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Stomata play a critical role in plant physiology by balancing gas exchange and water conservation. Their development is driven by a precisely orchestrated sequence of cell divisions and differentiation events, regulated by basic helix-loop-helix (bHLH) transcription factors such as MUTE. Previous research reports stomidazolone, a doubly sulfonylated imidazolone derivative, as an effective inhibitor of stomatal development which has been shown to bind strongly to MUTE, interfering with its interaction with SCRM, effectively suppressing stomatal differentiation. The ACTL domain, a conserved structural feature in plant bHLH proteins, acts as a potential site for chemical inhibition, enabling selective disruption of stomatal formation. This suggests a promising approach for enhancing drought resilience in plants by reducing water loss through transpiration. While experimental data support stomidazolone’s inhibitory role, the molecular details of its binding to MUTE remain inadequately characterized. To address this gap, a comprehensive in silico analysis combining molecular docking and density functional theory (DFT) was performed to elucidate the binding interactions, electronic properties, and reactive potential of stomidazolone, thereby uncovering the molecular features that underpin its affinity and specificity toward MUTE. An all-atom molecular dynamics (MD) simulations was then carried out to provide mechanistic insights beyond static binding models, followed by a number of post-simulation analyses assessing system stability and dynamics to gain deeper insight into the Stomidazolone-Mediated Inhibition of MUTE. Our results reveal the formation of a stable and compact stomidazolone–MUTE complex, characterized by a lower average RMSD (1.45 ± 0.12 nm) compared to the Apo state (1.65 ± 0.18 nm), while hydrogen bonding analysis further demonstrated persistent interactions involving Arg62 and Ser69, along with a stabilizing contribution from Glu163, collectively supporting the strong binding affinity of stomidazolone within the MUTE active site. Principal component analysis further highlighted the conformational coherence and coordinated atomic motion, while the free energy landscape showed well-defined energy minima, underscoring the stability of the interaction and energetic favorability of the complex. Together, these findings provide a molecular framework for understanding the inhibitory mechanism of stomidazolone on MUTE, offering a basis for the rational design of next-generation agrochemicals targeting stomatal development. The study also highlights the conceptual novelty of small-molecule modulation of lineage-specific transcription factors as a potential strategy for the synthetic control of plant developmental plasticity. While the results are computational, they outline clear directions for experimental validation and scaffold optimization, paving the way for future efforts to translate these insights into practical applications for improving crop resilience and water-use efficiency. Importantly, this study provides the first mechanistic, residue-level insight into how stomidazolone engages the ACT-Like (ACTL) domain of MUTE, revealing the specific molecular interactions and dynamic features that underpin its inhibitory effect.

In vivo astrocyte-to-neuron (AtN) conversion suffers low efficiency due to pre-existing intrinsic barriers. However, it is unclear whether astrocytes have inducible barriers to reprogramming. Here, we identify Olig2, a basic helix-loop-helix (bHLH) transcription factor, as an inducible barrier to Ngn2-mediated AtN conversion. Olig2 is strongly upregulated in cortical astrocytes following the ectopic expression of bHLH neuronal reprogramming factors such as Ngn2, NeuroD1, and Ascl1, but is barely expressed in normal astrocytes. Knocking down Olig2 in Ngn2-transduced astrocytes reduces astrocyte-specific gene expression, enhances neurogenesis-related gene expression, and increases AtN conversion efficiency by approximately threefold. Further multi-omics analysis shows that astrocytic Olig2 directly binds to regulatory regions of pro-neurogenic genes, including Ngn2, inhibiting their expression and impeding the expression of neural progenitor genes. Collectively, our findings reveal Olig2 as an inducible barrier to AtN conversion, providing insights into the regulation of neuronal reprogramming. Astrocyte-to-neuron (AtN) conversion is limited by intrinsic barriers, hindering its efficiency. Here, the authors show that Olig2 acts as an inducible barrier to in vivo Ngn2-mediated AtN conversion, reducing reprogramming efficiency.

