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A hydrophobic residue stabilizes dimers of regulatory ACT-like domains in plant basic helix-loop-helix transcription factors.

ABSTRACT: About a third of the plant basic helix-loop-helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (R^ACT) and GL3 (GL3^ACT). We show that R^ACT dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3^ACT. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in R^ACT, is important: when mutated to the Ser residue present in GL3^ACT, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3^ACT significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.

SUBMITTER: Lee YS

PROVIDER: S-EPMC8202348 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Regulatory switch enforced by basic helix-loop-helix and ACT-domain mediated dimerizations of the maize transcription factor R.

Project description:The maize R2R3-MYB regulator C1 cooperates with the basic helix-loop-helix (bHLH) factor R to activate the expression of anthocyanin biosynthetic genes coordinately. As is the case for other bHLH factors, R harbors several protein-protein interaction domains. Here we show that not the classical but rather a briefly extended R bHLH region forms homodimers that bind canonical G-box DNA motifs. This bHLH DNA-binding activity is abolished if the C-terminal ACT (aspartokinase, chorismate, and TyrA) domain is licensed to homodimerize. Then the bHLH remains in the monomeric form, allowing it to interact with R-interacting factor 1 (RIF1). In this configuration, the R-RIF1 complex is recruited to the promoters of a subset of anthocyanin biosynthetic genes, such as A1, through the interaction with its MYB partner C1. If, however, the ACT domain remains monomeric, the bHLH region dimerizes and binds to G-boxes present in several anthocyanin genes, such as Bz1. Our results provide a mechanism by which a dimerization domain in a bHLH factor behaves as a switch that permits distinct configurations of a regulatory complex to be tethered to different promoters. Such a combinatorial gene regulatory framework provides one mechanism by which genes lacking obviously conserved cis-regulatory elements are regulated coordinately.

| S-EPMC3409794 | biostudies-literature

Regulatory module network of basic/helix-loop-helix transcription factors in mouse brain.

Project description:BackgroundThe basic/helix-loop-helix (bHLH) proteins are important components of the transcriptional regulatory network, controlling a variety of biological processes, especially the development of the central nervous system. Until now, reports describing the regulatory network of the bHLH transcription factor (TF) family have been scarce. In order to understand the regulatory mechanisms of bHLH TFs in mouse brain, we inferred their regulatory network from genome-wide gene expression profiles with the module networks method.ResultsA regulatory network comprising 15 important bHLH TFs and 153 target genes was constructed. The network was divided into 28 modules based on expression profiles. A regulatory-motif search shows the complexity and diversity of the network. In addition, 26 cooperative bHLH TF pairs were also detected in the network. This cooperation suggests possible physical interactions or genetic regulation between TFs. Interestingly, some TFs in the network regulate more than one module. A novel cross-repression between Neurod6 and Hey2 was identified, which may control various functions in different brain regions. The presence of TF binding sites (TFBSs) in the promoter regions of their target genes validates more than 70% of TF-target gene pairs of the network. Literature mining provides additional support for five modules. More importantly, the regulatory relationships among selected key components are all validated in mutant mice.ConclusionOur network is reliable and very informative for understanding the role of bHLH TFs in mouse brain development and function. It provides a framework for future experimental analyses.

| S-EPMC2258200 | biostudies-literature

Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling.

Project description:The Hairy-related transcription-factor (HRT) genes encode three related basic helix-loop-helix transcription factors that show sequence similarity to the Hairy and Enhancer of split family of transcriptional repressors. HRT proteins are expressed in specific regions of the developing heart, vasculature, pharyngeal arches and somites, and the periodicity of their expression in somitic precursors mirrors that of Notch signaling-related molecules. In the present study, we show that the intracellular domain of the Notch1 receptor (Notch1 IC), which is constitutively active, up-regulates HRT2 expression in 10T(1/2) fibroblasts. Luciferase reporter assays using the regulatory regions of the mouse HRT genes revealed that transcription of all three genes is stimulated by Notch1 IC. The promoters of the HRT genes share homology in a binding site for Suppressor of Hairless [Su(H)], a transcriptional mediator of Notch signaling. A dominant-negative Su(H) mutant abolished Notch-activated HRT2 expression, and mutation of the conserved Su(H) consensus site in the HRT2 promoter attenuated transcriptional activation by Notch. Ectopic expression of HRT proteins also blocked activation of HRT2 expression by Notch1 IC through a mechanism requiring the basic region, but not the conserved carboxyl-terminal YQPW-TEVGAF motif of HRT2. These findings identify HRT genes as downstream targets for Notch signaling and reveal a negative autoregulatory loop whereby HRT proteins repress their own expression through interference with Notch signaling.

