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Results for “Basic helix-loop-helix (bHLH)”

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PubMed2001

Ectopic expression of negative ARNT2 factor disrupts fish development.

ARNT factors are a cluster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.

Amino Acid Sequence↗

PubMed2001

Expression and characterization of hypoxia-inducible factor (HIF)-3alpha in human kidney: suppression of HIF-mediated gene expression by HIF-3alpha.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS superfamily. Here we examined the expression of HIF-alpha subunit proteins in the human kidney and found that, in addition to HIF-1alpha and -2alpha, HIF-3alpha was also expressed. The sequence analysis revealed that, like mouse HIF-3alpha, human HIF-3alpha has high similarity with HIF-1alpha and -2alpha in the bHLH and PAS domains, but lacks structures for transactivation found in the C-terminus of HIF-1alpha and -2alpha. Furthermore, we performed reporter gene analysis and showed that HIF-3alpha suppresses hypoxia-inducible HIF-mediated gene expression. HIF-3alpha might be a negative regulator of hypoxia-inducible gene expression in the human kidney.

Amino Acid Sequence↗

PubMed2002

The basic helix-loop-helix domain of the E47 transcription factor requires other protein regions for full DNA binding activity.

Most transcription factors are believed to be composed of independently functioning modules [A. D. Frankel and P. S. Kim (1991) Cell 65, 717-719]. Basic helix-loop-helix (bHLH) transcription factors appear to fit this model, with the HLH domains mediating dimerization, the basic regions mediating DNA binding, and other modules controlling other functions such as transcriptional activation. We tested predictions of this model using forced dimers of bHLH proteins including E47 homodimers and MyoD:E47 heterodimers and found that protein dimers containing complete bHLH domains but lacking other regions of E47 have only 20% of the DNA binding ability and transcriptional transactivation activity of wild-type dimers. These results demonstrate that the bHLH domains do not function as completely independent DNA binding modules. In addition, these results demonstrate that the transcriptional activation domains from a single bHLH protein are sufficient to activate transcription.

3T3 Cells↗

PubMed2002

Dominant-negative HMGA1 blocks mu enhancer activation through a novel mechanism.

The immunoglobulin mu intronic enhancer is a potent B cell-specific transcriptional activator. The enhancer is activated by the appropriate combination of transcription factors, amongst which are ets and bHLH proteins. HMGA1 (formerly HMG-I(Y)) is a demonstrated co-activator of the mu enhancer. HMGA1 functions through direct interaction with PU.1, one of the ets proteins critical for enhancer activation. New data demonstrates dominant negative HMGA1 dramatically decreases enhancer activity in B cells. EMSA analysis demonstrated that DN HMGA1 disrupts established PU.1/mu enhancer binding. Similarly, DN HMGA1 blocks mu enhancer binding by Ets-1. In sharp contrast, DN HMGA1 had no effect on binding activity of the ETS DNA binding domains of either PU.1 or Ets-1, or the bHLH-zip protein TFE3, suggesting specificity. Taken together, the data suggest that DN HMGA1 utilizes a novel mechanism to specifically block interaction between ets proteins and mu enhancer DNA, suggesting DN HMGA1 represents a new, highly specific means of regulating mu enhancer activity.

Animals↗

PubMed1994

The E box is essential for activity of the cardiac actin promoter in skeletal but not in cardiac muscle.

The sequence CANNTG (E box) is frequently found in the promoters of muscle-specific genes and binds members of the basic helix-loop-helix (bHLH) family of transcription factors. We compared the need for the E box in the expression of a muscle-specific promoter normally expressed in both cardiac and skeletal muscle. The E box was mutated in a construct of the cardiac alpha-actin promoter driving the Escherichia coli lacZ gene. The wild-type and mutant constructs were transfected and stably integrated into the genomes of P19 embryonal carcinoma cells. The wild-type promoter was expressed in both cardiac and skeletal myocytes. The promoter lacking an E box was expressed in cardiac but not in skeletal muscle. Neither promoter was active in nonmuscle cells. Thus the E box is not necessary for the cardiac actin promoter activity in P19-derived cardiac muscle but is essential for its activity in skeletal muscle. This result is consistent with our inability to detect cardiac muscle-specific members of the MyoD family of bHLH transcription factors.

Actins↗

PubMed1995

Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family have been shown to control differentiation of a variety of cell types. Tissue-specific bHLH proteins dimerize preferentially with ubiquitous bHLH proteins to form heterodimers that bind the E-box consensus sequence (CANNTG) in the control regions of target genes. Using the yeast two-hybrid system to screen for tissue-specific bHLH proteins, which dimerize with the ubiquitous bHLH protein E12, we cloned a novel bHLH protein, named Dermo-1. Within its bHLH region, Dermo-1 shares extensive homology with members of the twist family of bHLH proteins, which are expressed in embryonic mesoderm. During mouse embryogenesis, Dermo-1 showed an expression pattern similar to, but distinct from, that of mouse twist. Dermo-1 was expressed at a low level in the sclerotome and dermatome of the somites, and in the limb buds at Day 10.5 post coitum (p.c.), and accumulated predominantly in the dermatome, prevertebrae, and the derivatives of the branchial arches by Day 13.5 p.c. As differentiation of prechondrial cells proceeded, Dermo-1 expression became restricted to the perichondrium. Expression of Dermo-1 increased continuously in the dermis through Day 17.5 p.c. and was also detected in the dermis of neonates, but became downregulated in adult tissues. The Dermo-1 protein bound the E-box consensus sequence in the presence of E12, but transcriptional activity was not detectable. Instead, Dermo-1 repressed transcriptional activity of myogenic bHLH proteins. The expression pattern of Dermo-1 suggests that it functions as a regulator of gene expression in a subset of mesenchymal cell lineages including developing dermis.

Amino Acid Sequence↗

PubMed1995

Paraxis: a basic helix-loop-helix protein expressed in paraxial mesoderm and developing somites.

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome, and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic helix-loop-helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. Here we report the cloning of a bHLH protein, called paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in its amino and carboxyl termini. During mouse embryogenesis, paraxis transcripts are first detected at about Day 7.5 postcoitum within primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated in the myotome. Paraxis and scleraxis are coexpressed in the sclerotome, but paraxis expression declines soon after sclerotome formation, whereas scleroaxis expression increases in the sclerotome and its derivatives. The sequential expression of paraxis and scleraxis in the paraxial mesoderm and somites suggests that these bHLH proteins may comprise part of a regulatory pathway involved in patterning of the paraxial mesoderm and in the establishment of somitic cell lineages.

Amino Acid Sequence↗

PubMed1995

Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development.

We employed the yeast two-hybrid technique to screen a mouse embryo cDNA library for novel tissue-specific Class B basic helix-loop-helix (bHLH) transcription factors, which heterodimerize with the ubiquitously expressed Class A bHLH protein E12. From this screen, we cloned a novel bHLH protein, which we named eHAND. Its low sequence identity with other bHLH family members and unique expression pattern during development suggest that eHAND defines a new subclass of Class B bHLH proteins. eHAND was expressed at high levels in trophoblast cells and extraembryonic membranes throughout development. The first site of eHAND expression in embryos was the heart, where it was expressed at high levels between 8.5 and 10.5 days post coitum (d.p.c.), after which transcript levels declined abruptly. By 13.5 d.p.c., eHAND expression in the heart was localized to regions of valve formation. Expression in other regions of the embryo was confined to tissues with a substantial neural crest component. eHAND was expressed in the first branchial arch and its derivatives, in the sympathoadrenal lineage, and in the enteric systems. The expression pattern of eHAND during development is distinct from that of other bHLH genes and suggests that it has a role in formation of extraembryonic tissues, heart, and neural crest derivatives.

Amino Acid Sequence↗

PubMed1996

daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression.

Initiation of Drosophila peripheral nervous system (PNS) development requires the achaete-scute complex (AS-C) and the atonal (ato) genes. The AS-C and ato encode basic helix-loop-helix (bHLH) transcription factors that dimerize in vitro with another bHLH protein, daughterless (da). da has many functions during Drosophila embryonic development, as it is required for proper sex determination, oogenesis, and neurogenesis. Here, we examine the expression and function of da within the developing Drosophila eye. The use of a monoclonal antibody to the Da protein revealed that Da levels are modulated across the developing eye disc. Within the morphogenetic furrow (MF) and photoreceptor cell R8, there is a cell-by-cell correspondence between high levels of Da protein expression and Ato protein expression. Mosaic analysis of adult tissue demonstrates that da function is cell autonomous and required within R2, R3, R4, R5, and R8. Examination of gene expression in da- imaginal disc clones reveals that da regulates Ato expression in the MF, affects the progression of the MF, and is necessary for the reestablishment of the G2 and M phases of the synchronized cell cycle posterior to the MF.

Animals↗

PubMed1996

Identification of novel paired homeodomain protein related to C. elegans unc-4 as a potential downstream target of MASH1.

A novel paired homeodomain protein, PHD1, that is most closely related to C. elegans unc-4 has been identified by a differential RT-PCR method. PHD1 is expressed in a narrow layer adjacent to the ventricular zone of the dorsal spinal cord, immediately following expression of MASH1 but preceding overt neuronal differentiation. Some cells coexpressing MASH1 and PHD1 can be seen, suggesting that these two genes are sequentially activated within the same lineage. In the olfactory sensory epithelium, PHD1 expression not only follows but is dependent upon MASH1 function, suggesting that PHD1 acts downstream of MASH1. A sequential action of bHLH and paired homeodomain proteins is apparent in other neurogenic lineages and may be a general feature of both vertebrate and invertebate neurogenesis.

Amino Acid Sequence↗

PubMed1996

Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors.

Tissue-specific bHLH proteins play important roles in the specification and differentiation of neural cell lineages in invertebrate and vertebrate organisms. Two groups of bHLH proteins, atonal and achaete-scute, have proneural activities in Drosophila, and the mouse achaete-scute homolog MASH1 is required for the differentiation of several neural lineages. In a screen for proteins interacting with MASH1, we have isolated a novel bHLH protein related to atonal, named MATH4A, which is broadly expressed in neural precursor cells in the mouse embryonic CNS and PNS. Interaction assays in yeast and in vitro demonstrate that MATH4A interacts efficiently with both MASH1 and the ubiquitous bHLH protein E12. MATH4A-E12 heterodimers, but not MATH4A-MASH1, bind to a consensus E-box sequence. Math4A expression is restricted to undifferentiated neural precursors and is complementary to that of Mash1 in most regions of the nervous system. In particular, Math4A is transcribed at high levels in the cerebral cortex, dorsal thalamus, and epibranchial placodes, which present little or no Mash1 expression. However, expression of the two genes shows limited overlap in certain CNS regions (retina, preoptic area of the hypothalamus, midbrain, hindbrain). Its structure and expression pattern suggest that MATH4A may regulate an early step of neural development, either as a partner of ubiquitous bHLH proteins or associated with other neural-specific bHLH proteins.

Amino Acid Sequence↗

PubMed1997

Misexpression of nautilus induces myogenesis in cardioblasts and alters the pattern of somatic muscle fibers.

nautilus (nau), one member of the myogenic regulatory family of bHLH-encoding genes, is expressed in a subset of muscle precursors and differentiated fibers in the Drosophila embryo. To elucidate the role of nautilus in myogenesis, we have misexpressed it using the GAL4-targeted system. We find that ectopic expression results in lethality throughout Drosophila development. We analyzed the effects of embryonic expression in mesodermal tissues that include the cardioblasts of the dorsal vessel as well most, if not all, of the presumptive somatic muscle precursors. Immunohistochemical staining for muscle MHC revealed abnormalities that include an absence of cardial cells, coincident with the appearance of novel muscle fibers adjacent to the dorsal vessel. Moreover, many cardioblasts express increased levels of muscle-specific genes such as myosin, actin 57B, and Mlp60A, a protein that is restricted to the somatic, visceral, and pharyngeal muscles. These data suggest that the missing cardial cells have been transformed into cells with properties similar to those of the somatic muscles. In addition, ubiquitous expression of nautilus in somatic muscle cells of these embryos resulted in muscle pattern defects. Specifically, muscles that do not normally express nautilus were frequently absent, and novel fibers were observed in positions reminiscent of nau-expressing muscles. These data imply that nautilus can alter the developmental program of muscle precursors. In summary, we suggest that nautilus induces myogenic differentiation in vivo when ectopically expressed in developing cardioblasts and may affect the myogenic differentiation program of specific muscle fibers.

Animals↗

PubMed1997

Mist1: a novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern.

Basic helix-loop-helix (bHLH) proteins often belong to a family of transcription factors that bind to the DNA target sequence -CANNTG- (E-box) that is present in the promoter or enhancer regions of numerous developmentally regulated genes. In this study, we report the isolation and initial characterization of a novel bHLH factor, termed Mist1, that was identified by virtue of its ability to interact with E-box regulatory elements in a yeast "one-hybrid" screening procedure. Northern analysis revealed that Mist1 transcripts are expressed in several adult tissues, including stomach, liver, lung, and spleen but no expression is detected in the heart, brain, kidney, or testis. During mouse embryogenesis, Mist1 mRNA is first observed at E10.5 in the primitive gut and in the developing lung bud. Expression persists through E16.5 and remains restricted primarily to the epithelial lining. Mist1 also is detected in skeletal muscle tissues beginning at E12.5, persisting throughout all embryonic stages examined although in older embryos and in the adult expression becomes severely reduced. At later developmental times, Mist1 transcripts also are found in the pancreas, submandibular gland, and adult spleen. As predicted, the Mist1 protein is nuclear and binds efficiently to E-box sites as a homodimer. Mist1 also is capable of binding to E-box elements when complexed as a heterodimer with the widely expressed E-proteins, E12 and E47. Surprisingly, although Mist1 binds to E-boxes in vivo, the Mist1 protein lacks a functional transcription activation domain. These observations suggest that Mist1 may function as a unique regulator of gene expression in several different embryonic and postnatal cell lineages.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of chicken Paraxis: a regulator of paraxial mesoderm development and somite formation.

To investigate the molecular regulation of embryonic somite formation and development, we have cloned the full-length cDNA and characterized the embryonic expression profile of chicken Paraxis, a member of a novel family of basic helix-loop-helix (bHLH) proteins, which has been suggested to play a role in paraxial mesoderm development. Chicken Paraxis encodes a 1.35-kb mRNA and contains a 53-amino-acid residue bHLH domain, identical in sequence to that found in the mammalian Paraxis genes of mouse, hamster, and human. Northern analysis revealed significant Paraxis expression in the early embryo up to the 30- to 35-somite stage, declining from Incubation Day 4 on and becoming undetectable by Day 5. By whole-mount in situ hybridization, Paraxis expression is first seen distinctly in the emerging paraxial mesoderm of the primitive streak stage chick embryo. During gastrulation, Paraxis expression in the mesoderm defines bilaterally symmetric crescents located immediately rostral to Hensen's node and appears to pre-configure the emerging somitic mesoderm. During somite development, Paraxis expression is evident in the rostral segmental plate and the newly formed somites, although the level of expression clearly decreases in the more mature somites. By the 10-12th pair of somites, counting from the caudal end, Paraxis expression appears to be preferentially localized to the medial aspect of individual somites. Histological analysis showed that Paraxis expression is evenly distributed in the newly formed caudal epithelial somites, then localized to the medial portion of maturing somites, and preferentially localized in the dermomyotome of more rostral somites before diminishing to undetectable levels in the most cranial somites. The functional involvement of Paraxis in somite development was assessed by perturbing its expression in somitic stage chick embryos using a Paraxis-specific antisense oligonucleotide. Disruption of somite formation from the paraxial mesoderm was observed in 67% of the surviving topically treated embryos, whereas control embryos treated with sense or random sequence oligonucleotides did not show similar effects. In addition, direct injection of Paraxis-specific antisense oligonucleotide into the paraxial mesoderm produced discrete segmentation anomalies which correlated spatially with the site of injection. Whole-mount in situ hybridization revealed that the regions defective in somite formation displayed perturbed Paraxis expression and a reduction of Pax-1 expression, a marker for epithelial somites and sclerotome. Histological analysis indicated poor condensation and/or epithelization of the somitic mesoderm. Finally, embryos treated with valproic acid, a known teratogen which affects somite segmentation, showed perturbed Paraxis expression, suggesting that the mechanism of action of this teratogen involves a pathway(s) requiring Paraxis activity. These data provide evidence that Paraxis acts as an important regulator of paraxial mesoderm and somite development and functions in axial patterning of the chick embryo.

Amino Acid Sequence↗

PubMed1997

Faithful expression of the Myf-5 gene during mouse myogenesis requires distant control regions: a transgene approach using yeast artificial chromosomes.

Myf-5, a member of the family of four muscle-specific basic helix-loop-helix (bHLH) transcription factors is the first to be expressed in somites, branchial arches, and limb buds during prenatal mouse development. However, little is known about control mechanisms which actually regulate Myf-5 gene activity within these various muscle-forming domains. To identify control regions that contribute to the correct spatiotemporal activity pattern of the Myf-5 gene during mouse embryogenesis, here we report the characterization of yeast artificial chromosomes (YACs) which faithfully direct muscle-specific expression of the gene in chimeric mouse embryos. Forty-five kilobases of sequence 5' to the Myf-5 gene together with 500 kb of 3' flanking DNA drives the correct Myf-5 expression in the mesenchyme of the visceral arches and in somites but not in the hypaxial muscles of limb buds. An additional 50 kb of DNA at the 5' end is required to activate Myf-5 gene expression in developing limbs. These results demonstrate for the first time that unexpectedly distant regions of the Myf-5 gene are necessary to recapitulate its precise developmental expression pattern. We also show that Myf-5 expression in hypaxial muscles and in somites and visceral arches is regulated by separate and distinct far upstream regions. The identification of these remote regulatory elements on YACs carrying the mouse Myf-5 gene constitutes the first important step toward further dissection of the complex mechanisms by which cell-autonomous and external cues control Myf-5 expression during skeletal muscle formation in the mouse embryo.

Animals↗

PubMed1998

The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness.

dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Here we show that although eHAND is asymmetrically expressed along the anterior-posterior and dorsal-ventral embryonic axes, it is symmetrically expressed along the left-right axis at early stages of embryonic and cardiac development. After cardiac looping, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. Previously we showed that dHAND-null mice fail to form a right-sided pulmonary ventricle. Here mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle which is morphologically a left (systemic) ventricle. These data suggest that the HAND genes are involved in development of segments of the heart tube which give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals.

Animals↗

PubMed1998

Mammalian BarH homologue is a potential regulator of neural bHLH genes.

Vertebrate neurogenesis involves sequential actions of transcription factors. neurogenins, encoding Atonal-related bHLH transcription factors, function as neuronal determination genes in Xenopus. neurogenins and antother bHLH factor gene, Mash1, are expressed in distinct subsets or areas of cells giving rise to neurons, suggesting that these genes play important roles to generate distinct populations of neurons. A mammalian homologue of BarH (MBH1) is expressed in a complementary pattern to Mash1 expression in the developing nervous system like neurogenins. Forced expression of MBH1 down-regulates expression of Mash1 and up-regulates neurogenin2/Math4A, a member of neurogenins, in P19 cells during neuronal differentiation. This suggests that MBH1 is a potential regulator of mammalian neural bHLH genes, thereby establishing distinct pathways of neuronal differentiation.

Amino Acid Sequence↗

PubMed1998

cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos.

The segmentation of somites from the paraxial mesoderm is a crucial event in vertebrate embryonic development; however, the mechanisms underlying this process are not well understood. In a yeast two-hybrid screen we have identified the novel basic-helix-loop-helix (bHLH) protein cMeso-1 which is expressed in the presomitic mesoderm of early chicken embryos. Initially the gene is activated in the epiblast and transcripts concentrate later in and around the primitive streak. When the segmental plate is laid down the cMeso-1 expression domain successively retracts toward the caudal end but a second domain appears in bilateral stripes in the anterior paraxial mesoderm. This highly dynamic domain of cMeso-1 transcripts demarcates the area immediately posterior to the next prospective pair of somites in cyclic waves which apparently correspond to the formation of new somites. Loss of cMeso-1 function by antisense RNA or oligonucleotides results in severe attenuation of somitogenesis suggesting that it plays an important role in setting up the segmentation process. The dynamic and periodically reiterated expression of cMeso-1 along the anteroposterior axis is not dependent on anterior structures or the propagation of a signal along the anteroposterior axis but seems to follow an intrinsic patterning program which is already set up in the segmental plate.

Amino Acid Sequence↗