Hormone receptor-like in 46
A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3, now known as Hr46, is in the same subclass as C. elegans "CNR-3" and human RORalpha. Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: (1) C. elegans rhr-2, (2) Human RARalpha, beta and gamma, (3) Human thyroid hormone receptor alpha and beta, and (4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).
Hr46/DHR3 encodes one of a growing number of "orphan" receptors for which ligands have not yet
been identified. The structure of the Hr46 protein is strikingly similar to that of the MHR3 protein (e.g.,
97% amino acid identity for the DNA binding domains), another orphan receptor encoded by an
ecdysone-inducible early gene from another insect, Manduca sexta (Koelle, 1992).
Using cDNAs for the human retinoic acid receptor alpha (hRAR alpha) and Drosophila hormone receptor
46 (Hr46/DHR3), a cDNA was isolated encoding a member of the steroid hormone receptor superfamily from the
tobacco hornworm, Manduca sexta. This cDNA is most closely related to Hr46
(97 and 68% amino acid identity in the DNA and ligand binding regions, respectively) followed by hRAR
alpha (65 and 20% identity, respectively) and therefore is named MHR3. The cDNA hybridized to two
mRNAs (3.8 and 4.5 kb) found in the epidermis during the ecdysteroid rises for the embryonic, larval,
and pupal molts. Culture of fourth instar larval epidermis with ecdysone causes the appearance of MHR3 mRNA within 3 hr and maximal expression by 6
hr; after 12 hr continuous exposure to ecdysone, the mRNA level declines. The 4.5-kb mRNA appears first,
both mRNAs are present in equal amounts by 12 hr, and by 20 hr the predominant transcript is 3.8 kb. Similar
ecdysone-induced expression is seen in epidermis explanted 1 day after the onset of wandering, although
with a slower time course. The induction is largely independent of protein synthesis, but the
subsequent decline requires protein synthesis as is typical of the "early" puffs in Drosophila. Continuous
exposure to ecdysone is necessary for MHR3 expression; in its absence, the mRNA declines with a
half-life of 2 hr. Thus, MHR3 is an ecdysteroid-inducible DNA binding protein that likely is a transcription
factor involved in the cascade of gene activation and inactivation caused by ecdysteroids during the insect
molt (Palli, 1992).
The responsiveness of several nuclear transcription factor genes to
20-hydroxyecdysone (20E) was characterized in an embryonic cell line, GV1, from
Manduca sexta. The mRNA for the Manduca ecdysone receptor (MsEcR) is
present in the GV1 cells and transiently increases 2.3-fold by 5 h after the addition of
20-hydroxyecdysone (20E). In contrast, Manduca
ultraspiracle (MsUSP) mRNA level in the GV1 cells decreases slowly to half of its
initial level by 12 h when exposed to 20E. The mRNAs for
two putative transcription factors, MsE75 and MHR3, are induced in the GV1 cells
by 20E; the mRNA for E75 appears within 1 hour whereas that for MHR3 appears within 2 hours (Lan, 1997).
CHR3 cDNA codes for 546 amino
acids. The deduced amino acid sequence of this open reading frame contains all five
regions typical of a steroid hormone nuclear receptor. The C domain shows the
highest identity to Manduca hormone receptor 3 (MHR3), Drosophila hormone
receptor 3 (Hr46/DHR3) and Galleria hormone receptor 3 (GHR3). The A/B, D and E
domains also showed significant amino acid similarity with MHR3, Hr46 and GHR3.
The 683 bp CHR3 cDNA probe detects two mRNAs of 3.8 and 4.5 kb present
during the ecdysteroid peaks for embryonic, larval, pupal and adult molts but not during the intermolt periods. In sixth instar larvae, the 3.8 and 4.5 kb mRNA
are detected in the epidermis, fat body and midgut tissues and the maximum
expression was observed during the prepupal peak of ecdysteroids in the hemolymph.
CHR3 mRNA is induced in 20-hydroxyecdysone treated CF-203 cells as well as in
the midgut, fat body and epidermis of larvae when fed the non-steroidal molting
hormone agonist, RH-5992. In vitro transcription and translation of the CHR3 cDNA
yields a 61 kDa protein that binds to the retinoid related orphan receptor response
element (Palli, 1996).