Background Basic helix-loop-helix family, member e22 (BHLHE22), a basic helix loop helix transcription factor family member, functions vary in different types of cancer. Currently, its function in triple-negative breast cancer (TNBC) is unclear. Methods The GSE45827 and GSE113865 datasets were used to screen potential TNBC marker. Biological websites were used to analyze BHLHE22 expression in TNBC, its relationship with the prognosis of patients with TNBC, and its potential function. A series of in vitro and in vivo experiments was performed to investigate the function of BHLHE22 in TNBC. The regulatory relationship between OTU domain-containing protein 3 (OTUD3) and BHLHE22 was verified by Co-Immunoprecipitation, ubiquitination assay, and site-specific mutation experiments. The mRNA-seq analysis was performed to identify potential genes for the anti-cancer role of BHLHE22. The transcriptional regulation of DNA replication factor 1 (CDT1) by BHLHE22 was confirmed by dual luciferase reporter assay. Results We identified the downregulation of BHLHE22 in TNBC tissues based on the GSE45827 and GSE113865 datasets. The expression of BHLHE22 in TBNC patients was lower than that in non-TNBC patients, and patients at stages 3 and 4 tended to express lower BHLHE22 expression than patients at stages 1 and 2. Patients with high expression of BHLHE22 had better survival prognosis than those with BHLHE22 low expression. Functional studies revealed that BHLHE22 overexpression impaired cell growth in vitro and in vivo . However, BHLHE22 silencing enhanced the malignant behaviors of cancer cells. OTUD3, a deubiquitinase known to suppress TNBC progression, was found to increase the stability of BHLHE22 protein through deubiquitination regulation. The C76 site mutation of OTUD3 eliminated the catalytic activity of OTUD3 and failed to regulate the protein stability of BHLHE22. Furthermore, BHLHE22 medicated the antitumor effect of OTUD3 in TNBC. The mRNA-seq analysis identified potential genes for the anti-cancer role of BHLHE22, involving CDT1. BHLHE22 was proved to reduce CDT1 RNA expression by blocking CDT1 transcription. The anti-proliferative effect of BHLHE22 overexpression was reversed by CDT1 overexpression. Conclusions Our observations demonstrate that BHLHE22 may be a promising target for TNBC therapy. Graphical abstract

Background NeuroD1 and NeuroD2, members of the bHLH transcription factors family, are key regulators of nervous system development and function. While they share similar roles in neuronal regulation, their divergent expression patterns and specialized functions suggest the complexity of transcriptional control they perform. The activity of bHLH TFs depends on tightly regulated intracellular trafficking orchestrated by NLSs and/or NESs located within the protein’s sequences. A detailed characterization of these molecular motifs is essential to understand the mechanisms regulating NeuroD1 and NeuroD2 functions. Methods We prepared cDNA vector that enabled expression of the full-length and truncated variants of NeuroD1 and NeuroD2 fused to YFP in COS-7 and N2a cells. Confocal microscopy was used to assess intracellular localization and to identify the location of motifs presenting NLS, NES, and NoLS activity. Results We demonstrated that previously documented NLS (NLS1), conserved both in NeuroD1 and NeuroD2 also presents NoLS (NoLS1) activity, revealing unexpected dual functionality. Additionally, we identified overlapping NLS2 and NES1 within the bHLH domains of both proteins. Notably, NeuroD2 harbours distinct NLS3 and a second NoLS (NoLS2) motifs, located in the C-terminal region. These elements, suggest differentiated regulation and specialization between the homologs. Conclusions Our study reveals a surprisingly complex network of overlapping localization signals in NeuroD1 and NeuroD2 that regulate their cyto-nuclear trafficking. The presence of multiple, potentially competing signals suggests that their activity may be fine-tuned by specific ligands or interacting partners, adding further complexity to their regulation. These findings provide novel insight into how subcellular localization contributes to the functional divergence of homologous transcription factors.

Background Transcription factor 21 (TCF21) encodes a basic-helix-loop-helix transcription factor that is thought to play a vital role in epicardial progenitor cell development, with these cells differentiating into coronary artery smooth muscle cells and cardiac fibroblasts. Reduced TCF21 expression has been linked to coronary artery disease severity; however, its relationship with coronary artery calcium (CAC) and genetic determinants of circulating TCF21 levels has not been examined. This study investigated the associations between plasma TCF21 levels, CAC burden, and TCF21 gene polymorphisms in patients with chronic schizophrenia, a population at increased cardiometabolic risk. Methods A total of 185 consecutive patients with chronic schizophrenia were enrolled. Plasma TCF21 concentrations were measured using enzyme-linked immunosorbent assay. CAC was quantified using cardiac multislice computed tomography, with independent assessment by two blinded radiologists. The rs12190287 (G/C) polymorphism of the TCF21 gene was genotyped. Simple linear regression analyses were performed to explore associations between TCF21 levels and clinical variables, followed by multiple linear regression adjusted for age and sex. Multiple testing was addressed using false discovery rate (FDR) correction as a sensitivity analysis. Multivariable logistic regression analyses were conducted to assess the independent association between TCF21 levels and severe CAC after adjustment for established cardiovascular risk factors. Results After adjustment for age and sex, plasma TCF21 levels were positively associated with triglyceride levels and circulating inflammatory cell counts, including white blood cell, neutrophil, and monocyte counts, and inversely associated with both calcium score-Agatston and calcium score-volume. Following FDR correction, the associations with CAC measures, triglycerides, and inflammatory cell counts remained statistically significant. Lower plasma TCF21 levels were independently associated with severe CAC defined by both Agatston and calcium volume thresholds after adjustment for established cardiovascular risk factors. In genotype-based analyses, patients carrying the CC genotype exhibited significantly lower plasma TCF21 levels and higher CAC burden compared with GG and GC genotypes, and these differences remained significant after FDR correction. Conclusions Lower plasma TCF21 levels are robustly associated with greater CAC burden in patients with chronic schizophrenia, independent of major cardiovascular risk factors. Genetic variation in TCF21 is associated with reduced circulating TCF21 levels and higher coronary calcification. These findings suggest that TCF21 may represent a potential biomarker related to cardiovascular risk in this vulnerable population. Clinical trial number Not applicable.

