Helix-loop-helix Research Articles

Evolutionary studies of the basic helix–loop–helix (bHLH) IVc gene family in plants and the role of AtILR3 in Arabidopsis response to ABA stress

AbstractThe basic helix–loop–helix (bHLH) IVc transcription factors (TFs) play central roles in controlling iron (Fe) homeostasis and biotic stress responses. However, their evolutions and functions in other abiotic stresses are poorly understood. In this study, the IAA‐LEUCINE RESISTANT3 (ILR3) homologs were traced roughly back to before the early origin of land plants and divided into six main clades (Clade A‐F). Further analysis found that the ILR3 orthologs were angiosperm‐specific, suffering from motif‐acquisition events and loose purifying selection. Synteny analysis displayed that the whole genome duplications (WGDs) contributed to the establishment of the IRON DEFICIENCY TOLERANT1 (IDT1, also called bHLH34)/bHLH104 lineage prior to the divergence of angiosperms. Sequence analysis revealed that the ILR3 homologs had some novel and conserved motifs except bHLH and leucine zipper (ZIP) domains. Particularly, Arabidopsis thaliana ILR3 (AtILR3) was a nuclear protein and greatly activated by ABA and CdCl2 stresses. Simultaneously, the molecular and genetic analyses suggested that the AtILR3 acted as a positive regulator in the ABA stress response through enhancing the ability of reactive oxygen species (ROS)‐scavenging, such as the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). The inductively coupled plasma mass spectrometry (ICP‐MS) analyses exhibited that the AtILR3 affected the absorption of nutrient elements, especially iron elements, under ABA stress. Collectively, our findings could shed deep light on the origin and evolution of the plant ILR3s, as well as the functions of the AtILR3 under ABA stress.

Genome-wide analysis of the bHLH family and identification of bHLH genes involved in fruit development and ripening of cultivated octoploid strawberry

Abstract Objectives The basic helix–loop–helix (bHLH) transcription factors (TFs) regulate fruit growth in many plants. However, there is no available study on the bHLH gene family in the haplotype-resolved genome of cultivated strawberry (Fragaria × ananassa). Materials and Methods The 131 FabHLH genes identified in the strawberry cultivar ‘Yanli’ haplotype-resolved genome were classified into 24 subfamilies according to their phylogenetic relationships. Gene structure, conserved motifs, and chromosomal locations were investigated using bioinformatics. Results In total, 15 FabHLH genes potentially involved in fruit development were screened based on transcriptome analysis of different stages of fruit development. We also identified the cis-regulatory elements of these 15 FabHLH genes, predicted upstream transcription factors, and identified protein–protein interactions. Conclusions The findings of this study improve our understanding of the regulation mediated by bHLH TFs during strawberry fruit growth and maturation.

A light responsive transcription factor CsbHLH89 positively regulates anthocyanidin synthesis in tea (Camellia sinensis)

Anthocyanins are a class of secondary metabolites with rich physiological functions and many benefits for the human body. Although there have been numerous reports on anthocyanin synthesis in tea plants, the mechanism by which bHLH transcription factors regulate anthocyanin synthesis in tea plants is still unclear. In this study, we identified CsbHLH89, a basic helix–loop–helix (bHLH) transcription factor that is located in the nucleus and cytoplasm and has transcriptional activation activity. CsbHLH89 directly binds to G-box elements and activates the CsCHS, CsFLS, and CsDFR promoters in vitro and in vivo. Further experiments demonstrated that CsbHLH89 could be involved in the synthesis of tea plant anthocyanins as homodimers or heterodimers. Our research results show that CsbHLH89 can positively regulate anthocyanin synthesis in tea. In addition, it was found that CsHY5, a light signal transduction factor, could bind to the promoter of CsbHLH89 to indirectly promote anthocyanin accumulation. Silencing of CsHY5 and CsbHLH89 by antisense oligonucleotide, respectively, revealed that the expression of structural genes and transcription factors were down-regulated. Shading treatment of tea plants revealed a significant reduction in anthocyanin content and down-regulation of the expression of genes involved in anthocyanin synthesis. Our research provides new insight into the regulatory network of anthocyanin synthesis in tea and a new strategy for breeding rich anthocyanin.

Study of glutathione S-transferase (CqGSTs) gene expression patterns, the response of basic helix–loop–helix (bHLH) transcription factor and genome-wide identification gene family in quinoa (Chenopodium quinoa Willd.) and its mechanisms of salt stress tolerance

Study of glutathione S-transferase (CqGSTs) gene expression patterns, the response of basic helix–loop–helix (bHLH) transcription factor and genome-wide identification gene family in quinoa (Chenopodium quinoa Willd.) and its mechanisms of salt stress tolerance

Functional analysis of sweet cherry PavbHLH106 in the regulation of cold stress.

