ftz-f1
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 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).
From a database containing sequences of published nuclear hormone receptors (NRs), an alignment of the C, D and E domains of NR transcription factors was constructed. Using this alignment, tree reconstruction was performed using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses six NR subfamilies were derived: (I) a large clustering of thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (II) retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (III) steroid receptors; (IV) NGFIB orphan receptors; (V) FTZ-F1 orphan receptors; and finally (vi) only one gene (to date), the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV, which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or hetero-dimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. It is suggested that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, two major steps of NR diversification have been defined: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. In vertebrate species, the various groups of NRs accumulated mutations at very different rates (Laudet, 1997).
Nuclear receptors are essential players in the development of all
metazoans. The nematode C. elegans possesses more than 200 putative
nuclear receptor genes, several times more than the number known in any other
organism. Very few of these transcription factors are conserved with components
of the steroid response pathways in vertebrates and arthropods. Ftz-F1, one of
the evolutionarily oldest nuclear receptor types, is required for
steroidogenesis and sexual differentiation in mice and for segmentation and
metamorphosis in Drosophila. Two complementary approaches,
direct mutagenesis and RNA interference, were used to explore the developmental role of nhr-25, a C.
elegans ortholog of Ftz-F1. Deletion mutants show that nhr-25 is essential for
embryogenesis. RNA interference reveals additional requirements throughout the
postembryonic life, namely in molting and differentiation of the gonad and
vulva. All these defects are consistent with the nhr-25 expression pattern,
determined by in situ hybridization and GFP reporter activity. These data link the C. elegans Ftz-F1 ortholog with a number of developmental
processes. Significantly, its role in the periodical replacement of cuticle
(molting) appears to be evolutionarily shared with insects and thus supports
the monophyletic origin of molting (Asahina, 2000).
Inactivation of nhr-25 function, either by a mutation
deleting the essential regions of nhr-25 or by RNAi,
causes embryonic lethality. The introduction of an
nhr-25 plasmid into the (delta2389) mutants partially
restores hatching, indicating that the embryonic lethal
phenotype is due to a loss-of-function of nhr-25.
Incomplete rescue may be due to the detrimental
effects of excessive copies of a functional gene or to
the mosaic partition of the extrachromosomal array
during development. nhr-25 is expressed in embryonic
epithelial cells. Although early descendants of the E
blastomere are the first cells to express nhr-25, this
expression seems to be transient and is soon followed
by lasting expression, mainly in the progeny of ABp
and C cells, forming the hyp7 syncytium, seam and
other hypodermal cells. Consistent with the nhr-25 expression in hypodermal cells, both the nhr-25 mutant and RNAi affected
embryos fail to elongate, arresting at the 1.5-fold
stage. These embryos are not properly enclosed,
showing extruding tissues, particularly at their posterior. This suggests that nhr-25 is required for the secretion of cuticle by the hypodermis (Asahina, 2000).
In situ hybridization shows strong expression in the germ-line and maternal deposition of the mRNA. Although dsRNA is known to interfere efficiently with maternal genes, defects earlier than those seen in nhr-25 mutant embryos were not seen using RNAi. It is assumed that either sufficient nhr-25 protein is also deposited into the eggs or that the maternal RNA is not essential (Asahina, 2000).
Post-embryonic expression of nhr-25 suggests that this
gene plays a role in larvae and adults. RNAi causes
lethality in L1-L2 but not in older larvae, corresponding
with nhr-25 expression and its decline in the hypodermis past L2 stage. Defects in cuticle replacement (molting) and morphogenesis, consistent with the
spatial expression, again point to an nhr-25 requirement
in the hypodermis. Lesions and irregularities of the
integument show that not only the shedding of the
old cuticle, but also synthesis of a new one are compromised (Asahina, 2000).
Those larvae that reached L3-L4 instars usually formed sterile adults, invariably with tumorous gonads and often with a missing or abnormal vulva and deformed tail. Since genetic data are lacking with respect to the involvement of nhr-25 in pathways controlling gonadal
and vulval differentiation, it is difficult at this point to
discuss how nhr-25 participates in these events. The
hyperplasia of the RNAi-affected gonad shows that
excessive mitoses occur in the germ-line. Similar gonad
appearance results from loss of cul-1, a cullin gene
necessary for cell cycle exit in C. elegans. Unlike the case with cul-1, however, no extra neurons were found in RNAi treated worms carrying the
mec-7::GFP marker, suggesting that nhr-25 is not a general regulator of the cell cycle. Similar defects of gonadal differentiation also result from
mutations in the gon-1 metalloprotease gene, implicated
in the shaping of extracellular matrix. Gonadal hyperplasia is also caused by a
constitutive activation of the Notch relative GLP-1, required for signaling between the distal tip cell and the germ-line. It is speculated that
nhr-25, which is expressed strongly in the germ-line,
might be a downstream component of such a pathway (Asahina, 2000).
