Specificity of MYB interactions relies on motifs in ordered and disordered contexts.

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB-bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB-bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB-bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB-bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix-Loop-Helix Transcription Factors.

The basic helix-loop-helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell's fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

Identification, Molecular Characteristic, and Expression Analysis of PIFs Related to Chlorophyll Metabolism in Tea Plant (Camellia sinensis).

The phytochrome-interacting factors (PIFs) proteins belong to the subfamily of basic helix-loop-helix (bHLH) transcription factors and play important roles in chloroplast development and chlorophyll biosynthesis. Currently, knowledge about the PIF gene family in Camellia sinensis remains very limited. In this study, seven PIF members were identified in the C. sinensis genome and named based on homology with AtPIF genes in Arabidopsis thaliana. All C. sinensis PIF (CsPIF) proteins have both the conserved active PHYB binding (APB) and bHLH domains. Phylogenetic analysis revealed that CsPIFs were clustered into four groups-PIF1, PIF3, PIF7, and PIF8-and most CsPIFs were clustered in pairs with their corresponding orthologs in Populus tremula. CsPIF members in the same group tended to display uniform or similar exon-intron distribution patterns and motif compositions. CsPIF genes were differentially expressed in C. sinensis with various leaf colors and strongly correlated with the expression of genes involved in the chlorophyll metabolism pathway. Promoter analysis of structural genes related to chlorophyll metabolism found DNA-binding sites of PIFs were abundant in the promoter regions. Protein-protein interaction networks of CsPIFs demonstrated a close association with phytochrome, PIF4, HY5, TOC1, COP1, and PTAC12 proteins. Additionally, subcellular localization and transcriptional activity analysis suggested that CsPIF3b was nuclear localized protein and possessed transcriptional activity. We also found that CsPIF3b could activate the transcription of CsHEMA and CsPOR in Nicotiana benthamiana leaves. This work provides comprehensive research of CsPIFs and would be helpful to further promote the regulation mechanism of PIF on chlorophyll metabolism in C. sinensis.

Tracheophytes Contain Conserved Orthologs of a Basic Helix-Loop-Helix Transcription Factor That Modulate ROOT HAIR SPECIFIC Genes.

ROOT HAIR SPECIFIC (RHS) genes, which contain the root hair-specific cis-element (RHE) in their regulatory regions, function in root hair morphogenesis. Here, we demonstrate that an Arabidopsis thaliana basic helix-loop-helix transcription factor, ROOT HAIR DEFECTVE SIX-LIKE4 (RSL4), directly binds to the RHE in vitro and in vivo, upregulates RHS genes, and stimulates root hair formation in Arabidopsis. Orthologs of RSL4 from a eudicot (poplar [Populus trichocarpa]), a monocot (rice [Oryza sativa]), and a lycophyte (Selaginella moellendorffii) each restored root hair growth in the Arabidopsis rsl4 mutant. In addition, the rice and S. moellendorffii RSL4 orthologs bound to the RHE in in vitro and in vivo assays. The RSL4 orthologous genes contain RHEs in their promoter regions, and RSL4 was able to bind to its own RHEs in vivo and amplify its own expression. This process likely provides a positive feedback loop for sustainable root hair growth. When RSL4 and its orthologs were expressed in cells in non-root-hair positions, they induced ectopic root hair growth, indicating that these genes are sufficient to specify root hair formation. Our results suggest that RSL4 mediates root hair formation by regulating RHS genes and that this mechanism is conserved throughout the tracheophyte (vascular plant) lineage.

Regulation Mechanism of MYC Family Transcription Factors in Jasmonic Acid Signalling Pathway on Taxol Biosynthesis.

Paclitaxel is an important anticancer drug. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in Taxus, but the molecular mechanism has not yet been resolved. To establish the jasmonic acid signalling pathway of Taxus media, based on the gene of the jasmonic acid signalling pathway of Arabidopsis thaliana, sequence analysis was performed to isolate the jasmonic acid signal from the transcriptome, a transcriptional cluster of pathway gene homologs and the full length of 22 genes were obtained by RACE PCR at 5' and 3': two EI ubiquitin ligase genes, COI1-1 and COI1-2;7 MYC bHLH type transcription factor (MYC2, MYC3, MYC4, JAM1, JAM2, EGL3, TT8); 12 JAZ genes containing the ZIM domain; and MED25, one of the components of the transcriptional complex. The protein interaction between each were confirmed by yeast two hybridization and bimolecular fluorescence complementation based on similar genes interaction in Arabidopsis. A similar jasmonate signaling pathway was illustrated in T. media. All known paclitaxel biosynthesis genes promoters were isolated by genome walker PCR. To investigate the jasmonate signaling effect on these genes' expression, the transcription activity of MYC2, MYC3 and MYC4 on these promoters were examined. There are 12, 10 and 11 paclitaxel biosynthesis genes promoters that could be activated by MYC2, MYC3 and MYC4.

