Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer.

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.

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FIGalpha, a germ cell specific transcription factor involved in the coordinate expression of the zona pellucida genes

Development ◽

10.1242/dev.124.24.4939 ◽

1997 ◽

Vol 124 (24) ◽

pp. 4939-4947 ◽

Cited By ~ 13

Author(s):

L. Liang ◽

S.M. Soyal ◽

J. Dean

Keyword(s):

Transcription Factor ◽

Zona Pellucida ◽

Reporter Genes ◽

Single Copy ◽

Luciferase Reporter ◽

Embryonic Fibroblasts ◽

Transcription Start Sites ◽

Helix Loop Helix ◽

E Box ◽

Dna Complex

The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3, encoded by single-copy genes whose expression is temporally and spatially restricted to oocytes. All three proteins are required for the formation of the extracellular zona matrix and female mice with a single disrupted zona gene lack a zona and are infertile. An E-box (CANNTG), located approximately 200 bp upstream of the transcription start sites of Zp1, Zp2 and Zp3, forms a protein-DNA complex present in oocytes and, to a much lesser extent, in testes. It has been previously shown that the integrity of this E-box in Zp2 and Zp3 promoters is required for expression of luciferase reporter genes microinjected into growing oocytes. The presence of the ubiquitous transcription factor E12 in the complex was used to identify a novel basic helix-loop-helix protein, FIGalpha (Factor In the Germline alpha) whose expression was limited to oocytes within the ovary. The ability of FIGalpha to transactivate reporter genes coupled to each of the three mouse zona promoters in heterologous 10T(1/2) embryonic fibroblasts suggests a role in coordinating the expression of the three zona pellucida genes during oogenesis.

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The Basic-Region Helix-Loop-Helix Transcription Factor DevR Significantly Affects Polysaccharide Metabolism inAspergillus oryzae

Applied and Environmental Microbiology ◽

10.1128/aem.00089-19 ◽

2019 ◽

Vol 85 (8) ◽

Cited By ~ 3

Author(s):

Miao Zhuang ◽

Zhi-Min Zhang ◽

Long Jin ◽

Bao-Teng Wang ◽

Yasuji Koyama ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Oryzae ◽

Bhlh Transcription Factor ◽

Regulation Mechanism ◽

Starch Metabolism ◽

Poor Growth ◽

Content Type ◽

Helix Loop Helix ◽

Amylase Production ◽

Basic Region

ABSTRACTBasic-region helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that are often involved in the control of growth and differentiation. Recently, it was reported that the bHLH transcription factor DevR is involved in both asexual and sexual development inAspergillus nidulansand regulates the conidial melanin production inAspergillus fumigatus. In this study, we identified and characterized anAspergillus oryzaegene that showed high similarity withdevRofA. nidulansandA. fumigatus(AodevR). In the AodevR-disrupted strain, growth was delayed and the number of conidia was decreased on Czapek-Dox (CD) minimal agar plates, but the conidiation was partially recovered by adding 0.6 M KCl. Simultaneously, the overexpression of AodevRwas induced and resulted in extremely poor growth when the carbon source changed from glucose to polysaccharide (dextrin) in the CD agar plate. Scanning electron microscopy (SEM) indicated that the overexpression of AodevRresulted in extremely thin aberrant hyphal morphology. Conversely, the deletion of AodevRresulted in thicker hyphae and in more resistance to Congo red relative to the control strain. Quantitative reverse transcriptase PCR (RT-PCR) further indicated that AoDevR significantly affects chitin and starch metabolism, and importantly, the overexpression of AodevRinhibited the expression of genes related to starch degradation. A yeast one-hybrid assay suggested that the DevR protein possibly interacted with the promoter ofamyR, which encodes a transcription factor involved in amylase production. Importantly, AoDevR is involved in polysaccharide metabolism and affects the growth of theA. oryzaestrain.IMPORTANCEAspergillus oryzaeis an industrially important filamentous fungus; therefore, a clear understanding of its polysaccharide metabolism and utilization is very important for its industrial utilization. In this study, we revealed that the basic-region helix-loop-helix (bHLH) transcription factor AoDevR is importantly involved in chitin and starch metabolism inA. oryzae. The overexpression of AodevRstrongly suppressed the expression of amylase-related genes. The results of a yeast one-hybrid assay suggested that the DevR protein potentially interacts with the promoter ofamyR, which encodes a transcription factor involved in amylase production and starch utilization. This study provides new insight for further revealing the regulation mechanism of amylase production inA. oryzae.

