Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “bHLH domain”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1992

The centromere and promoter factor 1 of yeast contains a dimerisation domain located carboxy-terminal to the bHLH domain.

CPF1 is a basic helix-loop-helix (bHLH) protein required for optimal centromere function and for maintaining methionine independent growth in yeast. In this work, we show that the region carboxy-terminal to the bHLH domain of CPF1 is essential for CPF1 function in the cell and for dimerisation of CPF1 in solution. The C-terminus of CPF1 contains a potential long amphipathic helix with a hydrophobic face which could provide a suitable protein:protein interface. Point mutations in residues forming this hydrophobic face are sufficient to weaken the interaction between the protein and DNA. By fusing the DNA binding domain or the transcriptional activation domain of GAL4 to the C-terminal 87 amino acids of CPF1, we show that this region is sufficient for mediating protein:protein interactions in vivo. The C-terminal domain of CPF1 can be replaced by the leucine repeat region of the bHLH-ZIP protein USF and the hybrid CPF1-USF protein functions in vivo to provide normal centromere function and methionine independent growth. However, the CPF1-USF hybrid protein is unable to interact with CPF1 suggesting that a dimer of CPF1 is sufficient for maintaining methionine independent growth and normal centromere function.

Amino Acid Sequence↗
PubMed1996

Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila.

The Enhancer of split and achaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis in Drosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs. Also, the pattern of interactions of the bHLH domains of certain proteins encoded by the two gene complexes may explain their functional redundancy.

Animals↗
PubMed1999

Characterization of the DNA binding properties of the bHLH domain of Deadpan to single and tandem sites.

The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCl) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (Kd = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCl and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.

Amino Acid Sequence↗
PubMed1995

Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

Amino Acid Sequence↗
PubMed2004

Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners.

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Amino Acid Sequence↗
PubMed1996

Monoclonal antimyogenin antibodies define epitopes outside the bHLH domain where binding interferes with protein-protein and protein-DNA interactions.

We have developed a panel of monoclonal antibodies against rat myogenin, a skeletal muscle regulatory protein of the bHLH family. Some of these monoclonals have been widely used by others, and details of their production are presented. Mapping the epitopes by immunoprecipitation of myogenin deletion mutants demonstrates that this panel recognizes epitopes spanning the entire molecule outside the HLH region. Four antibodies against epitopes outside the bHLH region interfere with the binding of myogenin/E-protein heterodimers to DNA sequences containing the myogenin heterodimer consensus recognition site. Three of these epitopes are partially masked in the heterodimers; antibody binding to these epitopes reduces the interactions between myogenin and E12. This suggests that surfaces outside the HLH dimerization domain may contribute to the stability of myogenin/E12 complexes. The binding of one antibody to its epitope did not appear to affect the myogenin/E12 interaction but nonetheless interfered with the binding of the complex to DNA, suggesting that this epitope lies near to a surface occupied by DNA.

Animals↗
PubMed1997

USF2/FIP associates with the b-Zip transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf DNA binding activity.

In screening for proteins that interact with the basic zipper (bZip) transcription factor, c-Maf, we isolated USF2/FIP. USF2 is a member of the bHLH-Zip protein family, possessing a basic (b) DNA binding region, a helix-loop-helix (HLH) motif, and a leucine zipper (Zip) structure. Mutants of USF2 that lacked a Zip formed heterodimers with c-Maf, but did not homodimerize. Deletion of the USF2 basic region or mutation of its helices abrogated its binding to c-Maf, but had no effect on homodimerization. A functional c-Maf bZip motif was necessary for both homodimerization and heterodimerization with USF2. These data suggest a tetrameric configuration for Maf-USF2 complexes. In the presence of USF2, the DNA binding activity of c-Maf was markedly reduced. Therefore, USF2 and c-Maf may interact to regulate gene expression.

Basic-Leucine Zipper Transcription Factors↗
PubMed1998

A cysteine residue in helixII of the bHLH domain is essential for homodimerization of the yeast transcription factor Pho4p.

The yeast transcription factor Pho4p is required for expression of the phosphate-repressible acid phosphatase encoded by the PHO5 gene. Functional studies have shown that the molecule is composed of an N-terminal acidic activation domain, a central region which is necessary for interaction with a negative regulatory factor (the cyclin Pho80) and a C-terminal basic helix-loop-helix domain, which mediates DNA binding and homodimerization. In this study the homodimerization domain maps specifically to helixII of this region and a cysteine residue within this region is essential for this function. Experiments support the role of an intermolecular disulfide bond in stabilization of homodimerization, which is critical for DNA binding.

Amino Acid Sequence↗
PubMed2000

Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR.

Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co-crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T. Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15: 4689-4697). The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics. We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue. These results are discussed in relation to the mechanism of sequence specific recognition and binding.

