Invertebrate Arnt homologs

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the 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 other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, 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 that the 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 level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently 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-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene 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) during normal 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 signaling are 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 and fertile 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. elegans hermaphrodite 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 cultured on 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 with similarity to the mammalian aryl hydrocarbon nuclear translocator (Arnt) protein were isolated from RTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human Arnt. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence of an 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 the sequence 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 glutamine and asparagine. mRNAs for both rtARNT splice variants are detected in RTG-2 gonad cells, trout liver, 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 Arnt function (type II) results in rtARNT protein expression localized to the nucleus. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appears to be due to inefficient binding of rtARNTa.AhR complexes to DNA. The presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro. 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 rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1). Whereas Arnt2 gene expression occurs selectively in brain and kidney, the expression of Arnt1 is ubiquitous, suggesting that the two proteins play distinct 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 novel basic-helix-loop-helix-PAS (bHLH-PAS) proteins that interact with either the Ah receptor (AhR) or the Ah receptor nuclear translocator (Arnt). Five new "members of the PAS superfamily" (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. MOP5 contain the characteristic PAS domain and a variable C terminus; it is possible that the cDNA contains 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 are transcriptionally active at defined DNA enhancer sequences in vivo. MOP3 is found to associate with the AhR in vitro but not in vivo. This observation, coupled with the fact that MOP3 forms tighter associations with the 90-kDa heat shock protein than the human AhR, suggests that MOP3 may be a conditionally active bHLH-PAS protein that requires activation by an unknown ligand. The expression profiles of the AhR, MOP1, and MOP2 mRNAs, coupled with the observation that they all share Arnt as a common dimeric partner, suggest that the cellular pathways mediated by MOP1 and MOP2 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 public concern due to their accumulation in the food chain and resistance to metabolism. The most well known dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxins exert their effects through a ligand activated transcription factor termed the dioxin or aryl hydrocarbon receptor (AhR), which acts in concert with another structurally related protein: the aryl hydrocarbon nuclear translocator (Arnt). In situ hybridization was used to study the localization of the mRNAs for these two proteins in the rat brain. mRNAs for both AhR and Arnt are found predominantly in the same neuronal populations: the olfactory bulb, the hippocampus, and the cerebral and cerebellar cortices. Arnt, however, has a more widespread expression than AhR in the brain. The present results demonstrate that dioxins may act directly in the brain and that the effects of dioxin may occur in discrete neuronal populations. However, in some parts of the brain, e.g. the hypothalamus, that are thought to be targets of the toxic effects of dioxins, detectable levels of AhR mRNA are not observed. Furthermore, it appears that Arnt may have additional functions in the brain, apart from being the heterodimerization partner of AhR, possibly through heterodimerizing with other transcription factors (Kainu, 1995).

The AhR is a ligand-activated partner of the Arnt protein. Both proteins are required to transcriptionally regulate gene expression. Arnt must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR-ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and Arnt are expressed in human embryonic palatal cells and AhR has a specific developmental pattern of expression in the mouse embryo. In the present study, expression of Arnt is characterized in C57Bl/6N mouse embryos from gestation day 10-16. The day 10-11 embryos show highest levels of Arnt in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also has significant expression of Arnt, with strong nuclear localization. After day 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 in adrenal gland and liver, although Arnt is also detected in the submandibular gland, the ectoderm, the tongue, and in bone and muscle. In all of these tissues Arnt is cytoplasmic as well as nuclear, except in some of the 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 of AhR, 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 the basic-helix-loop-helix-PAS family of heterodimeric transcription factors, which includes the arylhydrocarbon receptor (AhR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila Single-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-Arnt heterodimers regulate genes involved in the metabolism of xenobiotics, whereas Arnt-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, it is shown that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that Arnt is crucial in the response to hypoxia and to hypoglycemia. Arnt-/- embryos are not viable past embryonic day 10.5 and show defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for 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 of hypoxic/nutrient-deprived cells, the subsequent activation of Arnt, and a concomitant increase in the expression of genes (including the gene encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues (Maltepe, 1997).

Homologous recombination was used in embryonic stem cells to generate mice heterozygous for an aryl hydrocarbon nuclear translocator (Arnt) null mutation. These mice were intercrossed, but no live homozygous Arnt-/- knockout mice were produced among 64 newborns. Homozygotes die in utero between 9.5 and 10.5 days of gestation. Abnormalities include neural tube closure defects, forebrain hypoplasia, 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 the placenta to vascularize and form the labyrinthine spongiotrophoblast. This may be related to Arnt's known role in hypoxic induction of angiogenesis. No defects were found in yolk sac circulation (Kozak, 1997).

Domain structure of Arnt