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PubMed1996

Molecular cloning of a novel basic helix-loop-helix protein from the rat brain.

In the mammalian nervous system, several basic helix-loop-helix (bHLH) proteins have been identified that may play essential roles in neurogenesis or neural development. This paper describes a novel bHLH protein in rat brain, which has a bHLH domain highly homologous with that of MATH-2 and NeuroD. On the other hand, the amino-acid sequence of the other regions had little homology with those of the known proteins. This protein consists of 381 amino acids and was detected only in neural tissue, indicating that it has an important role specific to neuronal activities.

Amino Acid Sequence↗

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↗

PubMed1997

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage.

Basic helix-loop-helix (bHLH)/PAS proteins, such as Sim, act as transcriptional factors, playing a critical role in the control of central nervous system (CNS) development. To isolate novel bHLH/PAS factors in the CNS an iterative search of a database for expressed sequence tags (ESTs) resulted in the location of several bHLH/PAS protein-like sequences. The rapid amplification of cDNA end (RACE) method was applied to isolate full-length cDNAs of these ESTs. Several 5' and 3' terminal sequences were isolated using primers derived from an EST from the human brain cDNA library. The predicted novel factor polypeptide had bHLH and PAS domains that were highly homologous with those of Ah receptor nuclear translocator (Arnt) and Arnt2. Combination of the isolated cDNA fragments revealed the existence of several alternatively spliced variants. The distribution of the novel bHLH/PAS factor message was analyzed by Northern blot hybridization. This detected only one transcript, which was 2.9 kb in size. Strong hybridization was found in the brain, skeletal muscle and heart. Expression of the novel bHLH/PAS factor, brain and muscle Arnt-like protein 1 (BMAL1), was different from that of Arnt and Arnt2, suggesting that BMAL1 has a different function in the CNS and muscle than Arnt and Arnt2.

ARNTL Transcription Factors↗

PubMed1997

cDNA cloning and expression analysis of NeuroD mRNA in human retina.

We have shown that bHLH proteins are involved in mammalian retinal development. Here we report the identification and analysis of the expression of a neurogenic differentiation gene, NeuroD, in human retina. In situ hybridization and immunocytochemical analyses of adult retina showed that NeuroD transcripts and NeuroD immunoreactivity are predominantly localized to the outer nuclear layer which contains the photoreceptors. Southern analysis of PCR-amplified cDNA revealed that NeuroD mRNA is also expressed in fetal human retina. Fetal monkey retina was used to analyse the spatial distribution of NeuroD in the developing retina. Both NeuroD transcripts and immunoreactivity are largely detected in the outer neuroblastic layer. Therefore, NeuroD may be involved in the differentiation as well as maintenance of the differentiated properties of photoreceptors.

Adult↗

PubMed1997

Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes.

The differentiation of human embryo chondrocytes was markedly induced by the addition of Bt2cAMP to the culture medium. Using this culture system, a novel human cDNA for a basic helix-loop-helix (bHLH) protein (named DEC1) expressed primarily in the chondrocytes in response to Bt2cAMP was cloned by the subtractive hybridization method. DEC1 protein consists of 412 amino acids and exhibits structural similarities to the mammalian HES family, Drosophila hairy, and Enhancer of split m7 in the bHLH region. Northern blot analysis showed that DEC1 mRNA was expressed in various tissues including the cartilage, lung, spleen, and intestine, but not in the brain. These findings suggest that the bHLH factor DEC is involved in the control of cell differentiation in several tissues including cartilage.

Amino Acid Sequence↗

PubMed1998

Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock.

We isolated a cDNA clone encoding a polypeptide of 626 amino acids containing basic helix-loop-helix (bHLH) and PAS domains from a mouse cDNA library of P19 cells. This protein, termed Arnt3, showed the highest similarity to Arnt and Arnt2 in the bHLH and PAS regions. Arnt3 mRNA was expressed in brain, skeletal muscle, 13.5-day embryos, and P19 cells treated with retinoic acid. The partner PAS proteins of Arnt3 were searched for by the two-hybrid system in yeast, and HIF-1 alpha, HLF, and Clock among various bHLH/PAS proteins were found. Gel mobility shift analysis using nuclear extracts from 293T cells cotransfected with Arnt3 and HIF-1 alpha (or HLF) expression plasmids revealed that these complexes specifically bound the hypoxia-response element (HRE). Coexpression of Arnt3 and HIF-1 alpha (or HLF) in Arnt-deficient c4 cells enhanced transcription of a reporter gene driven by the HRE sequences. We also showed that Arnt3 contained an activation domain at the C-terminal region and a repression domain between the PAS-A and PAS-B regions.