Transcriptome-wide identification and expression profile analysis of the bHLH family genes in Camellia sinensis.

The tea plant is an important commercial horticulture crop cultivated worldwide. Yield and quality of this plant are influenced by abiotic stress. The bHLH family transcription factors play a pivotal role in the growth and development, including abiotic stress response, of plants. A growing number of bHLH proteins have been functionally characterized in plants. However, few studies have focused on the bHLH proteins in tea plants. In this study, 120 CsbHLH TFs were identified from tea plants using computational prediction method. Structural analysis detected 23 conservative residues, with over 50% identities in the bHLH domain. Moreover, 103 CsbHLH proteins were assumed to bind DNA and encompassed 98 E-Box binders and 85 G-Box binders. The CsbHLH proteins were grouped into 20 subfamilies based on phylogenetic analysis and a previous classification system. A survey of transcriptome profiling screened 22 and 39 CsbHLH genes that were upregulated under heat and drought stress. Nine CsbHLH genes were validated using qRT-PCR. Results were approximately in accordance with transcriptome data. These genes could be induced by one or more abiotic stresses.

Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus.

The basic helix-loop-helix (bHLH) transcription factors (TFs) form a huge superfamily and play crucial roles in many essential developmental, genetic, and physiological-biochemical processes of eukaryotes. In total, 109 putative bHLH TFs were identified and categorized successfully in the genomic databases of cattle, Bos Taurus, after removing redundant sequences and merging genetic isoforms. Through phylogenetic analyses, 105 proteins among these bHLH TFs were classified into 44 families with 46, 25, 14, 3, 13, and 4 members in the high-order groups A, B, C, D, E, and F, respectively. The remaining 4 bHLH proteins were sorted out as 'orphans.' Next, these 109 putative bHLH proteins identified were further characterized as significantly enriched in 524 significant Gene Ontology (GO) annotations (corrected P value ≤ 0.05) and 21 significantly enriched pathways (corrected P value ≤ 0.05) that had been mapped by the web server KOBAS 2.0. Furthermore, 95 bHLH proteins were further screened and analyzed together with two uncharacterized proteins in the STRING online database to reconstruct the protein-protein interaction network of cattle bHLH TFs. Ultimately, 89 bHLH proteins were fully mapped in a network with 67 biological process, 13 molecular functions, 5 KEGG pathways, 12 PFAM protein domains, and 25 INTERPRO classified protein domains and features. These results provide much useful information and a good reference for further functional investigations and updated researches on cattle bHLH TFs.

The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.

The Antirrhinum AmDEL gene enhances flavonoids accumulation and salt and drought tolerance in transgenic Arabidopsis.

MAIN CONCLUSION: A basic helix-loop-helix (bHLH) transcription factor gene from Antirrhinum, AmDEL , increases flavonoids accumulation and enhances salt and drought tolerance via up-regulating flavonoid biosynthesis, proline biosynthesis and ROS scavenging genes in transgenic Arabidopsis. In plants, transcriptional regulation is the most important tools for increasing flavonoid biosynthesis. The AmDEL gene, as a basic helix-loop-helix transcription factor gene from Antirrhinum, has been shown to increase flavonoids accumulation in tomato. However, its role in tolerance to abiotic stresses has not yet been investigated. In this study, the codon-optimized AmDEL gene was chemically synthesized. Subcellular localization analysis in onion epidermal cells indicated that AmDEL protein was localized to the nucleus. Expression analysis in yeast showed that the full length of AmDEL exhibited transcriptional activation. Overexpression of AmDEL significantly increased flavonoids accumulation and enhanced salt and drought tolerance in transgenic Arabidopsis plants. Real-time quantitative PCR analysis showed that overexpression of AmDEL resulted in the up-regulation of genes involved in flavonoid biosynthesis, proline biosynthesis and ROS scavenging under salt and drought stresses. Meanwhile, Western blot and enzymatic analyses showed that the activities of phenylalanine ammonia lyase, chalcone isomerase, dihydroflavonol reductase, pyrroline-5-carboxylate synthase, superoxide dismutase and peroxidase were also increased. Further components analyses indicated that the significant increase of proline and relative water content and the significant reduction of H2O2 and malonaldehyde content were observed under salt and drought stresses. In addition, the rates of electrolyte leakage and water loss were reduced in transgenic plants. These findings imply functions of AmDEL in accumulation of flavonoids and tolerance to salt and drought stresses. The AmDEL gene has the potential to be used to increase the content of valuable flavonoids and improve tolerance to abiotic stresses in plants.