Amino Acid Sequence↗
PubMed2001

REF-1, a protein with two bHLH domains, alters the pattern of cell fusion in C. elegans by regulating Hox protein activity.

Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. Here we identify a gene, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis.

Amino Acid Sequence↗
PubMed1995

Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions.

The basic helix-loop-helix domain (bHLH) is present in a large class of transcriptional regulators involved in developmental processes and oncogenesis. It determines DNA binding and specific homo- and heterodimeric protein associations, crucial for protein function. Myc and Max belong to a subset of HLH proteins, containing a leucine zipper (LZ) adjacent to the bHLH domain. They differ in dimerization and functional properties such as DNA binding and transcriptional activation, and their association is required for malignant transformation by Myc. To analyze the interaction specificity of Myc and Max bHLH-LZ domains, we developed a simple Escherichia coli genetic system, which uses the amino-terminal lambda phage cI repressor as a reporter for dimerization and allows an easy detection of dimeric interactions. By reciprocal exchanges of different Myc and Max subdomains (helix 1, helix 2 and leucine zipper), we showed that the recognition specificity of Max homodimers as well as of Myc/Max heterodimers is entirely determined by the helix 2-leucine zipper region, the major role being played by the leucine zipper. The Myc LZ was found to prevent homodimeric interactions, thus explaining Myc inability to homodimerize efficiently. Moreover, we showed that the system is valid as well for reproducing the interaction of HLH proteins not containing a leucine zipper and that the chimerical proteins maintain sequence-specific DNA binding.

Bacteriophage lambda↗
PubMed1999

Positional dependence, cliques, and predictive motifs in the bHLH protein domain.

Quantitative analyses were carried out on a large number of proteins that contain the highly conserved basic helix-loop-helix domain. Measures derived from information theory were used to examine the extent of conservation at amino acid sites within the bHLH domain as well as the extent of mutual information among sites within the domain. Using the Boltzmann entropy measure, we described the extent of amino acid conservation throughout the bHLH domain. We used position association (pa) statistics that reflect the joint probability of occurrence of events to estimate the "mutual information content" among distinct amino acid sites. Further, we used pa statistics to estimate the extent of association in amino acid composition at each site in the domain and between amino acid composition and variables reflecting clade and group membership, loop length, and the presence of a leucine zipper. The pa values were also used to describe groups of amino acid sites called "cliques" that were highly associated with each other. Finally, a predictive motif was constructed that accurately identifies bHLH domain-containing proteins that belong to Groups A and B.

Amino Acid Sequence↗
PubMed2000

Correlations among amino acid sites in bHLH protein domains: an information theoretic analysis.

An information theoretic approach is used to examine the magnitude and origin of associations among amino acid sites in the basic helix-loop-helix (bHLH) family of transcription factors. Entropy and mutual information values are used to summarize the variability and covariability of amino acids comprising the bHLH domain for 242 sequences. When these quantitative measures are integrated with crystal structure data and summarized using helical wheels, they provide important insights into the evolution of three-dimensional structure in these proteins. We show that amino acid sites in the bHLH domain known to pack against each other have very low entropy values, indicating little residue diversity at these contact sites. Noncontact sites, on the other hand, exhibit significantly larger entropy values, as well as statistically significant levels of mutual information or association among sites. High levels of mutual information indicate significant amounts of intercorrelation among amino acid residues at these various sites. Using computer simulations based on a parametric bootstrap procedure, we are able to partition the observed covariation among various amino acid sites into that arising from phylogenetic (common ancestry) and stochastic causes and those resulting from structural and functional constraints. These results show that a significant amount of the observed covariation among amino acid sites is due to structural/functional constraints, over and above the covariation arising from phylogenetic constraints. These quantitative analyses provide a highly integrated evolutionary picture of the multidimensional dynamics of sequence diversity and protein structure.

Amino Acids↗
PubMed1993

E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

Adenovirus E1A Proteins↗
PubMed2005

Role of DNA sequence in the binding specificity of synthetic basic-helix-loop-helix domains.

The basic helix-loop-helix (bHLH) domain defines a class of transcription factors that are essential for the regulation of many genes involved in cell differentiation and development. To determine the role of the DNA sequence in driving dimerization specificity of bHLH transcription factors, we analyzed the DNA sequence in and around a consensus hexanucleotide binding site (E-box). The bHLH domains of two transcription factors, E12 and TAL1, were chemically synthesized. The minimal DNA binding domain for both the E12 homodimer and the E12-TAL1 heterodimer was determined, thereby extending the E-box by two base pairs. Additional studies indicate that the presence of a thymine in the first flanking position 5' to the E-box prevents DNA binding of both dimer complexes. The presence of a thymine or cytosine in a flanking position two bases 5' to the E-box decreases the affinity for the E12 homodimer twofold but completely inactivates DNA binding for the E12-TAL1 heterodimer. Access to synthetic DNA and protein enabled the analysis of specific interactions between a conserved arginine residue in the basic helix of each bHLH domain and adenine in a flanking position two bases 5' to the E-box. Our results indicate a key role of the DNA sequence in driving dimerization specificity among bHLH transcription factors.

