Basic helix-loop-helix (bHLH) proteins comprise a large superfamily of transcription factors, which are involved in the regulation of various developmental processes. bHLH family members are widely distributed in various eukaryotes including yeast, fruit fly, zebrafish, mouse, and human. In this study, we identified 55 bHLH motifs encoded in genome sequence of the human body louse, Pediculus humanus corporis (Phthiraptera: Pediculidae). Phylogenetic analyses of the identified P. humanus corporis bHLH (PhcbHLH) motifs revealed that there are 23, 11, 9, 1, 10, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Examination to GenBank annotations of the 55 PhcbHLH members indicated that 29 PhcbHLH proteins were annotated in consistence with our analytical result, 8 were annotated different with our analytical result, 12 were merely annotated as hypothetical protein, and the rest 6 were not deposited in GenBank. A comparison on insect bHLH gene composition revealed that human body louse possibly has more hairy and E(spl) genes than other insect species. Because hairy and E(spl) genes have been found to negatively regulate the differentiation of insect preneural cells, it is suggested that the existence of additional hairy and E(spl) genes in human body louse is probably the consequence of its long period adaptation to the relatively dark and stable environment. These data provide good references for further studies on regulatory functions of bHLH proteins in the growth and development of human body louse.

Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites are dependent on the activity of additional signal-activated transcription factors that prime chromatin toward context-specific organization. We hypothesized a stress context dependency for GR binding in hippocampus as a consequence of rapidly induced stress mediators priming chromatin accessibility. Using chromatin immunoprecipitation sequencing to interrogate GR binding, we found no effect of restraint stress context on GR binding, although analysis of sequences underlying GR binding sites revealed mechanistic detail for hippocampal GR function. We note enrichment of GR binding sites proximal to genes linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix-loop-helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding sites and is proposed as a candidate lineage-determining transcription factor directing hippocampal chromatin access for GRs. Of our GR binding sites, 54% additionally contained half-sites for nuclear factor (NF)-1 that we propose as a collaborative or general transcription factor involved in hippocampal GR function. Our findings imply a dose-dependent and context-independent action of GRs in the hippocampus. Alterations in the expression or activity of NF-1/basic helix-loop-helix factors may play an as yet undetermined role in glucocorticoid-related disease susceptibility and outcome by altering GR access to hippocampal binding sites.

Basic helix-loop-helix (bHLH) proteins are a class of transcription factors found throughout eukaryotic organisms. Classification of the complete sets of bHLH proteins in the sequenced genomes of Arabidopsis thaliana and Oryza sativa (rice) has defined the diversity of these proteins among flowering plants. However, the evolutionary relationships of different plant bHLH groups and the diversity of bHLH proteins in more ancestral groups of plants are currently unknown. In this study, we use whole-genome sequences from nine species of land plants and algae to define the relationships between these proteins in plants. We show that few (less than 5) bHLH proteins are encoded in the genomes of chlorophytes and red algae. In contrast, many bHLH proteins (100-170) are encoded in the genomes of land plants (embryophytes). Phylogenetic analyses suggest that plant bHLH proteins are monophyletic and constitute 26 subfamilies. Twenty of these subfamilies existed in the common ancestors of extant mosses and vascular plants, whereas six further subfamilies evolved among the vascular plants. In addition to the conserved bHLH domains, most subfamilies are characterized by the presence of highly conserved short amino acid motifs. We conclude that much of the diversity of plant bHLH proteins was established in early land plants, over 440 million years ago.

The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH) family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281) of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

The basic helix-loop-helix (bHLH) transcription factors form a large superfamily that plays an important role in numerous physiological processes, including development and response to environmental stresses. In this study, the distribution of bHLH genes in nine molluscs was systematically investigated (including five bivalves, three gastropods and one cephalopod). Finally, 53-85 bHLH genes were identified from each genome and classified into corresponding families by using phylogenetic analysis. The results of gene structure and conserved motif analysis illustrated the hereditary conservation of bHLH transcription factors during evolution but showed low similarity in group C. Through transcription profile analysis of C. gigas and T. granosa, we found a important role of bHLH genes in responding to multiple external challenges and development; meanwhile, they also exhibited tissue-specific expression. Interestingly, we were also surprised to find different bHLH genes from the same group generally possess similar patterns expression that tends to simultaneously present high or lower expression of multiple challenges and different tissues in this study. In summary, this study lays the foundation for further investigation of the biological functions and evolution of molluscan bHLH genes.