Sweet cherry PavbHLH106 was up-regulated under cold induction and overexpressed to enhance the cold resistance in tobacco by mediating the scavenging of ROS through increasing of antioxidant enzyme activity. Sweet cherry (Prunus avium L.) is an economically important fruit. Chilling requirements are critical during dormancy, but abnormally low temperatures unfavorably affect fruit growth and development. Differences were found in the transcript level of PavbHLH106 under salt, dehydration, and low-temperature treatments, especially in response to cold stress, suggesting that this gene is involved in the regulation of different abiotic stresses. PavbHLH106 is homologous to Arabidopsis thaliana AtbHLH106 with a conserved bHLH domain, and transient expression in tobacco suggests that the protein is localized in the nucleus and has transcriptional activity in yeast. The PavbHLH106 overexpression in tobacco resulted in weaker electrolyte leakages, lower malondialdehyde, and higher proline content than the wild type at low-temperature treatment. Reactive oxygen species accumulation was significantly reduced in the overexpressed lines, negatively correlated with the antioxidant enzyme activity. In addition, overexpression of PavbHLH106 delayed the germination of tobacco seeds and promoted plant growth. Resistance-related genes were expressed more in the overexpressed plants compared to the wild type. PavbHLH106 bound to the PavACO promoter in yeast and potentially interacted with a bHLH162-like transcription factor. These results indicate that PavbHLH106 has various functions and is particularly active in controlling low-temperature stress.

Automatic annotation of the bHLH gene family in plants

BackgroundThe bHLH transcription factor family is named after the basic helix-loop-helix (bHLH) domain that is a characteristic element of their members. Understanding the function and characteristics of this family is important for the examination of a wide range of functions. As the availability of genome sequences and transcriptome assemblies has increased significantly, the need for automated solutions that provide reliable functional annotations is emphasised.ResultsA phylogenetic approach was adapted for the automatic identification and functional annotation of the bHLH transcription factor family. The bHLH_annotator, designed for the automated functional annotation of bHLHs, was implemented in Python3. Sequences of bHLHs described in literature were collected to represent the full diversity of bHLH sequences. Previously described orthologs form the basis for the functional annotation assignment to candidates which are also screened for bHLH-specific motifs. The pipeline was successfully deployed on the two Arabidopsis thaliana accessions Col-0 and Nd-1, the monocot species Dioscorea dumetorum, and a transcriptome assembly of Croton tiglium. Depending on the applied search parameters for the initial candidates in the pipeline, species-specific candidates or members of the bHLH family which experienced domain loss can be identified.ConclusionsThe bHLH_annotator allows a detailed and systematic investigation of the bHLH family in land plant species and classifies candidates based on bHLH-specific characteristics, which distinguishes the pipeline from other established functional annotation tools. This provides the basis for the functional annotation of the bHLH family in land plants and the systematic examination of a wide range of functions regulated by this transcription factor family.

Cloning, phylogenetic and expression analysis of two MyoDs in yellowtail kingfish (Seriola lalandi)

Yellowtail kingfish (Seriola lalandi) is a pelagic piscivore distributed circumglobally. Owing to its great market value, the growth mechanism of S. lalandi, including muscle development and growth, is a hot research topic. The myoblast determination protein (MyoD) gene has been shown to play an important role in formation of myoblasts and the function of somites in fish. The open reading frame (ORF) sequences of MyoD1 and MyoD2 in S. lalandi encoded 298 and 263 amino acids possessing three common characteristic domains, respectively, containing a myogenic basic domain, a bHLH domain, and a ser-rich region (helix III). S. lalandi MyoDs shared the highest identity with the MyoDs of S. dumerili. MyoDs are highly expressed in white muscle (P < 0.05) in S. lalandi. The expression level of MyoD1 mRNA was higher than that of MyoD2 mRNA during embryonic and early developmental stages, indicating that the two MyoD isoforms may have different roles in muscle formation. Moreover, the mRNA expression of MyoDs in the brain, pituitary, liver and muscle of endocrine growth axis were analyzed in the various sizes and ages stages. The expression levels of MyoDs in the different sizes and ages of S. lalandi showed that expression of both these genes was particularly high in 400-g fish and 2-year-old fish (P < 0.05). Moreover, the increases in the mRNA expression and plasma levels of growth hormone (GH) and insulin-like growth factor (IGF-I) were accompanied by an increase in mRNA expression of MyoDs, indicating the roles of GH and IGF-I in muscle development and growth of S. lalandi. Overall, the expression profiles of genes associated with muscle development are the first step taken towards deciphering fast growth mechanism in this important Seriola fish.