ARNTL Transcription Factors↗

PubMed1998

Circadian oscillation of BMAL1, a partner of a mammalian clock gene Clock, in rat suprachiasmatic nucleus.

A superfamily gene which encodes a bHLH (basic helix-loop-helix)/PAS transcription factor, BMAL1, was cloned and sequenced from rat cDNA. A robust circadian rhythm of rat BMAL1 expression was detected by in situ hybridization in the suprachiasmatic nucleus (SCN), the site of the circadian clock, with the highest level at the subjective night. Less prominent and completely reversed circadian rhythms of rBMAL1 mRNA were observed in the piriform and parietal cortices. The hybridization signals of rBMAL1 mRNA were also detected in the olfactory bulb, hippocampus, and cerebellum. Since the product of rBMAL1 was recently demonstrated to dimerize with the protein of a mammalian clock gene, Clock, and the protein complex was shown to bind the E Box in the promoter region of mPer1 (a mouse homologue to Drosophila clock gene, Per), rBMAL1 possibly plays a critical role in the clock mechanism generating the circadian oscillation in rats.

ARNTL Transcription Factors↗

PubMed1999

Expression of the dominant-negative regulator Id4 is induced during adipocyte differentiation.

Expression of the Id4 gene was investigated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Id4 is a member of a family encoding non-DNA binding helix-loop-helix proteins proposed to inhibit the activity of basic HLH (bHLH) proteins important in many developmental processes. We show here that Id4 expression is low in confluent preadipocytes and rapidly induced by treatment with the combination of hormones which causes differentiation into mature adipocytes. Id4 expression is also induced by treatment with individual hormones, especially dexamathasone. Id4 mRNA can be detected in mouse and human adipose tissue. Genes encoding E-proteins (bHLH proteins known to interact with and be regulated by Id proteins) are expressed and regulated during differentiation in 3T3-L1 cells. These data suggest that the Id4 transcriptional regulator is playing a role in adipose cell differentiation and suggest that DNA-binding HLH proteins may also be important in regulation of differentiation of these cells.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.

In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.

Amino Acid Sequence↗

PubMed1999

Characterization of three splice variants and genomic organization of the mouse BMAL1 gene.

The BMAL1 gene encodes a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) family of transcription factors. It is a key regulator of circadian rhythms. Using sequence information from human BMAL1 (hBMAL1) cDNAs previously reported by our laboratory, we have isolated and characterized cDNAs encoding three splice variants of the mouse BMAL1 (mBMAL1) gene. Of the three splice variants, mBMAL1b extends for 1878 bp in the coding sequence, which is 91% identical to that of hBMAL1b; its deduced amino acid sequence is 626 residues long and is 98% identical to that of hBMAL1b, and sequence identities in the bHLH, PAS-A, and PAS-B regions are 98, 100, and 100%, respectively. mBMAL1b' arises from alternative usage of exon 2, which results in a 7-amino-acid insertion and alternative splice acceptor usage at the intron 9/exon 10 splice junction, which causes an alanine residue deletion. mBMAL1b' encodes 632 amino acids and contains the bHLH/PAS domains. mBMAL1g' is generated by alternative splice acceptor usage at the intron 6/exon 7 splice junction, which results in a 28-bp deletion adjacent to the 5' end of the PAS domain. Since the 28-bp deletion shifts the reading frame, mBMAL1g' is predicted to encode a product of only 222 amino acids that lacks the PAS domain. The tissue distributions of the three splice variants showed some variation. The variations in the tissue distributions and predicted amino acid sequences suggest that the three splice variants may have different functions. Direct sequencing of the genomic mBMAL1 clones indicated that the coding sequence of mBMAL1 spans 32 kb and includes 17 exons. An unusual exon/intron donor sequence was found in intron 14, which begins with GC at the 5' end. Comparison with the bHLH/PAS family genes revealed that the intron/exon splice pattern of mBMAL1 most closely matches that of the mAhr, which suggests that BMAL1 and Ahr belong to the same subclass and may be derived from a common primordial gene.