As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae.

Members of the basic helix-loop-helix (bHLH) family of transcription factors play important roles in a wide range of developmental processes. In this study, we conducted a genome-wide survey using the chicken (Gallus gallus) genomic database, and identified 104 bHLH sequences belonging to 42 gene families in an effort to characterize the chicken bHLH transcription factor family. Phylogenetic analyses revealed that chicken has 50, 21, 15, 4, 8, and 3 bHLH members in groups A, B, C, D, E, and F, respectively, while three members belonging to none of these groups were classified as ''orphans". A comparison between chicken and human bHLH repertoires suggested that both organisms have a number of lineage-specific bHLH members in the proteomes. Chromosome distribution patterns and phylogenetic analyses strongly suggest that the bHLH members should have arisen through gene duplication at an early date. Gene Ontology (GO) enrichment statistics showed 51 top GO annotations of biological processes counted in the frequency. The present study deepens our understanding of the chicken bHLH transcription factor family and provides much useful information for further studies using chicken as a model system.

Basic helix-loop-helix (bHLH) transcription factors play essential roles in regulating eukaryotic developmental and physiological processes such as neuron generation, myocyte formation, intestinal tissue development, and response to environmental stress. In this study, the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), genome was found to encode 52 bHLH genes. All 52 P. xylostella bHLH (PxbHLH) genes were classified into correspondent bHLH families according to their orthology with bHLHs from fruit fly and other insect species. Among these 52 PxbHLH genes, 19 have been annotated consistently with our classification in GenBank database. The remaining 33 PxbHLH genes are either annotated as general bHLH genes or as hypothetical genes. Therefore, our data provide useful information for updating annotations to PxbHLH genes. P. xylostella has four stem cell leukemia (SCL) genes (one of them has three copies), two Dys genes, two copies of MyoR, Mitf, and Sima genes, and three copies of Sage genes. Further studies may be conducted to elucidate functions of these specific bHLH genes in regulating P. xylostella growth and development.

The giant panda (Ailuropoda melanoleuca) is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.

During development, transcription factors select distinct gene programs, providing the necessary regulatory complexity for temporal and tissue-specific gene expression. How related factors retain specificity, especially when they recognize the same DNA motifs, is not understood. We address this paradox using basic helix-loop-helix (bHLH) transcription factors ASCL1, ASCL2, and MYOD1, crucial mediators of lineage specification. In vivo, these factors recognize the same DNA motifs, yet bind largely different genomic sites and regulate distinct transcriptional programs. This suggests that their ability to identify regulatory targets is defined either by the cellular environment of the partially defined lineages in which they are endogenously expressed, or by intrinsic properties of the factors themselves. To distinguish between these mechanisms, we directly compared the chromatin binding properties of this subset of bHLH factors when ectopically expressed in embryonic stem cells, presenting them with a common chromatin landscape and cellular components. We find that these factors retain distinct binding sites; thus, specificity of binding is an intrinsic property not requiring a restricted landscape or lineage-specific cofactors. Although the ASCL factors and MYOD1 have some distinct DNA motif preference, it is not sufficient to explain the extent of the differential binding. All three factors can bind inaccessible chromatin and induce changes in chromatin accessibility and H3K27ac. A reiterated pattern of DNA binding motifs is uniquely enriched in inaccessible chromatin at sites bound by these bHLH factors. These combined properties define a subclass of lineage-specific bHLH factors and provide context for their central roles in development and disease.