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Sclerotome-related helix-loop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its level is enhanced by type-β transforming growth factor

Journal of Endocrinology ◽

10.1677/joe.0.1510491 ◽

1996 ◽

Vol 151 (3) ◽

pp. 491-499 ◽

Cited By ~ 20

Author(s):

Y Liu ◽

P Cserjesi ◽

A Nifuji ◽

E N Olson ◽

M Noda

Keyword(s):

Transcription Factor ◽

Growth Factor ◽

Transcriptional Activity ◽

Transforming Growth Factor ◽

Binding Activity ◽

The Novel ◽

Helix Loop Helix ◽

Nuclear Extracts ◽

E Box ◽

First Time

Abstract Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ. Journal of Endocrinology (1996) 151, 491–499

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A helix-loop-helix protein related to the immunoglobulin E box-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4384 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4384-4388 ◽

Cited By ~ 108

Author(s):

C S Carr ◽

P A Sharp

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Heavy Chain ◽

Binding Proteins ◽

Immunoglobulin E ◽

Expression Library ◽

Helix Loop Helix ◽

E Box ◽

Late Transcription ◽

Novel Protein

A human cDNA encoding a novel protein in the helix-loop-helix family has been isolated by screening a bacteriophage expression library with a probe containing the binding site for major late transcription factor. The protein encoded by this cDNA, TFEB, probably recognizes E-box sequences in the heavy-chain immunoglobulin enhancer.

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A helix-loop-helix protein related to the immunoglobulin E box-binding proteins

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4384-4388.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4384-4388

Author(s):

C S Carr ◽

P A Sharp

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Heavy Chain ◽

Binding Proteins ◽

Immunoglobulin E ◽

Expression Library ◽

Helix Loop Helix ◽

E Box ◽

Late Transcription ◽

Novel Protein

A human cDNA encoding a novel protein in the helix-loop-helix family has been isolated by screening a bacteriophage expression library with a probe containing the binding site for major late transcription factor. The protein encoded by this cDNA, TFEB, probably recognizes E-box sequences in the heavy-chain immunoglobulin enhancer.

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Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3449-3459.1992 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3449-3459

Author(s):

A L Nielsen ◽

N Pallisgaard ◽

F S Pedersen ◽

P Jørgensen

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Transcriptional Activator ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Nih 3T3 ◽

Murine Leukemia

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

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Intramolecular Regulation of MyoD Activation Domain Conformation and Function

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5478 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5478-5484 ◽

Cited By ~ 22

Author(s):

Jing Huang ◽

Hal Weintraub ◽

Larry Kedes

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Helix Loop Helix ◽

Dna Binding Specificity ◽

E Box ◽

And Function ◽

Bhlh Proteins ◽

Basic Region

ABSTRACT The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries a mutation of Ala-114. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that unmasks the activation domain. Here we demonstrate that an A114N mutant exhibits an altered conformation in the basic region and that this local conformational difference can lead to a more global change affecting the conformation of the activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins among many with diverse functions yet with DNA binding specificities known to be similar.

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Structural basis for preferential binding of human TCF4 to DNA containing 5-carboxylcytosine

Nucleic Acids Research ◽

10.1093/nar/gkz381 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8375-8387 ◽

Cited By ~ 6

Author(s):

Jie Yang ◽

John R Horton ◽

Jia Li ◽

Yun Huang ◽

Xing Zhang ◽

...

Keyword(s):

Transcription Factor ◽

Positive Impact ◽

Minor Groove ◽

Negative Influence ◽

Autism Spectrum ◽

Structural Basis ◽

Major Groove ◽

E Box ◽

Arginine Residues ◽

Basic Region

Abstract The psychiatric risk-associated transcription factor 4 (TCF4) is linked to schizophrenia. Rare TCF4 coding variants are found in individuals with Pitt-Hopkins syndrome—an intellectual disability and autism spectrum disorder. TCF4 contains a C-terminal basic-helix-loop-helix (bHLH) DNA binding domain which recognizes the enhancer-box (E-box) element 5′-CANNTG-3′ (where N = any nucleotide). A subset of the TCF4-occupancy sites have the expanded consensus binding specificity 5′-C(A/G)-CANNTG-3′, with an added outer Cp(A/G) dinucleotide; for example in the promoter for CNIH3, a gene involved in opioid dependence. In mammalian genomes, particularly brain, the CpG and CpA dinucleotides can be methylated at the 5-position of cytosine (5mC), and then may undergo successive oxidations to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) forms. We find that, in the context of 5′-0CG-1CA-2CG-3TG-3′(where the numbers indicate successive dinucleotides), modification of the central E-box 2CG has very little effect on TCF4 binding, E-box 1CA modification has a negative influence on binding, while modification of the flanking 0CG, particularly carboxylation, has a strong positive impact on TCF4 binding to DNA. Crystallization of TCF4 in complex with unmodified or 5caC-modified oligonucleotides revealed that the basic region of bHLH domain adopts multiple conformations, including an extended loop going through the DNA minor groove, or the N-terminal portion of a long helix binding in the DNA major groove. The different protein conformations enable arginine 576 (R576) to interact, respectively, with a thymine in the minor groove, a phosphate group of DNA backbone, or 5caC in the major groove. The Pitt-Hopkins syndrome mutations affect five arginine residues in the basic region, two of them (R569 and R576) involved in 5caC recognition. Our analyses indicate, and suggest a structural basis for, the preferential recognition of 5caC by a transcription factor centrally important in brain development.

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