In plants, an elevation in ambient temperature induces adaptive morphological changes including elongated hypocotyls, which is predominantly regulated by a bHLH transcription factor, PIF4. Although PIF4 is expressed in all aerial tissues including the epidermis, mesophyll, and vascular bundle, its tissue-specific functions in thermomorphogenesis are not known. Here, we show that epidermis-specific expression of PIF4 induces constitutive long hypocotyls, while vasculature-specific expression of PIF4 has no effect on hypocotyl growth. RNA-Seq and qRT-PCR analyses reveal that auxin-responsive genes and growth-related genes are highly activated by epidermal, but not by vascular, PIF4. Additionally, inactivation of epidermal PIF4 or auxin signaling, and overexpression of epidermal phyB suppresses thermoresponsive growth, indicating that epidermal PIF4-auxin pathways are essential for the temperature responses. Further, we show that high temperatures increase both epidermal PIF4 transcription and the epidermal PIF4 DNA-binding ability. Taken together, our study demonstrates that the epidermis regulates thermoresponsive growth through the phyB-PIF4-auxin pathway. The PIF4 transcription factor along with the phyB photoreceptor, regulates growth responses to elevated temperature in plants. Here the authors show that PIF4 expression in the epidermis, rather than the vasculature, stimulates auxin responses and thermoresponsive growth in Arabidopsis .

Transcription factor EB (TFEB), a member of the MiT/TFE family of basic helix-loop-helix leucine zipper transcription factors, is an established central regulator of the autophagy/lysosomal-to-nucleus signaling pathway. Originally described as an oncogene, TFEB is now widely known as a regulator of various processes, such as energy homeostasis, stress response, metabolism, and autophagy-lysosomal biogenesis because of its extensive involvement in various signaling pathways, such as mTORC1, Wnt, calcium, and AKT signaling pathways. TFEB is also implicated in various human diseases, such as lysosomal storage disorders, neurodegenerative diseases, cancers, and metabolic disorders. In this review, we present an overview of the major advances in TFEB research over the past 30 years, since its description in 1990. This review also discusses the recently discovered regulatory mechanisms of TFEB and their implications for human diseases. We also summarize the moonlighting functions of TFEB and discuss future research directions and unanswered questions in the field. Overall, this review provides insight into our understanding of TFEB as a major molecular player in human health, which will take us one step closer to promoting TFEB from basic research into clinical and regenerative applications.

Iron is an essential nutrient for plants, but excess iron is toxic due to its catalytic role in the formation of hydroxyl radicals. Thus, iron uptake is highly regulated and induced only under iron deficiency. The mechanisms of iron uptake in roots are well characterized, but less is known about how plants perceive iron deficiency. We show that a basic helix–loop–helix (bHLH) transcription factor Upstream Regulator of IRT1 (URI) acts as an essential part of the iron deficiency signaling pathway in Arabidopsis thaliana. The uri mutant is defective in inducing Iron-Regulated Transporter1 (IRT1) and Ferric Reduction Oxidase2 (FRO2) and their transcriptional regulators FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 in response to iron deficiency. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals direct binding of URI to promoters of many iron-regulated genes, including bHLH38/39/100/101 but not FIT. While URI transcript and protein are expressed regardless of iron status, a phosphorylated form of URI only accumulates under iron deficiency. Phosphorylated URI is subject to proteasome-dependent degradation during iron resupply, and turnover of phosphorylated URI is dependent on the E3 ligase BTS. The subgroup IVc bHLH transcription factors, which have previously been shown to regulate bHLH38/39/100/101, coimmunoprecipitate with URI mainly under Fe-deficient conditions, suggesting that it is the phosphorylated form of URI that is capable of forming heterodimers in vivo. We propose that the phosphorylated form of URI accumulates under Fe deficiency, forms heterodimers with subgroup IVc proteins, and induces transcription of bHLH38/39/100/101. These transcription factors in turn heterodimerize with FIT and drive the transcription of IRT1 and FRO2 to increase Fe uptake.

The basic helix–loop–helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members 1 . Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2 , 3 ). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry–exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A–H2B acidic patch 4 , the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes 5 – 7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors. Cryo-EM structures and analysis provide insight into the mechanisms by which basic helix–loop–helix transcription factors access E-box DNA sequences that are embedded within nucleosomes, and cooperate with other transcription factors.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F₁ population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase–encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait.