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Results for “Helix-Loop-Helix Motifs”

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PubMed1996

The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts.

Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.

Antigens, Polyomavirus Transforming↗
PubMed1996

Monoclonal antimyogenin antibodies define epitopes outside the bHLH domain where binding interferes with protein-protein and protein-DNA interactions.

We have developed a panel of monoclonal antibodies against rat myogenin, a skeletal muscle regulatory protein of the bHLH family. Some of these monoclonals have been widely used by others, and details of their production are presented. Mapping the epitopes by immunoprecipitation of myogenin deletion mutants demonstrates that this panel recognizes epitopes spanning the entire molecule outside the HLH region. Four antibodies against epitopes outside the bHLH region interfere with the binding of myogenin/E-protein heterodimers to DNA sequences containing the myogenin heterodimer consensus recognition site. Three of these epitopes are partially masked in the heterodimers; antibody binding to these epitopes reduces the interactions between myogenin and E12. This suggests that surfaces outside the HLH dimerization domain may contribute to the stability of myogenin/E12 complexes. The binding of one antibody to its epitope did not appear to affect the myogenin/E12 interaction but nonetheless interfered with the binding of the complex to DNA, suggesting that this epitope lies near to a surface occupied by DNA.

Animals↗
PubMed1997

Developmental restriction of Mash-2 expression in trophoblast correlates with potential activation of the notch-2 pathway.

Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development since overexpression of Mash-2 reduces giant cell formation. We have investigated the role that the Notch signaling pathway may play in trophoblast development. Mash-2 is a homologue of Drosophila achaete/scute complex genes. In Drosophila, activation of the Notch receptor induces transcriptional repressors encoded by the hairy/Enhancer of split (HES) genes, which interact with the Groucho protein to shut off achaete-scute transcription. In the developing mouse placenta, we found that all elements of the Notch pathway were expressed. In particular, the Notch-2, HES-2, and HES-3 genes were coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family were expressed in trophoblasts; TLE3 was expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 was limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development.

Animals↗
PubMed1998

Heart development in Drosophila and vertebrates: conservation of molecular mechanisms.

Vertebrate and insect (Drosophila) hearts look and function quite differently from each other. Nevertheless, during embryogenesis their mesodermal origin and initial assembly into a linear heart tube are comparable in many respects. In the past few years, numerous gene functions have been identified that are utilized by both vertebrates and Drosophila for the specification and differentiation of the heart progenitor cells. These studies have begun with the discovery of the homeobox gene tinman in Drosophila and its vertebrate counterparts. By now, there is also evidence that MEF2 transcription factors and TGF-beta signaling have cardiogenic functions in both these systems. Perhaps in a few years, the GATA and HAND transcription factors and Wnt signaling, which currently only have a demonstrated cardiogenic function in one of the systems, may also be part of this group. One of the pressing but still wide open questions is if the spectrum of targets for these transcription factors and signaling pathways is also conserved.

Animals↗
PubMed1998

Pair-rule gene runt restricts orthodenticle expression to the presumptive head of the Drosophila embryo.

Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizes transcriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid. Here we investigate the relevance of runt's activity as a repressor of otd in normal Drosophila embryogenesis otd expression is activated in the posterior region of embryos that are mutant for runt. This posterior expression domain of otd depends on the activity of the orphan nuclear receptor protein Tailless. Repression of otd by runt does not require the conserved VVVRPY motif that mediates interaction between Runt and the co-repressor protein Groucho. The observed functional interactions between runt and tailless on otd expression may indicate there are other contexts where members of these two families of transcriptional regulators interact to regulate gene expression during development.

Animals↗
PubMed1999

Sequence and expression analysis of Nhlh1: a basic helix-loop-helix gene implicated in neurogenesis.

