Invertebrate Arnt homologs

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effectivecontrol of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstratethe need to identify and understand genes for resistance to this class of insect growth regulators.Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to otherclasses of insecticide. Biochemical studies reveal a target-site resistance mechanism, that ofreduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. Thisproperty of reduced JH binding was cytogenetically localized to the Met region on the X chromosomeand can account for the resistance. Possible identities for this binding protein include either anaccessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein invertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

TheMet+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutantalleles, showing that underproduction of the normal gene product can lead to insecticide resistance.Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating thatthe cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix(bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene levelin insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known tobind a variety of environmental toxicants. Met shows three regions of homology to membersof a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PASproteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene hasrecently been cloned, and DARNT has higher homology to vertebrate ARNT than does Met,suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-Aregion (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Metgene is the same as for other bHLH-PAS genes (Ashok, 1998).

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) duringnormal mammalian development or tumor progression. Evidence is presented that a similar complex mediates response to hypoxia in C. elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) alpha and ß subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions,but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. The expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation.Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. It is concluded that the mechanisms of hypoxia signalingare likely conserved among metazoans. Additionally, it is found that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1 (Jiang, 2001).

Although mammalian HIF-1alpha has an essential role in embryonic development, C. elegans hif-1 mutants are viable andfertile when cultured in standard laboratory conditions. This reflects the relatively simple physiology of C. elegans. A mammalian embryo relies on hypoxia-induced angiogenesis to oxygenate tissues. Hif-1alpha -/- mice die by E9.0 with severe vascular defects. In contrast, an adult C. eleganshermaphrodite has no apparent need for specialized respiratory structures or a complex circulatory system. Any cell in the organism is only a few cell widths from the outer surface of the worm or the intestinal lumen. Oxygen sensing and hif-1 function is likely to be very important in the soil environment inhabited naturally by C. elegans. In the laboratory, C. elegans are culturedon top of an agar-based medium, and the ambient oxygen concentrations are relatively high. However, high concentrations of bacteria, the C. elegans food source, can deplete oxygen in a soil microenvironment. The nematodes must be able to sense and adapt to these hypoxic conditions (Jiang, 2001).

AHA-1 translocation to the nuclei of intestinal cells is inefficient in hif-1 mutants. This result was not predicted by the prevalent models for hypoxia signaling. In the mammalian cell lines commonly used to study hypoxia-inducible factor or aryl hydrocarbon receptor (AHR) signaling, ARNT is localized to the nucleus constitutively. After HIF-1alpha or activated AHR translocates to the nucleus, it forms a dimer with ARNT. However, in the Drosophila embryo, Drosophila ARNT (encoded by the tango gene) apparently remains cytoplasmic until a bHLH-PAS dimerization partner is expressed. It is concluded that the role of AHA-1 in the formation and nuclear localization of an active transcriptional complex may depend on cell type-specific factors, such as the expression of other bHLH-PAS proteins. This hypothesis will be explored by examining the expression of other bHLH-PAS genes in those cells that localize AHA-1 to the nucleus in the absence of hif-1 (Jiang, 2001).

Mammalian Arnts

cDNAs encoding two distinct basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) proteins withsimilarity to the mammalian aryl hydrocarbon nuclear translocator (Arnt) protein were isolated fromRTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identicalover the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical tohuman Arnt. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence ofan additional 373 base pairs of sequence that manifest the characteristics of an alternatively spliced exon.The presence of the 373-base pair region causes a shift in the reading frame. rtARNTa lacks thesequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine.rtARNTb contains the sequence and has a COOH-terminal domain of 190 residues rich in glutamineand asparagine. mRNAs for both rtARNT splice variants are detected in RTG-2 gonad cells, troutliver, and gonad tissue. rtARNTa and rtARNTb protein were identified in cell lysates from RTG-2 cells.Transfection of rtARNT expression vectors into murine Hepa-1 cells that are defective in Arntfunction (type II) results in rtARNT protein expression localized to the nucleus. Treatment of these cellswith 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteinseffectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appearsto be due to inefficient binding of rtARNTa.AhR complexes to DNA. The presence ofrtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and invitro. It is concluded that rtARNTa is a dominant negative activity in the bHLH/PAS family (Pollenz, 1995).

Arnt2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from ratbrain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the arylhydrocarbon receptor nuclear translocator (Arnt1). Whereas Arnt2gene expression occursselectively in brain and kidney, the expression of Arnt1 is ubiquitous, suggesting that the two proteins playdistinct roles, presumably via dimerization and DNA binding with different partners (Drutel, 1996).

In an effort to better understand the toxicity mechanism of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, an iterative search of sequence tags expressed in humans was employed to identify novelbasic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AhR) orthe Ah receptor nuclear translocator (Arnt). Five new "members of the PASsuperfamily" (MOPs 1-5), similar in size and structural organization to the AhR and Arnt, have been characterized.MOPs 1-4 have N-terminal bHLH and PAS domains and C-terminal variable regions. MOP5contain the characteristic PAS domain and a variable C terminus; it is possible that the cDNAcontains a bHLH domain, but the entire open reading frame has yet to be completed.Coimmunoprecipitation studies, yeast two-hybrid analysis, and transient transfection experiments all demonstrate that MOP1 and MOP2 dimerize with Arnt and that these complexes aretranscriptionally active at defined DNA enhancer sequences in vivo. MOP3 is found to associatewith the AhR in vitro but not in vivo. This observation, coupled with the fact that MOP3 forms tighterassociations with the 90-kDa heat shock protein than the human AhR, suggests that MOP3 may be aconditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expressionprofiles of the AhR, MOP1, and MOP2 mRNAs, coupled with the observation that they all shareArnt as a common dimeric partner, suggest that the cellular pathways mediated by MOP1 andMOP2 may influence or respond to the dioxin signaling pathway (Hogenesch, 1997).

Expression of Arnt, a mammalian Tango homolog

Dioxins are environmental pollutants, whose detrimental effects on health are the cause of wide publicconcern due to their accumulation in the food chain and resistance to metabolism. The most wellknown dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxins exert their effects through aligand activated transcription factor termed the dioxin or aryl hydrocarbon receptor (AhR), which acts inconcert with another structurally related protein: the aryl hydrocarbon nuclear translocator (Arnt). In situ hybridization was used to study the localization of the mRNAs forthese two proteins in the rat brain. mRNAs for both AhR and Arnt are found predominantly in thesame neuronal populations: the olfactory bulb, the hippocampus, and the cerebral and cerebellarcortices. Arnt, however, has a more widespread expression than AhR in the brain. The present resultsdemonstrate that dioxins may act directly in the brain and that the effects of dioxin may occur indiscrete neuronal populations. However, in some parts of the brain, e.g. the hypothalamus, that arethought to be targets of the toxic effects of dioxins, detectable levels of AhRmRNA are not observed. Furthermore, it appears that Arnt may have additional functions in the brain, apart from beingthe heterodimerization partner of AhR, possibly through heterodimerizing with other transcriptionfactors (Kainu, 1995).

The AhR is aligand-activated partner of the Arnt protein. Both proteins are required to transcriptionally regulategene expression. Arnt must be complexed to AhR to permit binding to the regulatory DNAsequence. The AhR-ligand complex is known to mediate a range of biological responses, such asdevelopmental toxicity, induction of cleft palate, and hydronephrosis. AhR and Arnt are expressed inhuman embryonic palatal cells and AhR has a specific developmental patternof expression in the mouse embryo. In the present study, expression of Arnt is characterized inC57Bl/6N mouse embryos from gestation day 10-16. The day 10-11 embryos show highest levels of Arnt inneuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglioniccomplexes. The heart also has significant expression of Arnt, with strong nuclear localization. Afterday 11, expression in heart and brain declines. In day 12-13 embryos expression is highest in the liver,where expression increases from day 12 to 16. At days 15-16 the highest levels of Arnt occurs inadrenal gland and liver, although Arnt is also detected in the submandibular gland, the ectoderm, the tongue, and inbone and muscle. In all of these tissues Arnt is cytoplasmic as well as nuclear, except in some ofthe cortical adrenal cells, in which Arnt is strongly cytoplasmic with little or no nuclear localization.These specific patterns of Arnt expression, which differ in certain tissues from the expression ofAhR, suggest that Arnt may have additional roles in normal embryonic development (Abbott, 1995).