Amino Acid Sequence↗
PubMed1992

HEN1 and HEN2: a subgroup of basic helix-loop-helix genes that are coexpressed in a human neuroblastoma.

An important family of regulatory molecules is made up of proteins that possess the DNA-binding and dimerization motif known as the basic helix-loop-helix (bHLH) domain. The bHLH family includes subgroups of closely related proteins that share common functional properties and overlapping patterns of expression (e.g., the MyoD1 and achaete-scute subgroups). In this report we describe HEN1 and HEN2, mammalian genes that encode a distinct subgroup of bHLH proteins. The HEN1 gene was identified on the basis of cross-hybridization with TAL1, a known bHLH gene implicated in T-cell acute lymphoblastic leukemia. In situ fluorescence hybridization was used to localize the human HEN1 gene to chromosome band 1q22. HEN1 and HEN2 are coexpressed in the IMR-32 human neuroblastoma cell line, and they encode highly related proteins of 133 and 135 residues, respectively, that share 98% amino acid identity in their hHLH domains. These data imply that the bHLH protein subgroup encoded by HEN1 and HEN2 may serve important regulatory functions in the developing nervous system.

Amino Acid Sequence↗
PubMed1994

Formation of in vivo complexes between the TAL1 and E2A polypeptides of leukemic T cells.

Tumor-specific activation of the TAL1 gene occurs in approximately 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). The TAL1 gene products possess a basic helix-loop-helix (bHLH) domain that interacts in vitro with the bHLH proteins (E12 and E47) encoded by the E2A locus. We have now applied two independent methods, the two-hybrid procedure and co-immunoprecipitation analysis, to demonstrate that TAL1 and E2A polypeptides also associate in vivo. These studies show that the bHLH domain of TAL1 selectively interacts with the bHLH domains of E12 and E47, but not with the Id1 helix-loop-helix protein. TAL1 does not self-associate to form homodimeric complexes, implying that the in vivo functions of TAL1 depend on heterologous interaction with other bHLH proteins such as E12 and E47. Co-immunoprecipitation analysis revealed the presence of endogenous TAL1/E2A complexes in Jurkat cells, a leukemic line derived from a T-ALL patient. Thus, the malignant properties of TAL1 may be due to obligate interaction with the E2A polypeptides.

Base Sequence↗
PubMed2026

Clinical and molecular characterization of TCF12 variants in an Asian pediatric cohort with craniosynostosis.

BACKGROUND: Craniosynostosis is a genetically heterogeneous craniofacial disorder caused by the premature fusion of one or more cranial sutures. Pathogenic variants in TCF12, encoding a basic helix-loop-helix (bHLH) transcription factor, represent a major cause of autosomal dominant coronal craniosynostosis and are characterized by incomplete penetrance and marked phenotypic variability. However, clinical and molecular data from Asian pediatric populations remain limited. METHODS: Trio-based whole-exome sequencing was performed on ten pediatric patients with cranial deformities and their parents. The identified TCF12 variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines and validated by Sanger sequencing. Detailed clinical and radiological data were collected. In addition, a comprehensive literature review was conducted to summarize previously reported TCF12 variants and associated phenotypes. RESULTS: Ten distinct heterozygous TCF12 variants were identified in ten unrelated pediatric patients, all of which were classified as pathogenic or likely pathogenic according to ACMG criteria. Six variants were inherited, and four occurred de novo. Seven patients had imaging-confirmed craniosynostosis, predominantly involving the coronal sutures (five bilateral and one unilateral), while one patient presented with multisuture craniosynostosis (left coronal and sagittal sutures). Three patients showed cranial deformities without radiographic evidence of suture fusion. Phenotypic heterogeneity and incomplete penetrance were observed, including a mildly affected parent. Most pathogenic variants were truncating variants distributed mainly across exons 14-19 and predicted to induce loss of function, either through nonsense-mediated mRNA decay or the production of truncated proteins lacking the entire C-terminal bHLH domain. Structural modeling analysis further indicated that the bHLH-domain-located missense variant p.Arg603Trp alters the local DNA-binding conformation of TCF12 and impairs its binding affinity to the E-box DNA motif. CONCLUSIONS: This study provides additional clinical and molecular data on TCF12-related craniosynostosis in a pediatric cohort from an Asian population. Our findings support haploinsufficiency as the central pathogenic mechanism, primarily driven by truncating variants affecting the C-terminal bHLH domain. The marked clinical heterogeneity, the presence of mild or evolving phenotypes, and incomplete penetrance observed in our cohort underscore the importance of early diagnosis and longitudinal clinical surveillance in affected families.

Humans↗