Nhlh1 is a basic helix-loop-helix (bHLH) gene that has been implicated in mouse neurogenesis. Previous studies have shown it to be expressed in regions in which there are differentiating neurons during late embryonic and fetal development, but detailed studies of the role of Nhlh1 earlier in embryonic development have not been performed. In this paper, we examine the expression of Nhlh1 transcripts at early embryonic stages (E8.5-E10.5), at the onset of neurogenesis, and compare the pattern of expression with that of Islet-1, a marker of postmitotic neurons. We show that Nhlh1 is expressed in early postmitotic neurons but is down-regulated as these cells migrate from the ventricular zone. We have also determined the genomic structure of mouse Nhlh1 and have characterised the promoter sequence, as a first step towards identifying factors that may control Nhlh1 expression. Nhlh1 has been implicated previously as a candidate for the neural tube defect mutant loop-tail (Lp); here, we present sequence and expression data indicating that Nhlh1 is unlikely to be responsible for the Lp mutation.

Amino Acid Sequence↗
PubMed1999

A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.

During human placental development, specialized cells allocated to the extraembryonic lineage (cytotrophoblasts) invade the uterus, anchoring the conceptus to the decidua and tapping a supply of maternal blood. This unusual behavior requires cytotrophoblasts to assume highly specialized characteristics; some are commonly associated with tumor cells, while others are typical of endothelia. Here we investigated the transcriptional mechanisms that control cytotrophoblast differentiation/invasion. Specifically, we examined the cells' expression of a number of transcription factors, at the RNA level, as they differentiated along the invasive pathway in vitro. Since basic helix-loop-helix (bHLH) proteins play important roles in murine trophoblast differentiation, we first examined their expression by cytotrophoblasts. As in murine placental development, expression of the human homologue of Mash-2 was confined to progenitor cells. But expression of Hand-1, which promotes differentiation of murine trophoblast giant cells, was not detected. We also found that cytotrophoblasts upregulated the expression of bHLH/PAS factors that function in adaptive responses to hypoxia, including hEPAS-1, which is expressed primarily in endothelial cells. Quite unexpectedly, we discovered that cytotrophoblasts express high levels of mRNA encoding the human homologue of the Drosophila neuronal fate gene, glial cells missing-1 (gcm-1). We also found evidence of crosstalk between the bHLH and GCM-1 regulatory networks. Together, these results offer insights into the transcriptional mechanisms that govern cytotrophoblast differentiation/invasion. Interestingly, these mechanisms suggest analogies with those that govern differentiation of murine stem cells allocated to both the intra- and extraembryonic lineages.

Animals↗
PubMed1999

Enhancer of split [E(spl)(D)] is a gro-independent, hypermorphic mutation in Drosophila.

Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl).

Alleles↗
PubMed1998

The function of hairy-related bHLH repressor proteins in cell fate decisions.

Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.

Animals↗
PubMed1999

Deconstructing cell determination: proneural genes and neuronal identity.

Vertebrates express scores of bHLH proteins during neural development. Earlier studies inspired by the established role of "proneural" genes in fly neurogenesis, as well as by the vertebrate bHLH myogenic program, focused on the reconstruction of bHLH gene cascades, which are thought to control successive steps leading to neuronal differentiation. Little attention has been paid thus far to the relationship between the diversity of neural bHLH genes and the diversity of neuronal phenotypes. This article reviews recent evidence that, akin to their fly counterparts, vertebrate neural bHLH genes probably confer not only "generic" neuronal properties, but also neuronal type-specific properties, inextricably linking neural determination and the specification of neuronal identity. We also speculate on the relations between positional information and gene activity, and on the evolutionary significance of the diversity of bHLH genes.

Animals↗
PubMed1999

3-methyladenine DNA glycosylases: structure, function, and biological importance.

The genome continuously suffers damage due to its reactivity with chemical and physical agents. Finding such damage in genomes (that can be several million to several billion nucleotide base pairs in size) is a seemingly daunting task. 3-Methyladenine DNA glycosylases can initiate the base excision repair (BER) of an extraordinarily wide range of substrate bases. The advantage of such broad substrate recognition is that these enzymes provide resistance to a wide variety of DNA damaging agents; however, under certain circumstances, the eclectic nature of these enzymes can confer some biological disadvantages. Solving the X-ray crystal structures of two 3-methyladenine DNA glycosylases, and creating cells and animals altered for this activity, contributes to our understanding of their enzyme mechanism and how such enzymes influence the biological response of organisms to several different types of DNA damage.

Animals↗
PubMed2000

The pan-neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes.

Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. Our analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo. genesis 26:77-85, 2000.

Animals↗
PubMed2001

Craniosynostosis and related limb anomalies.

Many genetically determined craniosynostosis syndromes feature limb anomalies, implying that pathways of cranial suture and limb morphogenesis share some identical components. Identification of heterozygous mutations in FGFR1, FGFR2, FGFR3, TWIST and MSX2 in craniosynostosis has focused particular attention on these genes. Here we explore two themes: use of clinical/molecular analysis to provide new clues to pathophysiology and the contrasting effects of loss- and gain-of-function mutations. Apert syndrome is a severe craniosynostosis/syndactyly disorder usually caused by specific substitutions (Ser252Trp or Pro253Arg) in FGFR2. The relative severity of cranial and limb malformations varies in opposite directions for the two mutations, suggesting that these phenotypes arise by different mechanisms. Clinical and biochemical evidence supports a model in which alternative splice forms of FGFR2 mediate these distinct effects. Pro-->Arg substitutions equivalent the Pro253Arg/FGFR2 mutation occur in both FGFR1 and FGFR3, and are also associated with craniosynostosis. This suggests a common pathological mechanism, whereby enhanced affinity for a limited repertoire of tissue-specific ligand(s) excessively prolongs signalling in the cranial suture. The first MSX2 mutation in craniosynostosis was described in 1993 but this remains the only example. We have recently identified three MSX2 mutations associated with a different cranial phenotype, parietal foramina. DNA binding studies show that the craniosynostosis and parietal foramina arise from gain and loss of function, respectively.

Acrocephalosyndactylia↗
PubMed2001

The HIF pathway: implications for patterns of gene expression in cancer.

Regulation of the growth and metabolism of large organisms is tightly constrained by the need for precise oxygen homeostasis. Work on control of the haematopoietic growth factor erythropoietin has led to the recognition of a widespread transcriptional response to hypoxia which provides insights into how this is achieved. The central mediator of this response is a DNA binding complex termed hypoxia inducible factor 1 (HIF-1), which plays a key role in the regulation by oxygen of a large and rapidly growing panel of genes. In cancer, activity of the HIF system is up-regulated both by microenvironmental hypoxia and by genetic changes. The clearest example of genetic activation is seen in the hereditary cancer syndrome von Hippel-Lindau (VHL) disease. In normal cells the product of the VHL tumour suppressor gene targets the regulatory HIF subunits (HIF-1alpha and HIF-2alpha) for oxygen-dependent proteolysis, acting as the substrate recognition component of an E3 ubiquitin ligase. In pVHL defective cells this process is blocked leading to constitutive up-regulation of HIF-1alpha subunits, activation of the HIF complex and overexpression of HIF target genes. Using gene array screens we have defined a large number of VHL-regulated genes. The majority of these show hypoxia-inducible responses, supporting the central involvement of pVHL in gene regulation by oxygen. In addition to known HIF target genes involved in angiogenesis, glucose metabolism and vasomotor control, these new targets include examples with functions in matrix metabolism, apoptosis, carbon dioxide metabolism and secondary cascades of transcriptional control. Thus activation of HIF provides insights into the classical metabolic alterations in cancer cells, and into the mechanisms by which microenvironmental hypoxia might influence tumour behaviour. In the case of VHL disease, this activation can be linked to mutations in a defined tumour suppressor gene. Equally regulation of the HIF-1alpha/pVHL interaction in normal cells should provide insights into the physiological mechanisms operating in cellular oxygen sensing.

Basic Helix-Loop-Helix Proteins↗
PubMed2001

Downregulation of hASH1 is associated with the retinoic acid-induced differentiation of human neuroblastoma cell lines.