Choristoneura (the spruce budworm) hormone receptor 3 (CHR3) is a 20E (20-hydroxyecdysone)-induced
delayed early gene that is homologous to Manduca hormone receptor 3 (MHR3),
Drosophila hormone receptor 3 (Hr46/DHR3), and Galleria hormone receptor 3 (GHR3).
Multiple isoforms are evident. CHR3C differs
from the CHR3B in two regions.
The deduced amino acid sequence of this CHR3C contais all five
domains that are typical for a steroid hormone nuclear receptor. The nucleotide
sequence of CHR3C cDNA is identical to the nucleotide sequence of CHR3B cDNA
except for two major differences in the A/B and D-domains. The CHR3C specific
probes detect two mRNAs 5.4 kb (CHR3C), and 6.4 kb (CHR3D), which are
present in the pupal stage. The CHR3C and CHR3D mRNAs are induced by the
stable ecdysteroid analog RH-5992. The CHR3C protein also binds to the response
element of the retinoic acid receptor-related orphan receptor (Palli, 1997).
A cDNA from Galleria mellonella prepupae has been identified that detects a 177 bp fragment that has 87% identity to the Manduca sexta gene MHR3 and 75% to the Drosophila melanogaster Hr46/DHR3 gene, and was therefore named "GHR3". The 557 amino acid sequence of GHR3 shows 92% overall identity with the MHR3 protein and 97 and 70% identity with Hr46 in the putative DNA- and ligand-binding domains, respectively. Hybridization of whole body RNA reveals high GHR3 mRNA levels during both the larval and pupal molts, coincident with the molt-inducing ecdysteroid pulses, and low or undetectable levels during the first half of the last instar. During the larval-pupal transformation, no GHR3 mRNA is found at the beginning of the stemmatal pigment retraction at the onset of the ecdysteroid rise; maximal levels are observed 4 h later, coincident with the peak ecdysteroid titer (over 2.3 micrograms 20E equivalents/ml hemolymph). Two mRNAs (4.6 and 3.6 kb) are detected when the ecdysteroid titer is high. Injection of 20E into isolated final instar larval abdomens induces the appearance of the 4.6 kb mRNA within 1.5 h; the mRNA level then reaches maximum by 3 h and declines by 6 h. No 3.6 kb mRNA is detectable during that time. A 10-fold lower 20E dose causes only trace induction by 3 h (Jindra, 1994).
MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5' flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter by 20E per ml in Manduca GV1 cells is similar to that of endogenous MHR3, with detectable response by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) are expressed at high levels under the control of a constitutive promoter, expression levels after a 3-h exposure to 20E increases two- to six-fold. In contrast, high expression of EcR-B1 and USP-2 cause little increase in reporter levels in response to 20E. Moreover, expression of USP-2 prevents activation by EcR-B1-USP-1. Deletion experiments show that the upstream region, including the three most proximal putative EcREs, is responsible for most of the 20E activation, with the EcRE3 at -671 and the adjacent GGGTCA being most critical. The EcRE1 at -342 is necessary but not sufficient for the activational response but is the only one of the three
putative EcREs to bind the EcR-B1-USP-1 complex in gel mobility shift assays and
is responsible for the silencing action of EcR-B1-USP-1 in the absence of
hormone. EcRE2 and EcRE3 each specifically bind other protein(s) in the cell
extract, but not EcR and USP, and so are not EcREs in this cellular context.
When cell extracts were used, the EcR-B1-USP-2 heterodimer shows no binding to
EcRE1, and the presence of excess USP-2 prevents the binding of EcR-B1-USP-1 to
this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both
form heterodimeric complexes with EcR-B1 and bind to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms (Lan, 1999).
Three distinct members of the steroid/thyroid hormone receptor (STR)
family have been cloned from the nematode Caenorhabditis elegans. All three belong to the retinoic acid
receptor (RAR), thyroid hormone receptor subfamily of genes. The cDNA of one of
these clones shows such a high homology to Hr46/DHR3, an early ecdysone response gene
found in Drosophila, and MHR3, identified in Manduca sexta, and it has been termed
CHR3. The C-terminal portion of the deduced protein sequence shows a
box containing eight identical amino acids among CHR3, Hr46, and MHR3,
suggesting an identical specific ligand for these proteins. CNR8 shows homology to
NAK1, and CNR14 has homology to both the RAR-gamma 1 gene and to another
ecdysone response gene, E78A. Neither of the latter two cDNAs is a clear
homolog of any known gene and each is distinctive. All of these genes are
expressed varyingly in both larval and adult stages of nematode development. These data demonstrate that the STR family of
genes is represented in a nematode whose ancestor appeared well before the
branching that gave rise to the Arthropoda and Chordata (Kostrouch, 1995).
