Per-ARNT-Sim (PAS) Domains in Basic Helix-Loop-Helix (bHLH)-PAS Transcription Factors and Coactivators: Structures and Mechanisms.

PAS domains are ubiquitous in biology. They perform critically important roles in sensing and transducing a wide variety of environmental signals, and through their ability to bind small-molecule ligands, have emerged as targets for therapeutic intervention. Here, we discuss our current understanding of PAS domain structure and function in the context of basic helix-loop-helix (bHLH)-PAS transcription factors and coactivators. Unlike the bHLH-PAS domains of transcription factors, those of the steroid receptor coactivator (SRC) family are poorly characterized. Recent progress for this family and for the broader bHLH-PAS proteins suggest that these domains are ripe for deeper structural and functional studies.

Decoding Allosteric Control in Hypoxia-Inducible Factors.

The mammalian family of basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors possess the ability to sense and respond to diverse environmental and physiological cues. These proteins all share a common structural framework, comprising a bHLH domain, two PAS domains, and transcriptional activation or repression domain. To function effectively as transcription factors, members of the family must form dimers, bringing together bHLH segments to create a functional unit that allows for DNA response element binding. The significance of bHLH-PAS family is underscored by their involvement in many major human diseases, offering potential avenues for therapeutic intervention. Notably, the clear identification of ligand-binding cavities within their PAS domains enables the development of targeted small molecules. Two examples are Belzutifan, targeting hypoxia-inducible factor (HIF)-2α, and Tapinarof, targeting the aryl hydrocarbon receptor (AHR), both of which have gained regulatory approval recently. Here, we focus on the HIF subfamily. The crystal structures of all three HIF-α proteins have been elucidated, revealing their bHLH and tandem PAS domains are used to engage their dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1ß). A broad range of recent findings point to a shared allosteric modulation mechanism among these proteins, whereby small-molecules at the PAS-B domains exert direct influence over the HIF-α transcriptional functions. As our understanding of the architectural and allosteric mechanisms of bHLH-PAS proteins continues to advance, the possibility of discovering new therapeutic drugs becomes increasingly promising.

Automatic annotation of the bHLH gene family in plants.

BACKGROUND: The 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. RESULTS: A 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. CONCLUSIONS: The 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.

Physics-based approach to extend a de novo TIM barrel with rationally designed helix-loop-helix motifs.

Computational protein design promises the ability to build tailor-made proteins de novo. While a range of de novo proteins have been constructed so far, the majority of these designs have idealized topologies that lack larger cavities which are necessary for the incorporation of small molecule binding sites or enzymatic functions. One attractive target for enzyme design is the TIM-barrel fold, due to its ubiquity in nature and capability to host versatile functions. With the successful de novo design of a 4-fold symmetric TIM barrel, sTIM11, an idealized, minimalistic scaffold was created. In this work, we attempted to extend this de novo TIM barrel by incorporating a helix-loop-helix motif into its ßα-loops by applying a physics-based modular design approach using Rosetta. Further diversification was performed by exploiting the symmetry of the scaffold to integrate two helix-loop-helix motifs into the scaffold. Analysis with AlphaFold2 and biochemical characterization demonstrate the formation of additional α-helical secondary structure elements supporting the successful extension as intended.

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.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. 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 patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 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.

Evolution and diversification of the ACT-like domain associated with plant basic helix-loop-helix transcription factors.

Basic helix-loop-helix (bHLH) proteins are one of the largest families of transcription factor (TF) in eukaryotes, and ~30% of all flowering plants' bHLH TFs contain the aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain at variable distances C-terminal from the bHLH. However, the evolutionary history and functional consequences of the bHLH/ACT-like domain association remain unknown. Here, we show that this domain association is unique to the plantae kingdom with green algae (chlorophytes) harboring a small number of bHLH genes with variable frequency of ACT-like domain's presence. bHLH-associated ACT-like domains form a monophyletic group, indicating a common origin. Indeed, phylogenetic analysis results suggest that the association of ACT-like and bHLH domains occurred early in Plantae by recruitment of an ACT-like domain in a common ancestor with widely distributed ACT DOMAIN REPEAT (ACR) genes by an ancestral bHLH gene. We determined the functional significance of this association by showing that Chlamydomonas reinhardtii ACT-like domains mediate homodimer formation and negatively affect DNA binding of the associated bHLH domains. We show that, while ACT-like domains have experienced faster selection than the associated bHLH domain, their rates of evolution are strongly and positively correlated, suggesting that the evolution of the ACT-like domains was constrained by the bHLH domains. This study proposes an evolutionary trajectory for the association of ACT-like and bHLH domains with the experimental characterization of the functional consequence in the regulation of plant-specific processes, highlighting the impacts of functional domain coevolution.