ARNTL Transcription Factors↗

PubMed1999

Transcriptional activation of the myogenin gene by MEF2-mediated recruitment of myf5 is inhibited by adenovirus E1A protein.

The basic helix-loop-helix (bHLH) transcription factor myogenin plays a crucial role in terminal differentiation of committed myoblasts into mature myocytes. Transcriptional activation of the myogenin gene requires coordinate action of myocyte enhancer factor 2 (MEF2) proteins and the myogenic bHLH regulators, MyoD or Myf5. Here we show that transcription of the myogenin gene in differentiated cells correlates with MEF2 and NF1 binding to their cognate sites in the proximal myogenin promoter but not with binding of Myf5 or MyoD to the E-box. The importance of MEF2 activity was further demonstrated by expression of antisense MEF2 RNA which repressed MEF2 and Myf5-mediated MEF2 site-dependent reporter gene activation and the synergistic transactivation of a myogenin CAT reporter by Myf5 and MEF2. Adenovirus E1A which has previously been shown to specifically interfere with myogenin gene transcription also inhibited the cooperative transactivation by Myf5/MEF2 and MEF2. Consistently, coimmunoprecipitation studies revealed impaired MEF2/Myf5 protein-protein interactions. These results support a model of transcriptional activation and stabilization of myogenin expression in which DNA-bound MEF2 recruits myogenic bHLH factors into an active but E1A-sensitive transcription factor complex.

Adenovirus E1A Proteins↗

PubMed2000

Rhythmic expression of BMAL1 mRNA is altered in Clock mutant mice: differential regulation in the suprachiasmatic nucleus and peripheral tissues.

BMAL1 is a putative clock gene which encodes a basic helix-loop-helix (bHLH)-PAS transcription factor. To examine whether the CLOCK protein is required for the circadian expression of BMAL1 mRNA, in situ hybridization and Northern blot analysis were performed in the suprachiasmatic nucleus (SCN) and peripheral tissues of homozygous Clock mutant mice. In the SCN of Clock mutants, BMAL1 mRNA did not oscillate significantly but apparently expressed with low levels, while in wild-type mice the mRNA was robustly oscillated in a circadian manner. The peak-trough amplitudes of BMAL1 mRNA levels were 6.5-, 8.6-, and 6.7-fold in liver, heart, and kidney of wild-type mice, respectively. In Clock mutants, the amplitudes were extremely damped to 1.2-, 2.1-, and 1.4-fold, respectively. Furthermore, expressions of BMAL1 mRNA in the peripheral of Clock mutant mice were close to the peak level in wild-type mice, whereas mPer2 mRNA levels were severely blunted at trough values. Daily expression of albumin site D-binding protein (DBP), a clock controlled output gene (CCG), was also abolished at trough values by the Clock mutation in all tissues examined. These observations suggest that the circadian expression of BMAL1 mRNA is affected by the CLOCK-induced transcriptional feedback loop in the SCN and peripheral tissues in a different way and that the regulation mechanism appeared to be different from those in mPer2 and DBP expressions in vivo.

ARNTL Transcription Factors↗

PubMed2000

A cysteine residue in the helix-loop-helix domain of Id2 is critical for homodimerization and function.

Id proteins are negative regulators of basic helix-loop-helix (bHLH) transcription factors. In this study, we compared the expression of Id2 mRNA in proliferating (fetal) and nonproliferating (adult) alveolar epithelial cells (AECs). The expression of Id2 was higher in adult AECs than in the corresponding fetal cells, suggesting that Id2 might play a functional role in developmental regulation of lung epithelial cell proliferation. By screening a mouse embryo cDNA library in the yeast two-hybrid system, Id2 was identified as a self-associating protein. Structural analysis by deletion and site-directed mutagenesis demonstrated that the HLH domain and a cysteine residue within the HLH domain are essential for Id2 homodimerization. Furthermore, in vitro synthesized Id2 homodimers became monomers under reducing conditions, indicating that the formation of an intermolecular disulfide bond is critical for Id2 homodimerization. Transient transfection assays in A549 cells showed that wild-type Id2 down-regulated the activity of the cyclin A promoter by 70%, while mutating the cysteine critical for Id2 homodimerization abolished the inhibitory effect of wild-type Id2.

Animals↗

PubMed2000

HASH-1 and E2-2 are expressed in human neuroblastoma cells and form a functional complex.