The poplar basic helix-loop-helix transcription factor BEE3 - Like gene affects biomass production by enhancing proliferation of xylem cells in poplar.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Genome-wide identification, classification and functional analyses of the bHLH transcription factor family in the pig, Sus scrofa.

The basic helix-loop-helix (bHLH) transcription factors are one of the largest families of gene regulatory proteins and play crucial roles in genetic, developmental and physiological processes in eukaryotes. Here, we conducted a survey of the Sus scrofa genome and identified 109 putative bHLH transcription factor members belonging to super-groups A, B, C, D, E, and F, respectively, while four members were orphan genes. We identified 6 most significantly enriched KEGG pathways and 116 most significant GO annotation categories. Further comprehensive surveys in human genome and other 12 medical databases identified 72 significantly enriched biological pathways with these 113 pig bHLH transcription factors. From the functional protein association network analysis 93 hub proteins were identified and 55 hub proteins created a tight network or a functional module within their protein families. Especially, there were 20 hub proteins found highly connected in the functional interaction network. The present study deepens our understanding and provided insights into the evolution and functional aspects of animal bHLH proteins and should serve as a solid foundation for further for analyses of specific bHLH transcription factors in the pig and other mammals.

bHLH-PAS heterodimer of methoprene-tolerant and Cycle mediates circadian expression of juvenile hormone-induced mosquito genes.

Juvenile hormone (JH) governs a great diversity of processes in insect development and reproduction. It plays a critical role in controlling the gonadotrophic cycles of female mosquitoes by preparing tissues for blood digestion and egg development. Here, we show that in female Aedes aegypti mosquitoes JH III control of gene expression is mediated by a heterodimer of two bHLH-PAS proteins-the JH receptor methoprene-tolerant (MET) and Cycle (CYC, AAEL002049). We identified Aedes CYC as a MET-interacting protein using yeast two-hybrid screening. Binding of CYC and MET required the presence of JH III. In newly eclosed female mosquitoes, the expression of two JH-responsive genes, Kr-h1 and Hairy, was dependent on both the ratio of light to dark periods and JH III. Their expression was compromised by in vivo RNA interference (RNAi) depletions of CYC, MET, and the steroid receptor coactivator SRC/FISC. Moreover, JH III was not effective in induction of Kr-h1 and Hairy gene expression in vitro in fat bodies of female mosquitoes with RNAi-depleted CYC, MET or SRC/FISC. A sequence containing an E-box-like motif from the Aedes Kr-h1 gene promoter specifically interacted with a protein complex, which included MET and CYC from the female mosquito fat body nuclear extract. These results indicate that a MET/CYC heterodimer mediates JH III activation of Kr-h1 and Hairy genes in the context of light-dependent circadian regulation in female mosquitoes during posteclosion development. This study provides an important insight into the understanding of the molecular basis of JH action.

A novel bHLH transcription factor PebHLH35 from Populus euphratica confers drought tolerance through regulating stomatal development, photosynthesis and growth in Arabidopsis.

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in a variety of physiological processes including the regulation of plant responses to various abiotic stresses. However, few drought-responsive bHLH family members in Populus have been reported. In this study, a novel bHLH gene (PebHLH35) was cloned from Populus euphratica. Expression analysis in P. euphratica revealed that PebHLH35 was induced by drought and abscisic acid. Subcellular localization studies using a PebHLH35-GFP fusion showed that the protein was localized to the nucleus. Ectopic overexpression of PebHLH35 in Arabidopsis resulted in a longer primary root, more leaves, and a greater leaf area under well-watered conditions compared with vector control plants. Notably, PebHLH35 overexpression lines showed enhanced tolerance to water-deficit stress. This finding was supported by anatomical and physiological analyses, which revealed a reduced stomatal density, stomatal aperture, transpiration rate, and water loss, and a higher chlorophyll content and photosynthetic rate. Our results suggest that PebHLH35 functions as a positive regulator of drought stress responses by regulating stomatal density, stomatal aperture, photosynthesis and growth.