The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, and human. This study identified 54 bHLH family members in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), genome. Phylogenetic analyses revealed that they belong to 37 bHLH families with 21, 13, 9, 1, 9, and 1 members in group A, B, C, D, E, and F, respectively. Through in-group phylogenetic analyses, all of the identified A. pisum bHLH members were assigned into their correspondent bHLH families with confidence, among which 51 were defined according to phylogenetic analyses with orthologs from Drosophila melanogaster Meigen (Diptera: Drosophilidae), and 3 of them were defined according to phylogenetic analyses with orthologs from Bombyx mori L. (Lepidoptera: Bombycidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Analyses on genomic coding regions revealed that the number and average length of introns in A. pisum bHLH motifs are higher than those in other insects. The present study provides useful background information for future studies on structure and function of bHLH proteins in the regulation of A. pisum development.

The basic helix-loop-helix (bHLH) transcription factors (TFs) have been identified and functionally characterized in many plants. However, no comprehensive analysis of the bHLH family in papaya (Carica papaya L.) has been reported previously. Here, a total of 73 CpbHLHs were identified in papaya, and these genes were classified into 18 subfamilies based on phylogenetic analysis. Almost all of the CpbHLHs in the same subfamily shared similar gene structures and protein motifs according to analysis of exon/intron organizations and motif compositions. The number of exons in CpbHLHs varied from one to 10 with an average of five. The amino acid sequences of the bHLH domains were quite conservative, especially Leu-27 and Leu-63. Promoter cis-element analysis revealed that most of the CpbHLHs contained cis-elements that can respond to various biotic/abiotic stress-related events. Gene ontology (GO) analysis revealed that CpbHLHs mainly functions in protein dimerization activity and DNA-binding, and most CpbHLHs were predicted to localize in the nucleus. Abiotic stress treatment and quantitative real-time PCR (qRT-PCR) revealed some important candidate CpbHLHs that might be responsible for abiotic stress responses in papaya. These findings would lay a foundation for further investigate of the molecular functions of CpbHLHs.

Huntington's disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type (wt) and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix (bHLH) family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the postmortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease.

Basic helix-loop-helix (bHLH) proteins, which are characterized by a conserved bHLH domain, comprise one of the largest families of transcription factors in both plants and animals, and have been shown to have a wide range of biological functions. However, there have been very few studies of bHLH proteins from perennial tree species. We describe here the identification and characterization of 175 bHLH transcription factors from apple (Malus × domestica). Phylogenetic analysis of apple bHLH (MdbHLH) genes and their Arabidopsis thaliana (Arabidopsis) orthologs indicated that they can be classified into 23 subgroups. Moreover, integrated synteny analysis suggested that the large-scale expansion of the bHLH transcription factor family occurred before the divergence of apple and Arabidopsis. An analysis of the exon/intron structure and protein domains was conducted to suggest their functional roles. Finally, we observed that MdbHLH subgroup III and IV genes displayed diverse expression profiles in various organs, as well as in response to abiotic stresses and various hormone treatments. Taken together, these data provide new information regarding the composition and diversity of the apple bHLH transcription factor family that will provide a platform for future targeted functional characterization.

Overactivation of osteoclasts due to altered osteoclastogenesis causes multiple bone metabolic diseases. However, how osteoclast differentiation is tightly regulated and involved in multiple pathophysiological states remains mystery. In this study, we noticed that the downregulation of BHLHE41 (basic-helix-loop-helix family member e41) was tightly associated with osteoclast differentiation and osteoporosis. Functionally, the upregulation or downregulation of BHLHE41 suppressed or promoted osteoclast differentiation, respectively, in vitro. A mechanism study indicated that the direct binding of BHLHE41 to the promoter region of NFATc1 that led to its downregulation. Notably, the inhibition of NFATc1 abrogated the enhanced osteoclast differentiation in BHLHE41-knockdown bone marrow macrophages (BMMs). Additionally, upregulation of BHLHE41 impeded bone destruction in OVX mice with osteoporosis. Therefore, our research reveals the mechanism by which BHLHE41 regulates osteoclast differentiation and bone resorption via NFATc1, and targeting BHLHE41 is a potential strategy for the treatment of osteoporosis.

The Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.

The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix-loop-helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

Polyphenols are the main active components of the anti-inflammatory compounds in dandelion, and chlorogenic acid (CGA) is one of the primary polyphenols. However, the molecular mechanism underlying the transcriptional regulation of CGA biosynthesis remains unclear. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT2) is the last rate-limiting enzyme in chlorogenic acid biosynthesis in Taraxacum antungense. Therefore, using the TaHQT2 gene promoter as a probe, a yeast one-hybrid library was performed, and a basic helix-loop-helix (bHLH) transcription factor, TabHLH1, was identified that shared substantial homology with Gynura bicolor DC bHLH1. The TabHLH1 transcript was highly induced by salt stress, and the TabHLH1 protein was localized in the nucleus. CGA and luteolin concentrations in TabHLH1-overexpression transgenic lines were significantly higher than those in the wild type, while CGA and luteolin concentrations in TabHLH1-RNA interference (RNAi) transgenic lines were significantly lower. Quantitative real-time polymerase chain reaction demonstrated that overexpression and RNAi of TabHLH1 in T. antungense significantly affected CGA and luteolin concentrations by upregulating or downregulating CGA and luteolin biosynthesis pathway genes, especially TaHQT2, 4-coumarate-CoA ligase (Ta4CL), chalcone isomerase (TaCHI), and flavonoid-3'-hydroxylase (TaF3'H). Dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays indicated that TabHLH1 directly bound to the bHLH-binding motifs of proTaHQT2 and proTa4CL. This study suggests that TabHLH1 participates in the regulatory network of CGA and luteolin biosynthesis in T. antungense and might be useful for metabolic engineering to promote plant polyphenol biosynthesis.

Dendrobium officinale Kimura et Migo is of great importance as a traditional Chinese herb due to its abundant metabolites. The family of basic helix-loop-helix (bHLH) transcription factors widely exists in plants and plays an essential role in plant growth and development, secondary metabolism as well as responses to environmental changes. However, there is limited information on bHLH genes in D. officinale. In the present study, a total of 98 putative DobHLH genes were identified at the genomic level, which could be classified into 18 clades. Gene structures and conserved motifs in DobHLH genes showed high conservation during their evolution. The conserved amino acids and DNA bindings of DobHLH proteins were predicted, both of which are pivotal for their function. Furthermore, gene expression from eight tissues showed that some DobHLH genes were ubiquitously expressed while other DobHLH genes were expressed in the specific tissues. Expressional changes of DobHLH genes under MeJA and ABA treatments were detected by qRT-PCR. The protein-protein interactions between DobHLHs were predicted and several interactions were confirmed by yeast two hybrid. Therefore, our results here contribute to the understanding of bHLH genes in D. officinale and lay a foundation for the further functional study of its biological processes.

The basic helix-loop-helix (bHLH) transcription factors in insects play essential roles in multiple developmental processes including neurogenesis, sterol metabolism, circadian rhythms, organogenesis and formation of olfactory sensory neurons. The identification and function analysis of bHLH family members of the most destructive insect pest of rice, Nilaparvata lugens, may provide novel tools for pest management. Here, a genome-wide survey for bHLH sequences identified 60 bHLH sequences (NlbHLHs) encoded in the draft genome of N. lugens. Phylogenetic analysis of the bHLH domains successfully classified these genes into 40 bHLH families in group A (25), B (14), C (10), D (1), E (8) and F (2). The number of NlbHLHs with introns is higher than many other insect species, and the average intron length is shorter than those of Acyrthosiphon pisum. High number of ortholog families of NlbHLHs was found suggesting functional conversation for these proteins. Compared to other insect species studied, N. lugens has the highest number of bHLH members. Furthermore, gene duplication events of SREBP, Kn(col), Tap, Delilah, Sim, Ato and Crp were found in N. lugens. In addition, a putative full set of NlbHLH genes is defined and compared with another insect species. Thus, our classification of these NlbHLH members provides a platform for further investigations of bHLH protein functions in the regulation of N. lugens, and of insects in general.