Mutation of Arnt

The arylhydrocarbon-receptor nuclear translocator (Arnt) is a member of thebasic-helix-loop-helix-PAS family of heterodimeric transcription factors, which includes thearylhydrocarbon receptor (AhR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the DrosophilaSingle-minded protein (Sim). Arnt forms heterodimeric complexes with the aryl hydrocarbon receptor,HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AhR-Arntheterodimers regulate genes involved in the metabolism of xenobiotics, whereas Arnt-HIF-1alphaheterodimers probably regulate those involved in the response to oxygen deprivation. By generating atargeted disruption of the Arnt locus in the mouse, it is shown that Arnt-/- embryonic stem cells failto activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond toa decrease in glucose concentration, indicating that Arnt is crucial in the response to hypoxia and tohypoglycemia. Arnt-/- embryos are not viable past embryonic day 10.5 and show defectiveangiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. Thedefect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reportedfor mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings,a model is proposed in which increasing tissue mass during organogenesis leads to the formation ofhypoxic/nutrient-deprived cells, the subsequent activation of Arnt, and a concomitant increase in theexpression of genes (including the gene encoding vascular endothelial growth factor) that promotevascularization of the developing yolk sac and solid tissues (Maltepe, 1997).

Homologous recombination was used in embryonic stem cells to generate mice heterozygous for an arylhydrocarbon nuclear translocator (Arnt) null mutation. These mice were intercrossed, but no livehomozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in uterobetween 9.5 and 10.5 days of gestation. Abnormalities include neural tube closure defects, forebrainhypoplasia, delayed rotation of the embryo, placental hemorrhaging, and visceral arch abnormalities.However, the primary cause of lethality appears to be failure of the embryonic component of theplacenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to Arnt'sknown role in hypoxic induction of angiogenesis. No defects were found in yolk sac circulation (Kozak, 1997).

Domain structure of Arnt

Gene regulation by dioxins is mediated by the dioxin receptor-Arnt heterodimer, a ligand generatedcomplex of two basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) transcription factors. By usingdioxin receptor chimeras where the dimerization and DNA binding bHLH motif has been replaced by aheterologous DNA binding domain, the ability of Arnt to interact with the dioxinreceptor via the PAS domain has been detected in a mammalian 'hybrid interaction' system. By coimmunoprecipitationassays, the ability of PAS domains of the dioxin receptor and Arnt to mediateindependent heterodimerization has been confirmed in vitro. Selectivity for PAS dimerization is noted in the hybridinteraction system, because neither the dioxin receptor nor Arnt PAS-mediated homodimers are detected.Surprisingly, however, the PAS domain of Drosophila Period can dimerize with both the dioxin receptor and Arntsubunits in vitro, and disrupt the ability of these subunits to form a DNA binding heterodimer.Ectopic expression of Per blocks dioxin signaling in mammalian cells. The PAS domains ofthe dioxin receptor and Arnt are therefore novel dimerizing regions critical to the formation of a functionaldioxin receptor-Arnt complex, while the PerPAS domain is a potential negative regulator ofbHLH/PAS factor function (Lindebro, 1995).

