorable flanking bases confer efficient TF occupancy and transcriptional activity, and DNA shape may explain how the flanks alter TF binding. Importantly, motif optimization within the 10-mer is strongly correlated with cell-type-independent recruitment of CEBPβ, providing key insight into how sequence sub-optimization affects genomic occupancy of widely expressed CEBPs across cell types.