Regulation of anthocyanin biosynthesis in peach fruits.

MAIN CONCLUSION: MYB10.1 and MYB10.3, with bHLH3, are the likely regulators of anthocyanin biosynthesis in peach fruit. MYB10.1/2/3 forms a cluster on the same genomic fragment where the Anther color ( Ag ) trait is located. Anthocyanins are bioactive compounds responsible for the pigmentation of many plant parts such as leaves, flowers, fruits and roots, and have potential benefits to human health. In peach [Prunus persica (L.) Batsch], peel color is a key determinant for fruit quality and is regulated by flavonoids including anthocyanins. The R2R3 MYB transcription factors (TFs) control the expression of anthocyanin biosynthetic genes with the help of co-activators belonging to the basic-helix-loop-helix (bHLH) and WD40 repeat families. In the peach genome six MYB10-like and three bHLH-like TFs were identified as candidates to be the regulators of the anthocyanin accumulation, which, in yellow flesh fruits, is highest in the peel, abundant in the part of the mesocarp surrounding the stone and lowest in the mesocarp. The expression of MYB10.1 and MYB10.3 correlates with anthocyanin levels of different peach parts. They also have positive correlation with the expression of key structural genes of the anthocyanin pathway, such as CHS, F3H, and UFGT. Functions of peach MYB10s were tested in tobacco and shown to activate key genes in the anthocyanin pathway when bHLHs were co-expressed as partners. Overexpression of MYB10.1/bHLH3 and MYB10.3/bHLH3 activated anthocyanin production by up-regulating NtCHS, NtDFR and NtUFGT while other combinations were not, or much less, effective. As three MYB10 genes are localized in a genomic region where the Ag trait, responsible for anther pigmentation, is localized, it is proposed they are key determinant to introduce new peach cultivars with higher antioxidant level and pigmented fruit.

A genome-wide identification and classification of basic helix-loop-helix genes in the jewel wasp, Nasonia vitripennis (Hymenoptera: Pteromalidae).

Basic helix-loop-helix (bHLH) proteins are highly conserved DNA-binding transcription factors of a large superfamily. Animal bHLH proteins play important regulatory roles in various developmental processes such as neurogenesis, myogenesis, heart development, and hematopoiesis. The jewel wasp (Nasonia vitripennis) is a good model organism of hymenoptera insects for studies of developmental and evolutionary genetics. In this study, we identified 48 bHLH genes in the genome of N. vitripennis. According to phylogenetic analysis, based on N. vitripennis bHLH (NvbHLH) motif sequences and structural domain distribution in their full-length protein sequences, the identified NvbHLH genes were classified into 36 bHLH families with 19, 12, 9, 1, 6, and 1 member(s) in groups A, B, C, D, E, and F, respectively. Our classification to the identified NvbHLH family members confirms GenBank annotations for 21 of the 48 NvbHLH proteins and provides useful information for further characterization and annotation of the remaining 27 NvbHLH proteins. Compared to other insect species, N. vitripennis has the lowest number of bHLH family members. No NvbHLH members have been found in the families Net, MyoRa, and PTFa, while all other insect species have at least one member in each of the families. These data constitute a solid basis for further investigations into the functions of bHLH proteins in developmental regulation of N. vitripennis.

Mammalian Per-Arnt-Sim proteins in environmental adaptation.

The Per-Arnt-Sim (PAS) domain is conserved across the kingdoms of life and found in an ever-growing list of proteins. This domain can bind to and sense endogenous or xenobiotic small molecules such as molecular oxygen, cellular metabolites, or polyaromatic hydrocarbons. Members of this family are often found in pathways that regulate responses to environmental change; in mammals these include the hypoxia, circadian, and dioxin response pathways. These pathways function in development and throughout life to regulate cellular, organ, and whole-organism adaptive responses. Remarkably, in the case of the clock, this adaptation includes anticipation of environmental change. In this review, we summarize the roles of PAS domain-containing proteins in mammals. We provide structural evidence that functionally classifies both known and unknown biological roles. Finally, we discuss the role of PAS proteins in anticipation of and adaptation to environmental change.

[Research progress of the regulation on active compound biosynthesis by the bHLH transcription factors in plants].