| S-EPMC17631 | biostudies-literature

Positioning a multifunctional basic helix-loop-helix transcription factor within the Ciona notochord gene regulatory network.

Project description:In a multitude of organisms, transcription factors of the basic helix-loop-helix (bHLH) family control the expression of genes required for organ development and tissue differentiation. The functions of different bHLH transcription factors in the specification of nervous system and paraxial mesoderm have been widely investigated in various model systems. Conversely, the knowledge of the role of these regulators in the development of the axial mesoderm, the embryonic territory that gives rise to the notochord, and the identities of their target genes, remain still fragmentary. Here we investigated the transcriptional regulation and target genes of Bhlh-tun1, a bHLH transcription factor expressed in the developing Ciona notochord as well as in additional embryonic territories that contribute to the formation of both larval and adult structures. We describe its possible role in notochord formation, its relationship with the key notochord transcription factor Brachyury, and suggest molecular mechanisms through which Bhlh-tun1 controls the spatial and temporal expression of its effectors.

| S-EPMC6788811 | biostudies-literature

A basic helix-loop-helix transcription factors network mediates the brassinosteroid signal

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-03-05 | E-GEOD-51121 | biostudies-arrayexpress

A basic helix-loop-helix transcription factors network mediates the brassinosteroid signal

Project description:This SuperSeries is composed of the SubSeries listed below.

2014-03-05 | GSE51121 | GEO

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2

Project description:Math2 (NEX-1/NeuroD6) is a member of the bHLH transcription factor family and is involved in neuronal differentiation and maturation. In the present study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. Examining the promoter region of rat Prg1, we found four E-boxes designated -E1 to -E4 (CANNTG) which were recognized by the bHLH transcription factor. Using chromatin immunoprecipitation (ChIP) assays, we found that Math2 directly bound to the E-box(es) in the Prg1 promoter. The reporter assay of Prg1 showed that -E1 was critical for the regulation of the Prg1 expression by Math2. Then, the functional role of Math2 and Prg1 was investigated in PC12 cells. Seventy-two hours after transfection of Math2 or Prg1, neurite length and number was significantly induced in PC12 cells. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

2008-07-27 | GSE11829 | GEO

An overview of the basic helix-loop-helix proteins.

Project description:The basic helix-loop-helix proteins are dimeric transcription factors that are found in almost all eukaryotes. In animals, they are important regulators of embryonic development, particularly in neurogenesis, myogenesis, heart development and hematopoiesis.

| S-EPMC463060 | biostudies-literature

Phylogenetic analysis of the human basic helix-loop-helix proteins.

Project description:BackgroundThe basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development, including that of fruitflies, nematodes and vertebrates. The identification of orthologous relationships among the bHLH genes from these widely divergent taxa allows reconstruction of the putative complement of bHLH genes present in the genome of their last common ancestor.ResultsWe identified 39 different bHLH genes in the worm Caenorhabditis elegans, 58 in the fly Drosophila melanogaster and 125 in human (Homo sapiens). We defined 44 orthologous families that include most of these bHLH genes. Of these, 43 include both human and fly and/or worm genes, indicating that genes from these families were already present in the last common ancestor of worm, fly and human. Only two families contain both yeast and animal genes, and no family contains both plant and animal bHLH genes. We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity, and that important diversification of the bHLH repertoire occurred independently in animals and plants.ConclusionsAs the last common ancestor of worm, fly and human is also that of all bilaterian animals, our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bHLH, highlighting its genomic complexity.

| S-EPMC116727 | biostudies-literature

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA.

Project description:CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7' base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.

| S-EPMC3567813 | biostudies-literature

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