CHR3 is a Caenorhabditis elegans orphan nuclear hormone receptor highly homologous to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. Related vertebrate factors include RORalpha/RZRalpha, RZRbeta and RevErb. Gel-shift studies show that CHR3 can bind the DR5-type hormone response sequence. CHR3 is a nuclear protein present in all blastomeres during early embryogenesis. During morphogenesis, both CHR3 protein and zygotically active reporter genes are detectable in epidermal cells and their precursors. CHR3 is present in all blastomeres of the embryo from at least the 2-cell stage until approximately the 200-cell stage. The presence of CHR3 in 2-cell embryos suggests a maternal contribution, a notion confirmed by in situ hybridization. In most blastomeres at about the 200-cell stage the ubiquitous distribution of CHR3 begins to fade and a more restricted pattern begins to emerge in which the most prominent staining nuclei are epidermal cells and their precursors. The epidermal staining pattern of CHR3 antibody is maintained throughout embryogenesis, while the staining of all other nuclei fades to background levels (Kostrouchova, 1998).
In C. elegans, epidermal cells originate from two founder blastomeres, AB and C. The early epidermal derivatives of these founder blastomeres are located in the dorsal region near the posterior of the embryo. At approximately the 350-cell stage (270 minutes postfertilization), these cells begin to migrate ventrally to enclose the developing embryo. Dorsal epidermal cells in the midbody, designated Hyp7, interdigitate and fuse to form a single large syncytium. Smaller multinucleate epidermal cells are also present in the head, whereas mostly mononucleate epidermal cells are present in the tail. Lateral and ventral epidermal cells of the embryo, designated V (seam cells) and P respectively, are stem cell-like. In addition to functioning as epidermis, the V and P cells divide postembryonically to generate additional epidermal and non-epidermal cell types. By the 1.5-fold stage of embryogenesis, the Hyp7, V and P nuclei are easily identified as three lateral rows on each side of the embryo. C. elegans CHR3 has been designated NHR-23, in keeping with C. elegans nomenclature. The expression of nhr-23 was assayed by in situ hybridization. nhr-23 RNA is easily detectable in the germline and oocytes; this confirms that nhr-23 is a maternal gene product. nhr-23 RNA is also detected in 2- and 4-cell embryos but then becomes undetectable in subsequent stages of early embryogenesis. By the comma stage of embryogenesis, a faint in situ signal for nhr-23 appears on the surface of the embryo, consistent with expression in the epidermis. Inhibition of the gene encoding CHR3 results in several larval defects associated with abnormal epidermal cell function, including molting and body size regulation, suggesting that CHR3 is an essential epidermal factor required for proper postembryonic development (Kostrouchova, 1998).
What is the role of CHR3 in the epidermis? CHR3 is not required for cells to adopt an epidermal cell fate; mutant embryos following nhr-23 inhibition are initially covered by an epidermis and cuticle and the epidermal cell number appears normal at hatching based on LIN-26 antibody staining. Two of many possible functions for CHR3 are (1) to regulate the onset of molting, perhaps by activating a collagenolytic pathway or (2) to regulate collagen gene expression (positively or negatively) at one or more stages of development. Defects in either one of these functions might result in the phenotypes that were observed. The inability of nhr-23(RNAi) animals to completely shed their old cuticles suggests that complete lysis of the matrix that attaches cuticle to the epidermal cells is a primary defect of loss of CHR3 function. The blistering of some larval cuticles in response to ectopic and/or the overproduction of CHR3 is consistent with a role of CHR3 in cuticle detachment. Further support for this model is that the close homolog of CHR3 in Drosophila has a role in ecdysis. However, with DHR3, the involvement appears to be indirect and the result of inhibition of some genes and the activation of other genes. Hence the role of CHR3 in C. elegans molting could also be indirect. Currently, this model is difficult to assess in C. elegans, as little is known about the genes and gene products responsible for breaking the attachment of the cuticle in this animal. However, the phenotype of nhr-23(RNAi) animals should serve as an entry point into the study of the molecular and genetic mechanisms of molting (Kostrouchova, 1998).