Transcription factor is one of the key factors in the regulation of gene expression at the transcriptional level. It plays an important role in plant growth, active components biosynthesis and response to environmental change. This paper summarized the structure and classification of bHLH transcription factors and elaborated the research progress of bHLH transcription factors which regulate the active components in plants, such as flavonoids, alkaloids, and terpenoids. In addition, the possibility of increasing the concentration of active substances by bHLH in medicinal plants was assessed. The paper emphasized great significance of model plants and multidisciplinary research fields including modern genomics, transcriptomics, metabolomics and bioinformatics, providing the contribution to improve the discovery and function characterization of bHLH transcription factors. Accelerating the research in the mechanism of bHLH transcription factors on the regulation of active components biosynthesis will promote the development of breeding and variety improvement of Chinese medicinal materials, also ease the pressure of resources exhaustion of traditional Chinese medicine home and abroad.

Somitogenesis in the anole lizard and alligator reveals evolutionary convergence and divergence in the amniote segmentation clock.

The axial skeleton is a defining feature of vertebrates and is patterned during somitogenesis. Cyclically expressed members of the notch and other signaling pathways, described as the 'segmentation clock', regulate the formation of somite boundaries. Comparisons among vertebrate model systems have revealed fundamental shifts in the regulation of expression among critical genes in the notch pathway. However, insights into the evolution of these expression differences have been limited by the lack of information from non-avian reptiles. We analyzed the segmentation clock of the first Lepidosaurian reptile sequenced, the green anole lizard, Anolis carolinensis, for comparison with avian and mammalian models. Using genomic sequence, RNA-Seq transcriptomic data, and in situ hybridization analysis of somite-stage embryos, we carried out comparative analyses of key genes and found that the anole segmentation clock displays features common to both amniote and anamniote vertebrates. Shared features with anamniotes, represented by Xenopus laevis and Danio rerio, include an absence of lunatic fringe (lfng) expression within the presomitic mesoderm (PSM), a hes6a gradient in the PSM not observed in the chicken or mouse, and EGF repeat structure of the divergent notch ligand, dll3. The anole and mouse share cycling expression of dll1 ligand in the PSM. To gain insight from an Archosaurian reptile, we analysed LFNG and DLL1 expressions in the American alligator. LFNG expression was absent in the alligator PSM, like the anole but unlike the chicken. In contrast, DLL1 expression does not cycle in the PSM of the alligator, similar to the chicken but unlike the anole. Thus, our analysis yields novel insights into features of the segmentation clock that are evolutionarily basal to amniotes versus those that are specific to mammals, Lepidosaurian reptiles, or Archosaurian reptiles.

Classification and evolutionary analysis of the basic helix-loop-helix gene family in the green anole lizard, Anolis carolinensis.

Helix-loop-helix (bHLH) proteins play essential regulatory roles in a variety of biological processes. These highly conserved proteins form a large transcription factor superfamily, and are commonly identified in large numbers within animal, plant, and fungal genomes. The bHLH domain has been well studied in many animal species, but has not yet been characterized in non-avian reptiles. In this study, we identified 102 putative bHLH genes in the genome of the green anole lizard, Anolis carolinensis. Based on phylogenetic analysis, these genes were classified into 43 families, with 43, 24, 16, 3, 10, and 3 members assigned into groups A, B, C, D, E, and F, respectively, and 3 members categorized as "orphans". Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with highly conserved patterns observed for introns and additional domains. Results from phylogenetic analysis of the H/E(spl) family suggest that genome and tandem gene duplications have contributed to this family's expansion. Our classification and evolutionary analysis has provided insights into the evolutionary diversification of animal bHLH genes, and should aid future studies on bHLH protein regulation of key growth and developmental processes.

Cath6, a bHLH atonal family proneural gene, negatively regulates neuronal differentiation in the retina.

Basic helix-loop-helix (bHLH) transcription factors play important roles in cell type specification and differentiation during the development of the nervous system. In this study, we identified a chicken homolog of Atonal 8/ath6 (Cath6) and examined its role in the developing retina. Unlike other Atonal-family proneural genes that induce neuronal differentiation, Cath6 was expressed in stem cell-like progenitor cells in the marginal region of the retina, and its overexpression inhibited neuronal differentiation. A Cath6 fused with a VP16 transactivation domain recapitulated the inhibitory effect of Cath6 on neuronal differentiation, indicating that Cath6 functions as a transcription activator. These results demonstrate that Cath6 constitutes a unique member of the Atonal-family of genes in that it acts as a negative regulator of neuronal differentiation.