High Intrinsic Oncogenic Potential in the Myc-Box-Deficient Hydra Myc3 Protein.

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver.

Intracellular Helix-Loop-Helix Domain Modulates Inactivation Kinetics of Mammalian TRPV5 and TRPV6 Channels.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca2+) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca2+ exerts a negative control over the activity of these channels by promoting inactivation. TRPV5 and TRPV6 inactivation has been divided into fast and slow phases based on their kinetics. While slow inactivation is common to both channels, fast inactivation is characteristic of TRPV6. It has been proposed that the fast phase depends on Ca2+ binding and that the slow phase depends on the binding of the Ca2+/Calmodulin complex to the internal gate of the channels. Here, by means of structural analyses, site-directed mutagenesis, electrophysiology, and molecular dynamic simulations, we identified a specific set of amino acids and interactions that determine the inactivation kinetics of mammalian TRPV5 and TRPV6 channels. We propose that the association between the intracellular helix-loop-helix (HLH) domain and the TRP domain helix (TDh) favors the faster inactivation kinetics observed in mammalian TRPV6 channels.

Genome-wide identification and characterisation of bHLH transcription factors in Artemisia annua.

BACKGROUND: A. annua (also named Artemisia annua, sweet wormwood) is the main source of the anti-malarial drug artemisinin, which is synthesised and stored in its trichomes. Members of the basic Helix-Loop-Helix (bHLH) family of transcription factors (TFs) have been implicated in artemisinin biosynthesis in A. annua and in trichome development in other plant species. RESULTS: Here, we have systematically identified and characterised 226 putative bHLH TFs in A. annua. All of the proteins contain a HLH domain, 213 of which also contain the basic motif that mediates DNA binding of HLH dimers. Of these, 22 also contained a Myc domain that permits dimerisation with other families of TFs; only two proteins lacking the basic motif contained a Myc domain. Highly conserved GO annotations reflected the transcriptional regulatory role of the identified TFs, and suggested conserved roles in biological processes such as iron homeostasis, and guard cell and endosperm development. Expression analysis revealed that three genes (AabHLH80, AabHLH96, and AaMyc-bHLH3) exhibited spatiotemporal expression patterns similar to genes encoding key enzymes in artemisinin synthesis. CONCLUSIONS: This comprehensive analysis of bHLH TFs provides a new resource to direct further analysis into key molecular mechanisms underlying and regulating artemisinin biosynthesis and trichome development, as well as other biological processes, in the key medicinal plant A. annua.

DNA-TCP complex structures reveal a unique recognition mechanism for TCP transcription factor families.

Plant-specific TCP transcription factors are key regulators of diverse plant functions. TCP transcription factors have long been annotated as basic helix-loop-helix (bHLH) transcription factors according to remote sequence homology without experimental validation, and their consensus DNA-binding sequences and protein-DNA recognition mechanisms have remained elusive. Here, we report the crystal structures of the class I TCP domain from AtTCP15 and the class II TCP domain from AtTCP10 in complex with different double-stranded DNA (dsDNA). The complex structures reveal that the TCP domain is a distinct DNA-binding motif and the homodimeric TCP domains adopt a unique three-site recognition mode, binding to dsDNA mainly through a central pair of ß-strands formed by the dimer interface and two basic flexible loops from each monomer. The consensus DNA-binding sequence for class I TCPs is a perfectly palindromic 11 bp (GTGGGNCCCAC), whereas that for class II TCPs is a near-palindromic 11 bp (GTGGTCCCCAC). The unique DNA binding mode allows the TCP domains to display broad specificity for a range of DNA sequences even shorter than 11 bp, adding further complexity to the regulatory network of plant TCP transcription factors.

Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains.

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.

Structural insight into the ligand binding mechanism of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors, plays important roles in regulating xenobiotic metabolism, cellular differentiation, stem cell maintenance, as well as immunity. More recently, AHR has gained significant interest as a drug target for the development of novel cancer immunotherapy drugs. Detailed understanding of AHR-ligand binding has been hampered for decades by the lack of a three-dimensional structure of the AHR PAS-B domain. Here, we present multiple crystal structures of the Drosophila AHR PAS-B domain, including its apo, ligand-bound, and AHR nuclear translocator (ARNT) PAS-B-bound forms. Together with biochemical and cellular assays, our data reveal structural features of the AHR PAS-B domain, provide insights into the mechanism of AHR ligand binding, and provide the structural basis for the future development of AHR-targeted therapeutics.

Structural basis of the bHLH domains of MyoD-E47 heterodimer.

The basic helix-loop-helix (bHLH) family is one of the most conserved transcription factor families that plays an important role in regulating cell growth, differentiation and tissue development. Typically, members of this family form homo- or heterodimers to recognize specific motifs and activate transcription. MyoD is a vital transcription factor that regulates muscle cell differentiation. However, it is necessary for MyoD to form a heterodimer with E-proteins to activate transcription. Even though the crystal structure of the MyoD homodimer has been determined, the structure of the MyoD heterodimer in complex with the E-box protein remains unclear. In this study, we determined the crystal structure of the bHLH domain of the MyoD-E47 heterodimer at 2.05 Å. Our structural analysis revealed that MyoD interacts with E47 through a hydrophobic interface. Moreover, we confirmed that heterodimerization could enhance the binding affinity of MyoD to E-box sequences. Our results provide new structural insights into the heterodimer of MyoD and E-box protein, suggesting the molecular mechanism of transcription activation of MyoD upon binding to E-box protein.

Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

SbbHLH85, a bHLH member, modulates resilience to salt stress by regulating root hair growth in sorghum.

bHLH family proteins play an important role in plant stress response. However, the molecular mechanism regulating the salt response of bHLH is largely unknown. This study aimed to investigate the function and regulating mechanism of the sweet sorghum SbbHLH85 during salt stress. The results showed that SbbHLH85 was different from its homologs in other species. Also, it was a new atypical bHLH transcription factor and a key gene for root development in sweet sorghum. The overexpression of SbbHLH85 resulted in significantly increased number and length of root hairs via ABA and auxin signaling pathways, increasing the absorption of Na+. Thus, SbbHLH85 plays a negative regulatory role in the salt tolerance of sorghum. We identified a potential interaction partner of SbbHLH85, which was phosphate transporter chaperone PHF1 and modulated the distribution of phosphate, through screening a yeast two-hybrid library. Both yeast two-hybrid and BiFC experiments confirmed the interaction between SbbHLH85 and PHF1. The overexpression of SbbHLH85 led to a decrease in the expression of PHF1 as well as the content of Pi. Based on these results, we suggested that the increase in the Na+ content and the decrease in the Pi content resulted in the salt sensitivity of transgenic sorghum.

RGS2 Suppresses Melanoma Growth via Inhibiting MAPK and AKT Signaling Pathways.

BACKGROUND/AIM: This study aimed to explore RGS2 as a regulator of melanoma cell growth. MATERIALS AND METHODS: Effect of RGS2 over-expression was analyzed in three melanoma cell lines, and Rgs2 knockdown was performed in zebrafish. RESULTS: RGS2 was differentially expressed among the cell lines. In B16F10 cells, RGS2 over-expression inhibited MAPK and AKT activation, and prevented cell growth. A similar outcome was observed in A375 cells, but the MAPK signals were not suppressed. In A2058 cells, RGS2 repressed AKT activation, but without affecting cell growth. Moreover, MAPK and AKT constitutive activation abolished the RGS2 inhibitory effect on B16F10 cell growth. Rgs2 knockdown caused ectopic melanocyte differentiation, and promoted MAPK and AKT activation in zebrafish embryos. CONCLUSION: RGS2 prevents melanoma cell growth by inhibiting MAPK and AKT, but this effect depends on the overall cell genetic landscape. Further studies are warranted to investigate the anticancer therapeutic potential of RGS2 for melanoma.