The aryl hydrocarbon (or dioxin) receptor (AhR) is a ligand-activated basic helix-loop-helix (bHLH)protein that heterodimerizes with the bHLH protein AhR nuclear translocator (Arnt) to form acomplex that binds to xenobiotic regulatory elements in the enhancers of target genes. Aseries of fusion proteins, with a heterologous DNA-binding domain, was used to independently study thetrans-activating function of the human AhR and Arnt proteins in yeast. The results confirm that boththe human AhR and Arnt contain carboxyl-terminal trans-activation domains. The AhR has acomplex trans-activation domain that is composed of multiple segments, which function independently andexhibit varying levels of activation. These regions within the AhR cooperate when linkedtogether, resulting in a synergistic activation of transcription. Fusion proteins of the AhR and Arnttrans-activation domains with the LexA DNA-binding domain, expressed in bacteria and purified tonear-homogeneity, stimulate transcription of a minimal promoter in vitro in yeast nuclear extracts.Using this in vitro transcription assay, it is also possible to demonstrate that the AhR and Arnttrans-activation domains, in the absence of a DNA-binding domain, inhibit activated and basaltranscription. In vitro, the receptor binds selectively to the basal transcription factors, theTATA-binding protein and TFIIF, whereas Arnt binds preferentially to TFIIF. Taken together,these results suggest that AhR and Arnt activate target gene expression, at least in part, throughdirect interactions with basal transcription factors (Rowlands, 1996).

Binding of Arnt to DNA

The Ah receptor (AhR) and its DNA binding partner, the Ah receptor nuclear translocator (Arnt),are basic helix-loop-helix proteins distinguished by their Per, AhR, Arnt, and Sim(PAS) homologyregions. To identify the amino acids of the AhR.Arnt heterodimer that contact the TNGCGTGrecognition sequence, deletion mapping and amino acid substitutions have been performed within the Ntermini of both the AhR and Arnt. The ability of the variant AhR and Arnt proteins to bind DNAand activate gene transcription was determined by gel shift analysis and transient transfectionassays. The amino acids of Arnt that contact DNA are similar to those of otherbasic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87.Although initial experiments indicate that DNA binding of the AhR may involve two regions (bordered by amino acids 9-17 and amino acids 34-42), further analysis demonstrates that only aminoacids 34-39 are critical for the AhR.TNGC interaction. These experiments indicate that while thestructural features of the Arnt.GTG complex may closely resemble that deduced for proteins such asMax, E47, and USF, the AhR.TNGC complex may represent a unique DNA binding form ofbasic/helix-loop-helix proteins (Swanson, 1996).

The Ah receptor (AhR), the Ah receptor nuclear translocator protein (Arnt), and single-mindedprotein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatoryproteins. The DNA half-site recognition and pairing rules for these proteins were examined using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotideselection-amplification reveals that a variety of bHLH-PAS protein combinations can interact, each one generating a unique DNA binding specificity. To validate the selection-amplification protocol, the preference of the AhR.Arnt complex was demonstrated for the sequence commonly found indioxin-responsive enhancers in vivo (TNGCGTG). The Arnt protein iscapable of forming a homodimer with a binding preference for the palindromic E-box sequence,CACGTG. Further examination indicates that Arnt may have a relaxed partner specificity, since itis also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG.Coprecipitation experiments using various PAS proteins and Arnt are consistent with the idea thatthe Arnt protein is capable of a broad range of interactions among the bHLH-PAS proteins, while the othermembers appear more restricted in their interactions. Comparison of this in vitro data with sites knownto be bound in vivo suggests that the high affinity half-site recognition sequences for the AhR, SIM,and Arnt are T(C/T)GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites), respectively (Swanson, 1995).

Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLHmotif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes ina ligand-dependent manner with the bHLH dioxin receptor, a process that enables thedioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxinresponse elements of target promoters. In the absence of dioxin, Arnt does not bind to this targetsequence motif. Arnt constitutively binds the E box motif CACGTG, also recognized by a number of distinctbHLH factors, including USF and Max. Amino acids that have been identified to be criticalfor E box recognition by Max and USF are conserved in Arnt. Consistent with these observations,full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain,constitutively activates CACGTG E box-driven reporter genes in vivo. These results indicate a role forArnt in the regulation of a network of target genes that is distinct from the regulatory role played by the Arnt-dioxinreceptor complex in dioxin-stimulated cells (Antonsson, 1995).

The aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (Arnt)belong to a novel subclass of basic helix-loop-helix transcription factors. The AhR.Arnt heterodimerbinds to the xenobiotic responsive element (XRE). Substitution of each of four amino acids in the basicregion of Arnt with alanine severely diminishes or abolishes XRE binding, intimating that these aminoacids contact DNA bases. Three of these amino acids are conserved among basic helix-loop-helixproteins, and the corresponding amino acids of Max and USF are known to contact DNA bases.Alanine scanning mutagenesis of the basic domain of AhR and substitution with conservative aminoacids at particular positions in this domain and in a more amino-proximal AhR segment previouslyshown to be required for XRE binding demonstrate that the most carboxyl-proximal amino acid position of the basic domain and aposition within the amino-proximal segment are intolerant to amino acid substitution with regard to XREbinding, suggesting that these two amino acids make base contacts. Amino acid positions in these AhRregions and in the Arnt basic region are less adversely affected by substitution are also identified. Theamino acids at these positions may contact the phosphodiester backbone. The apparent bipartite natureof the DNA binding region of AhR and the identity of those of its amino acids that apparently makeDNA contacts impute a novel protein-DNA binding behavior for AhR (Bacsi, 1996).

Expression of CYP1A1 gene is regulated in a substrate-inducible manner through at least two kinds ofregulatory DNA elements, in addition to the TATA sequence, XRE (xenobiotic responsive element),and BTE (basic transcription element), a GC box sequence. The trans-acting factor on the XRE is aheterodimer consisting of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt), whileSp1 acts as a regulatory factor on the BTE. An investigation was carried out of how these factors interact with oneanother to induce expression of the CYP1A1 gene. Both in vivo transfection assays using DrosophilaSchneider line 2 (SL2) cells, which is devoid of endogenous Sp1, AhR, and Arnt, and in vitrotranscription assays using baculovirus-expressed AhR, Arnt, and Sp1 proteins, reveal that thesefactors synergistically enhance expression of the reporter genes driven by a model CYP1A1promoter, consisting of four repeated XRE sequences and a BTE sequence. Both AhR and Arnt interact with the zinc finger domain of Sp1 via their basic HLH/PAS domains.When either the AhR.Arnt heterodimer of Sp1 is bound to its cognate DNA element, DNA bindingof the second factor is facilitated. A survey of DNA sequences in the promoter region shows that theXRE and GC box elements are commonly found in the genes whose expressions are induced bypolycyclic aromatic hydrocarbons, suggesting that the two regulatory DNA elements and their cognatetrans-acting factors constitute a common mechanism for induction of a group of drug-metabolizingenzymes (Kobayashi, 1996).

CBP/p300, an Arnt coactivator

A heterodimer of AhR (aryl hydrocarbon receptor) and Arnt (AhR nuclear translocator) conveys atransactivation signal of aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and3-methylcholanthrene to the genes for a group of drug-metabolizing enzymes. This inducible expressionof the genes is inhibited by adenovirus E1A, suggesting that CBP/p300 is somehow involved in thetransactivation of the genes by the AhR and Arnt heterodimer. Yeast and mammalian two hybridsystems revealed that CBP/p300 interacts with the transactivation domain of Arnt, but not with thatof AhR, via the CREB-binding domain. A pull down assay using GST-Arnt hybrid protein confirmsthe interaction between Arnt and CBP/p300. Considering these results and that Arnt or Arnt2functions as a common partner in the formation of transcriptional regulators with other bHLH/PASproteins (such as AhR, HLF, and HIF-1alpha), the possibility arises that CBP/p300 is extensivelyinvolved as a coactivator in the transactivation process by bHLH/PAS heterodimer transcription factors through the interaction with Arnt or Arnt2 (Kobayashi, 1997).

Evolutionary homologs continued: part 2/3 | part 3/3

tango: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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