Overexpression of GmPIF4b affects morpho-physiological traits to reduce heat-induced grain loss in soybean

Heat waves associated with climate change seriously threaten crop productivity. Crop seed yield depends on the success of reproduction. However, reproductive development is most vulnerable to heat stress conditions. Perception of heat and its conversion into cellular signals is a complex process. The basic helix loop helix (bHLH) transcription factor, Phytochrome Interacting Factor 4 (PIF4), plays a significant role in this process. However, studies on PIF4- mediated impacts on crop grain yield at a higher temperature are lacking. We investigated the overexpression of GmPIF4b in soybean to alleviate heat-induced damage and yield using a transgenic approach. Our results showed that under high-temperature conditions (38°C/28°C), overexpressing soybeans plants had higher chlorophyll a and b, and lower proline accumulation compared to WT. Further, overexpression of GmPIF4b improved pollen viability under heat stress and reduced heat-induced structural abnormalities in the male and female reproductive organs. Consequently, the transgenic plants produced higher pods and seeds per plant at high temperatures. Quantitative RT-PCR analysis showed that the overexpressing GmPIF4b soybeans had higher transcripts of heat shock factor, GmHSF-34, and heat-shock protein, GmHSP90A2. Collectively, our results suggest that GmPIF4b regulates multiple morpho-physiological traits for better yield under warmer climatic conditions.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

The Basic/Helix-Loop-Helix Transcription Factor Family Gene RcbHLH112 Is a Susceptibility Gene in Gray Mould Resistance of Rose (Rosa Chinensis).

The basic/helix-loop-helix (bHLH) family is a major family of transcription factors in plants. Although it has been reported that bHLH plays a defensive role against pathogen infection in plants, there is no comprehensive study on the bHLH-related defence response in rose (Rosa sp.). In this study, a genome-wide analysis of bHLH family genes (RcbHLHs) in rose was carried out, including their phylogenetic relationships, gene structure, chromosome localization and collinearity analysis. Via phylogenetic analysis, a total of 121 RcbHLH genes in the rose genome were divided into 21 sub-groups. These RcbHLHs are unevenly distributed in all 7 chromosomes of rose. The occurrence of gene duplication events indicates that whole-genome duplication and segmental duplication may play a key role in gene duplication. Ratios of non-synonymous to synonymous mutation frequency (Ka/Ks) analysis showed that the replicated RcbHLH genes mainly underwent purification selection, and their functional differentiation was limited. Gene expression analysis showed that 46 RcbHLHs were differentially expressed in rose petals upon B. cinerea infection. It is speculated that these RcbHLHs are candidate genes that regulate the response of rose plants to B. cinerea infection. Virus-induced gene silencing (VIGS) confirmed that RcbHLH112 in rose is a susceptibility factor for infection with B. cinerea. This study provides useful information for further study of the functions of the rose bHLH gene family.

Maize ZmBES1/BZR1-1 transcription factor negatively regulates drought tolerance

Drought stress is a common abiotic factor and restricts plant growth and development. Exploring maize stress-related genes and their regulatory mechanisms is crucial for ensuring agricultural productivity and food security. The BRI1-EMS1 suppressor (BES1)/brassinazole-resistant 1 (BZR1) transcription factors (TFs) play important roles in plant growth, development, and stress response. However, maize ZmBES1/BZR1s are rarely reported. In the present study, the ZmBES1/BZR1-1 gene was cloned from maize B73 and functionally characterized in transgenic Arabidopsis and rice in drought stress response. The ZmBES1/BZR1-1 protein possessed a conserved bHLH domain characterized by BES1/BZR1 TFs, localized in the nucleus, and showed transcription activation activity. The expression of ZmBES1/BZR1-1 exhibited no tissue specificity but drought-inhibitory expression in maize. Under drought stress, overexpression of ZmBES1/BZR1-1 resulted in the enhancement of drought sensitivity of transgenic Arabidopsis and rice with a lower survival rate, reactive oxygen species (ROS) level and relative water content (RWC) and a higher stomatal aperture and relative electrolyte leakage (REL). The RNA-seq results showed that 56 differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-1 by binding to E-box elements in their promoters. The GO analysis showed that the DEGs were significantly annotated with response to oxidative stress and oxygen level. The study suggests that the ZmBES1/BZR1-1 gene negatively regulates drought stress, which provides insights into further underlying molecular mechanisms in the drought stress response mediated by BZR1/BES1s.

A genome-wide CRISPR screen identifies USP1 as a novel regulator of the mammalian circadian clock.