CHR3 (nhr-23, NF1F4), the homolog of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode C. elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. The role of CHR3 during larval development was studied by using expression assays and RNA-mediated interference. The levels of expression of CHR3 (nhr-23) cycle during larval development. The reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a gene potentially acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function (Kostrouchova, 2001).
About 50% of young larvae exposed to CHR3 (nhr-23) RNAi by soaking or feeding show defects in gonad development if they survive to adulthood. The germ line is often misshapen in these animals, usually folded and constricted. The distal-tip cell position is misplaced as a result of improper migration. These animals often have incompletely shed the L4 cuticle during the L4/A molt, resulting in one or more constrictions of the body at the point at which the shedding cuticle had gotten stuck. In severely affected hermaphrodites, the germ line had an irregular shape and contains small and more numerous nuclei (Kostrouchova, 2001).
To see whether CHR3 (nhr-23) loss of function affected males, him-5 animals were treated with CHR3 (nhr-23) RNAi. The him-5 mutation alone results in about 16% of males among the population as opposed to less than 0.5% normally observed in N2 cultures. About 50% of him-5 males subjected to CHR3 (nhr-23) RNAi in feeding experiments had male tail defects in addition to molting defects. The main male tail phenotype includes problems in fan and sensory ray development, including a failure to retract the tail during morphogenesis. The fate of these cells is unaffected and the male tail defects arise primarily from problems in morphogenesis (Kostrouchova, 2001).
The let-7 microRNA (miRNA) gene of Caenorhabditis elegans
controls the timing of developmental events. let-7 is conserved
throughout bilaterian phylogeny and has multiple paralogs. The paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes
caused by mutations in let-7, whereas increased expression of
mir-84 suppresses a let-7 null allele. Adults with reduced
levels of mir-84 and let-7 express genes characteristic of
larval molting as they initiate a supernumerary molt. mir-84 and
let-7 promote exit from the molting cycle by regulating targets in
the heterochronic pathway and also nhr-23 and nhr-25, genes
encoding conserved nuclear hormone receptors essential for larval molting. The
synergistic action of miRNA paralogs in development may be a general feature
of the diversified miRNA gene family (Hayes, 2006).
The C. elegans genes nhr-23 and nhr-25 encode
orphan nuclear hormone receptors orthologous, respectively, to DHR3 and
ßFTZ-F1, which are related to mammalian ROR/RZR/RevErb and SF-1,
respectively. Both receptors are essential for completion of the larval molts,
suggesting that particular functions of nhr-23/DHR3 and
nhr-25/ ßFTZ-F1 might be conserved and, further, that
regulation by steroid hormones might be a common feature of molting in C.
elegans and Drosophila. However, a steroid hormone regulating
molting of C. elegans has not yet been identified and the genome
lacks orthologs of ECR or USP (Hayes, 2006).
A genetic model is presented for the function of mir-84 and let-7 in epithelial
differentiation, as related to the molting cycle. The let-7 miRNA
targets lin-41 mRNA and also hbl-1 mRNA, in combination with
paralogous miRNAs. During early larval development, LIN-41 and HBL-1 together
repress production of the zinc-finger transcription factor LIN-29.
Expression of let-7 and related miRNAs late in larval development
represses lin-41 and hbl-1, thereby activating LIN-29.
LIN-29 promotes expression of col-19 and possibly other collagen
genes characteristic of an adult cuticle and also represses expression of
col-17 and possibly other collagen genes characteristic of larval
cuticle. LIN-29 is likely to regulate additional genes that control
the molting cycle that have not yet been identified (Hayes, 2006).