Functional consequences of TCF4 missense substitutions associated with Pitt-Hopkins syndrome, mild intellectual disability, and schizophrenia.

Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor essential for neurocognitive development. The aberrations in TCF4 are associated with neurodevelopmental disorders including schizophrenia, intellectual disability, and Pitt-Hopkins syndrome, an autism-spectrum disorder characterized by developmental delay. Several disease-associated missense mutations in TCF4 have been shown to interfere with TCF4 function, but for many mutations, the impact remains undefined. Here, we tested the effects of 12 functionally uncharacterized disease-associated missense mutations and variations in TCF4 using transient expression in mammalian cells, confocal imaging, in vitro DNA-binding assays, and reporter assays. We show that Pitt-Hopkins syndrome-associated missense mutations within the basic helix-loop-helix domain of TCF4 and a Rett-like syndrome-associated mutation in a transcription activation domain result in altered DNA-binding and transcriptional activity of the protein. Some of the missense variations found in schizophrenia patients slightly increase TCF4 transcriptional activity, whereas no effects were detected for missense mutations linked to mild intellectual disability. We in addition find that the outcomes of several disease-related mutations are affected by cell type, TCF4 isoform, and dimerization partner, suggesting that the effects of TCF4 mutations are context-dependent. Together with previous work, this study provides a basis for the interpretation of the functional consequences of TCF4 missense variants.

Mnt Represses Epithelial Identity To Promote Epithelial-to-Mesenchymal Transition.

The multistep process of epithelial-to-mesenchymal transition (EMT), whereby static epithelial cells become migratory mesenchymal cells, plays a critical role during various developmental contexts, wound healing, and pathological conditions such as cancer metastasis. Despite the established function of basic helix-loop-helix (bHLH) transcription factors (TFs) in cell fate determination, only a few have been examined for their role in EMT. Here, using transcriptome analysis of distinct stages during stepwise progression of transforming growth factor beta (TGFß)-induced EMT in mammary epithelial cells, we revealed distinct categories of bHLH TFs that show differential expression kinetics during EMT. Using a short interfering RNA-mediated functional screen for bHLH TFs during EMT, we found Max network transcription repressor (MNT) to be essential for EMT in mammary epithelial cells. We show that the depletion of MNT blocks TGFß-induced morphological changes during EMT, and this is accompanied by derepression of a large number of epithelial genes. We show that MNT mediates the repression of epithelial identity genes during EMT by recruiting HDAC1 and mediating the loss of H3K27ac and chromatin accessibility. Lastly, we show that MNT is expressed at higher levels in EMT-High breast cancer cells and is required for their migration. Taken together, these findings establish MNT as a critical regulator of cell fate changes during mammary EMT. IMPORTANCE The bHLH TF Mnt promotes epithelial to mesenchymal transition through epigenetic repression of the epithelial gene expression program.

Structural basis for the dimerization mechanism of human transcription factor E3.

The transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) is a member of the microphthalmia (MiT/TFE) transcription factor family. Dysregulation of TFE3 due to chromosomal abnormalities is associated with a subset of human renal cell carcinoma. Little structural information of this key transcription factor has been reported. In this study, we determined the crystal structure of the helix-loop-helix leucine zipper (HLH-Lz) domain of human TFE3 to a resolution of 2.6 Å. The HLH-Lz domain is critical for the dimerization and function of TFE3. Our structure showed that the conserved HLH region formed a four-helix bundle structure with a predominantly hydrophobic core, and the leucine zipper region contributed to the function of TFE3 by promoting dimer interaction and providing partner selectivity. Together, our results elucidated the dimerization mechanism of this important transcription factor, providing the structural basis for the development of inhibiting strategies for treating TFE3 dysregulated diseases.