The circadian clock is generated by a molecular timekeeping mechanism coordinating daily oscillations of physiology and behaviors in mammals. In the mammalian circadian clockwork, basic helix-loop-helix ARNT-like protein 1 (BMAL1) is a core circadian component whose defects lead tocircadian disruption and elicit behavioral arrhythmicity. To identify previously unknown regulators for circadian clocks, we searched for genes influencing BMAL1 protein level by using a CRISPR/Cas9-based genome-wide knockout library. As a result, we found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (USP1) positively affects BMAL1 protein abundance. Overexpression of wild-type USP1, but not a deubiquitinase-inactive mutant USP1, upregulated BMAL1 protein level, whereas genetic ablation of USP1 downregulated BMAL1 protein level in U2OS cells. Furthermore, treatment with USP1 inhibitors led to significant downregulation of BMAL1 protein in U2OS cells as well as mouse tissues. Subsequently, genetic ablation or pharmacological inhibition of USP1 resulted in reduced mRNA levels of a panel of clock genes and disrupted circadian rhythms in U2OS cells. Mechanistically, USP1 was able to de-ubiquitinate BMAL1 and inhibit the proteasomal degradation of BMAL1. Interestingly, the expression of Usp1 was much higher than the other two deubiquitinases of BMAL1 (Usp2 and Usp9X) in the mouse heart, implying a tissue-specific function of USP1 in the regulation of BMAL1 stability. Our work thus identifies deubiquitinase USP1 as a previously unknown regulator of the mammalian circadian clock and highlights the potential of genome-wide CRISPR screens in the identification of regulators for the circadian clock.

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription.

A homologous gene of basic-helix-loop-helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv 'Tarocco'), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in 'Tarocco' than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15-TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in 'Tarocco'. The mRNA levels of Δ15-TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15-TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.

An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

An Encodable Scaffold for Sequence‐Specific Recognition of Duplex RNA

AbstractRNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence‐specifically target this biomolecule. We sought to develop ligands for double‐stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double‐stranded DNA is sequence‐specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix‐loop‐helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA‐binding proteins. We interrogated the high‐resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA‐binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence‐specific recognition of dsRNA.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

The MabHLH11 transcription factor interacting with MaMYB4 acts additively in increasing plant scopolin biosynthesis

The plant natural product scopolin, a coumarin secondary metabolite, has been extensively exploited in flavor, cosmetic, medicine, and other industrial fields. Melilotus albus, a leguminous rotation crop, contains high concentrations of coumarin. The transcriptional regulatory network that controls the flow through the scopolin biosynthesis pipeline in M. albus remains poorly understood. MabHLH11 encodes a basic helix–loop–helix (bHLH) transcription factor whose transcription is positively associated with scopolin accumulation and with the expression of MaMYB4, the bHLH partner of the MYB-bHLH complex. Phylogenetic analysis grouped MabHLH11 in the TRANSPARENT TESTA 8 (TT8) clade of the bHLH IIIf subgroup. The MabHLH11 protein contained an MYB-interacting region and physically interacted with MaMYB4 in yeast and tobacco leaves. Co-overexpression of MabHLH11 with MaMYB4 in M. albus additively increased the expression of UDP-glucosyltransferase (MaUGT79) and induced more scopolin accumulation than occurred under the expression of MabHLH11 alone. MabHLH11 directly targeted the promoter of MaUGT79 and the activation of MabHLH11 was strengthened by the presence of MaMYB4. Thus, MaMYB4 enhanced the function of MabHLH11 in upregulating scopolin biosynthesis in M. albus, providing a theoretical basis for scalable production of a high-value plant natural product.

Identification and characterization of the Cyamopsis tetragonoloba transcription factor MYC (CtMYC) under drought stress

The MYC transcription factor (TF) has a variety of roles in abiotic stress responses of plants. In the present work, MYC TF named CtMYC (Cymopsis tetragonoloba) from guar plant, which is induced by drought stress, was identified. The mature leaves of guar were employed to detect the full-length CtMYC TF on the 8th day of drought stress. The CtMYC gene showed tissue-specific expression and up regulated under drought stress conditions as compared to the control and maximum expression was observed in mature leaves. Additionally, CtMYC TF was cloned and expressed in E. coli Rosetta cells and CtMYC protein was purified. The circular dichroism (CD) analysis revealed the presence of helical content and beta sheets and in the presence of genomic DNA the conformational changes were observed in secondary structure, which showed DNA binding potential of CtMYC. These results were analyzed by CD and fluorescence studies. In silico studies reveal the presence of conserved bHLH domain and DNA-binding amino acid residues His, Glu and Arg in CtMYC. This is first report on CtMYC TF with DNA binding potential that is responsive to drought. This study provides the structure and characterization of CtMYC TF and DNA binding ability in drought tolerance mechanism in guar.

Cryo‐EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram‐positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton‐coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo‐EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate‐free QacA, complexed to two single‐domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward‐open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.