Inactivation of either one of the nuclear hormone receptor genes nhr-23 or nhr-25 is sufficient to prevent the aberrant supernumerary molt caused by reduced levels of mir-84 and
let-7. NHR-23 and NHR-25 thus serve as key downstream effectors of
the miRNAs in regulation of the molting cycle. One model is that
LIN-29, or a transcription factor regulated by LIN-29, represses
nhr-23 and nhr-25 following the fourth molt. Accordingly,
GFP expression from an nhr-23 reporter gene increases fourfold in the
hypodermis of let-7 mir-84 adults. The relationship between
nhr-23 and nhr-25 in C. elegans remains to be
determined; however, DHR3 stimulates transcription of ßFTZ-F1 in flies (Hayes, 2006).
The identification of sites in the 3' UTR of nhr-25 that are
complementary to let-7 family members and are also conserved in other
nematodes suggests that the let-7 family targets the nhr-25
message to negatively regulate production of NHR-25 in adults. Consistent with
this model, increasing the abundance of mir-84 partly suppresses the
supernumerary molt caused by a probable null mutation in the lin-29
gene. Also, in preliminary experiments RNA species
attributable to cleavage of the nhr-25 message upon binding of
let-7-like miRNAs were detected in extracts from wildtype adults. Steroid hormones and co-factors probably also regulate activity of NHR-23 and NHR-25 during the life cycle (Hayes, 2006).
Regulation by miRNAs thus converges on transcription factors upstream in
the genetic networks regulating molting. NHR-23 coordinates several aspects of
larval molting by promoting expression of genes required for patterning the
new cuticle and ecdysis, including, respectively, the collagen gene
dpy-7 and the collagenase gene nas-37. Inactivation of either nhr-23 or nhr-25 abrogates the reiterated expression of gfp reporters for
mlt-10 and nas-37 caused by mutation of let-7 and
mir-84. NHR-25 might promote expression of the corresponding genes
during larval development, even though RNAi of nhr-25 is not
sufficient to abrogate expression of the gfp reporters in wild-type
larvae. Interestingly, inactivation of nhr-23 or nhr-25 causes an
earlier blockade in the molting program in let-7 mir-84 adults than
in wild-type larvae, such that the mutant adults do not enter lethargus or attempt to ecdyse. Parallel pathways might drive early steps of molting during larval development (Hayes, 2006).
Intriguingly, adults with reduced levels of mir-84 and
let-7 are unable to shed their cuticle to complete the supernumerary
molt. One possibility is that particular genes required for ecdysis are not
induced. Whereas the hypodermis and seam cells retain some larval character in
let-7 mir-84 adults, other cells, perhaps particular neurons or
specialized epithelia, might be fully differentiated and therefore unable to
coordinate with the molting program. Consistent with this idea, let-7
mir-84 adults spend an atypically long time in lethargus, suggesting a
failure to exit the behavioral program. Alternatively, particular structural
features of the fifth cuticle might be physically incompatible with shedding
the exoskeleton (Hayes, 2006).
Considering an aberrant ecdysis as the terminal phenotype of let-7
mir-84 mutants, it is intriguing to speculate that the let-7
family and possibly other miRNAs regulate aspects of the larval molting cycle.
Indeed, increased expression of either mir-84 or let-7
causes some larvae to arrest development, trapped inside partly shed cuticle,
indicating that levels of let-7-like miRNAs can impact molting of larvae (Hayes, 2006).
Mechanisms that set the pace of the molting cycle are not well understood,
although physiologic cues such as nutritional status
and environmental cues such as temperature impact the duration of larval
stages. Interestingly, let-7 and let-7 mir-84 mutants
initiate the supernumerary molt in synchrony, rather than in a stochastic
fashion, relative to the time of hatching. Thus, a timing mechanism for
molting persists in these particular miRNA mutants (Hayes, 2006).
The let-7 gene is perfectly conserved throughout bilaterian
phylogeny, and vertebrate genomes specify many miRNAs homologous to
let-7. Vertebrate let-7 and protein-coding genes
orthologous to targets of let-7 identified in C. elegans
play crucial roles in development. Moreover, reduced expression of human let-7 correlates with shortened survival in lung cancer patients, and let-7 might regulate the RAS oncogene. The possibility of functional conservation among homologs of let-7 in
humans and worms intimates the importance of understanding how let-7 and its paralogs function in C. elegans. This work shows how analysis of double mutants can reveal how the many miRNAs that form paralogous families work together to regulate their targets (Hayes, 2006).
Continued: see Hr46 Evolutionary homologs part 2/2 Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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