BACKGROUND: MASH1, a transcription factor with basic helix-loop-helix domain, has a pivotal function to promote differentiation of neural crest cells into autonomic neurons. PROCEDURE: To investigate the functional significance of human MASH1 (hASH1) in the pathogenesis of neuroblastoma, which is originated from autonomic precursor cells, we studied hASH1 gene expression in primary neuroblastomas and human nueroblastoma cell lines. RESULTS: The follovving results were obtained: (i) hASH1 was expressed in 40 out of 61 (66%) primary neuroblastomas, (ii) hASH1 transcripts were downregulated in several cell lines prior to differentiation induced by all-trans retinoic acid (RA), (iii) a neuroblasotma cell line without expression of endogenous hASH1 did not respond to RA at all, and (iv) the analysis of the hASH1 genomic DNA revealed two possible transcription initiation sites, which may correspond to 3.0 kb and 3.5 kb transcripts. CONCLUSIONS: Our observations suggest that, although hASH1 may not preserve the growth capacity of neuroblastomas, downregulation of hASH1 may be necessary to promote neuronal differentiation of neuroblastoma.

5' Untranslated Regions↗
PubMed1996

Point mutations of the Mxil gene are rare in prostate cancers.

Overexpression of the Myc genes promote cellular transformation. Max protein exerts a pivotal function with the Myc family, and Mxi1/Mad proteins play a positive and negative activity, respectively, on transcription. The function of Mxi1 suggests that it might be a tumor suppressor protein. The Mxi1 gene map to 10p24-25 and deletions of this locus are frequently observed in prostate cancers. The N-terminal helical motif, helix-loop-helix (HLH) and leucine zipper (ZIP) regions of the Mxi1 gene are functionally important. We analyzed most of the coding region of the Mxi1 gene, including these three important regions in 32 prostate cancers and three cell lines by PCR-single strand conformational polymorphism (SSCP). To enrich neoplastic cells, the microdissection was performed on clinical samples. We detected a silent mutation in the HLH region, but no point mutations reflecting the functional change of Mxi1 were found in human prostate cancers. Point mutations of the Mxi1 gene, if they occur, must be minor events in primary prostate cancers.

Basic Helix-Loop-Helix Proteins↗
PubMed2001

Analysis of sequence signature defining functional specificity and structural stability in helix-loop-helix proteins.

Specific functional properties of many proteins directing developmental responses via transcriptional regulation are orchestrated by their characteristic helix-loop-helix (HLH) structural motif. The entire HLH motif in all these proteins assumes a common conformation irrespective of their individual biological effects. The motif controls the affinity of HLH proteins for homo- or heterodimerization, permitting mixing and matching of regulatory factors, and thereby expanding the functional repertoire. Systematic analysis of molecular contacts at the dimer interface using the models built for the functional dimers combined with the pattern of conserved/nonconserved residues within different categories of HLH proteins helped in understanding the differential role played by different residues at the dimer interface for expressing corresponding functions. The residues associated with the self and partner interactions were identified, and the signature residues contributing toward dimeric stability and functional specificity were defined. It is evident that most of the residues involved in self interactions are common among all the HLH proteins. However, while certain residues involved in partner interactions are common among all the HLH proteins, certain others are common within a category, and still others vary widely defining specificity signature at different levels.

Amino Acid Sequence↗
PubMed2000

Expression and subcellular localization of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein in mouse and chicken over developmental time.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic-helix-loop-helix/Per- ARNT-Sim (bHLH/PAS) transcription factor that is involved in multiple signaling pathways. This study focuses on the tissue distribution and subcellular localization of ARNT during embryological development of the mouse and chicken. Two different species were chosen to determine the consistency of the ARNT staining pattern. Immunohistochemical techniques were used to stain sections of embryos over three developmental time points for each species. Mouse tissues evaluated from embryonic day 10.5, 12.5, and 15, exhibited predominant nuclear staining with little change in expression patterns over time. Chicken tissues evaluated from embryonic day 2, 4, and 10 also showed predominant nuclear staining within all cells and little change in expression over developmental time, as well as, low levels of cytoplasmic ARNT staining in some cells. Importantly, in all tissues, the level of ARNT staining within the nuclear compartment was greater than staining observed in the cytoplasm. Thus, the overall conclusions from these studies are that i) the predominant subcellular localization of ARNT protein is nuclear, and ii) that mouse and chicken appear to maintain ARNT protein expression in many cell types over developmental time. These data support vertebrate ARNT as a nuclear transcription factor and a model in which dimerization partners require nuclear localization for interaction.

Animals↗