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homolog-1 (MASH-1 in mouse and HASH-1 in human) is essential for proper development of olfactory and most peripheral autonomic neurons, and for the formation of distinct neuronal circuits within the central nervous system. We have previously shown that HASH-1 is expressed in neuroblastoma tumors and cell lines, and in this study we have used the yeast two-hybrid system to isolate HASH-1 interacting proteins from a human neuroblastoma cDNA library. Two of the isolated clones contained cDNA from the E2-2 gene (also known as ITF2/SEF2-1). We show that E2-2 interacts with HASH-1 in both yeast and mammalian cells. The HASH-1/E2-2 complex binds an E-box (CACCTG) in vitro, and transactivates an E-box containing reporter construct in vivo. Furthermore, E2-2 seems to be one of the major HASH-1 interacting proteins in extracts from neuroblastoma cells. In conclusion, E2-2 forms a functional complex with HASH-1, and might therefore be involved in the development of specific parts of the central and peripheral nervous systems.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2000

cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2: its mRNA expression, subcellular distribution, and chromosomal localization.

We isolated a human cDNA encoding a novel member of the bHLH-PAS transcription factor superfamily, BMAL2, which is highly similar to, but distinct from, BMAL1. The composite cDNA covered a 1720-bp sequence consisting of a putative 1653-bp open reading frame encoding a polypeptide of 551 amino acids. The deduced BMAL2 product contains a bHLH-PAS domain in its N-terminal region and a variable C-terminus. The overall identity of BMAL2 polypeptide to that of human BMAL1 is 49%. RNA analysis revealed that expression of BMAL2 transcripts was restricted to the fetal brain and to the adult liver in human, while human BMAL1 mRNA was expressed in the brain and skeletal muscle. The chromosomal localization of the human BMAL2 gene was determined by fluorescent in situ hybridization to be localized on chromosome 12 at region p12.2-p11.2. These results suggest that BMAL2 may play different roles from BMAL1 in the embryonic brain and in adult mammals.

Adult↗

PubMed2000

Genomic organization, chromosomal localization, and the complete 22 kb DNA sequence of the human GCMa/GCM1, a placenta-specific transcription factor gene.

The genomic sequence of the human GCMa/GCM1 gene, a mammalian homologue of Drosophila melanogaster GCM, was determined. Drosophila GCM is a neural transcription factor that regulates glial cell fate. The mammalian homolog however, is a placenta-specific transcription factor that is necessary for placental development. The 22 kb DNA sequence spanning the GCMa gene contains six exons and five introns, encoding a 2.8 kb cDNA. Overall genomic organization is similar for the human and mouse. Several potential binding sites for transcription factors like GATA, Oct-1, and bHLH proteins were found in the 5'-flanking region of the human gene. A DNA motif for GCM protein binding exists in the 5'-flanking region that is highly homologous with that of the mouse gene. The location of this gene was mapped to chromosome 6 using fluorescence in situ hybridization.

Animals↗

PubMed2001

Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins.

DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.

Amino Acid Sequence↗

PubMed2001

Cloning of hypoxia-inducible factor 1alpha cDNA from chick embryonic ventricular myocytes.

Hypoxia-inducible factor 1 (HIF-1) is composed of HIF-1alpha and arylhydrocarbon nuclear receptor translocator (ARNT), which belong to the basic-helix-loop-helix-Per/ARNT/Sim (bHLH-PAS) family of transcription factors. HIF plays key roles in oxygen homeostasis and embryonic cardiovascular development. In this study, we have cloned cDNAs encoding the chick HIF-1alpha from cultured embryonic ventricular myocytes (CEVM) and then examined its expression in various embryonic tissues. The deduced amino acid sequence of the chick HIF-1alpha cDNA showed 79% identity with that of the human HIF-1alpha cDNA. In contrast, sequence homology between the chick HIF-1alpha and endothelial PAS protein 1 (EPAS1), another member of the bHLH-PAS proteins, was only low (49%). HIF-1alpha mRNA was expressed abundantly in CEVM, but scarcely in the liver, which was quite different from expression pattern of EPAS1 mRNA. These data suggest that HIF-1alpha may be involved in embryonic cardiovascular development. HIF-1alpha and EPAS1 may play distinct roles during developmental stages.

Amino Acid Sequence↗