Contrasting with nhr-25 is Drosophila Ftz-F1, expressed in perhaps all organs except for the gonads. Consistently, ftz-f1 mutant phenotypes do not suggest defects in reproduction. In the case of vulval development, nhr-25 expression in hypodermal vulva precursor cells may be required for their competence to form vulva. In this sense, ßFtz-F1 has been shown to render Drosophila tissues competent to undergo changes in response to the ecdysteroid signal. The posterior end defects possibly result from faulty expression of cuticle constituents, such as the sqt-1 and rol-6 collagen genes (Asahina, 2000).
Both arthropods and nematodes need steroids for
molting. In insects, precursors such as cholesterol are
converted into ecdysteroids and used as extracellular
signals to synchronize the molting of distant body
parts. In nematodes, the absence of dietary sterols compromises molting. More directly, both sterol starvation and mutations in a megalin-related protein LRP-1, which presumably mediates sterol endocytosis by hyp7 hypodermis, have been
shown to prevent the shedding and degradation of the old cuticle at all C. elegans molts. Whether ecdysteroids are required for nematode
molting is not clear, since their synthesis from cholesterol
has not been demonstrated (Asahina, 2000).
In the epidermis of insects, molting is regulated by
ecdysteroids acting through an ecdysone receptor complex EcR/USP and a cascade of transcription factors, including ßFtz-F1, on the expression of stage-specific genes. ßFtz-F1 is induced by an increase and a consequent decline of ecdysteroid titer
and is likely to control genes acting in cuticle formation. In Drosophila,
ßFtz-F1 is necessary for metamorphosis and for activation of at least
one pupal cuticle gene (EDG84A). The importance of ßFtz-F1 for the larval molt is
suggested by a rescue of ftz-f1 mutants with heat shock
induction of ßFtz-F1 around the molting period. Expression of
Drosophila ßFtz-F1 is activated by another nuclear
receptor, DHR3 (Asahina, 2000 and references therein).
How can nhr-25 act in the molting of C. elegans?
Since nhr-25 and NHR-23 are conserved with
Ftz-F1 and DHR3, it is possible that they are part of a
pathway analogous to that which exists in insects.
Significantly, RNAi targeting of nhr-23 causes molting
defects that were similar to those shown for nhr-25. An antibody is being prepared to test whether the genetic epistasis between nhr-23 and nhr-25 is the same as in Drosophila. However, the upstream part of this pathway is unclear, because C. elegans lacks orthologs of EcR and USP, which are thus far the only known receptors capable of conveying the ecdysteroid signal to gene
expression. There is an alternative possibility:
instead of being downstream of a steroid receptor, nhr-25 could mediate the conversion of dietary sterols to compounds active in molting. Encouraging this idea is the fact that Ftz-F1 type proteins direct steroid conversion in mammals (Asahina, 2000).
This analysis of nhr-25 function suggests that the
roles of ftz-f1 orthologs in Drosophila and C. elegans
molting are parallel and supports the monophyletic origin of molting. It is proposed that Ftz-F1/nhr-25 and DHR3/NHR-23 are the central, evolutionarily oldest regulators of genes executing cuticle replacement in all
Ecdysozoa. While these orphan receptors are sufficient
to control the molting program in small, simple forms,
such as C. elegans, the synchronized molting of distant
and diversified body parts in arthropods demands coordination by an extracellular signal (a molting hormone) and its receptor (Asahina, 2000).
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).
Coactivators MBF1 (Drosophila homolog: see Multiprotein bridging factor 1) and MBF2 mediate Bombyx mori FTZ-F1-dependent transcriptional activation in vitro by interconnecting
BmFTZ-F1, TATA binding protein TBP, and TFIIA. Temporal and spatial expression patterns of MBF2 have been examined
during embryonic and larval development of the silkworm Bombyx mori. MBF2 is detected in unfertilized eggs and
embryos until stage 26. In stage 22 embryos, MBF1, MBF2, and BmFTZ-F1 colocalize in neural cells. During the larval stage,
MBF2 is not expressed in the fat body and trachea. In the silk gland, MBF2 mRNA is constitutively expressed, but MBF2
protein appears in the period between the second day and the molting D3 stage in both the third and the fourth instars and
then disappears. MBF2 is also detected on the second and third days of the fifth instar. Immunostaining during the fourth
molt shows that MBF1, MBF2, and BmFTZ-F1 localize in the nucleus only at the D3 stage, while the two cofactors are
present in the cytoplasm at other stages. Immunoprecipitation experiments suggest that MBF1, MBF2, and BmFTZ-F1
form a complex at the D3 stage. Transient expression of these factors in Schneider cell line 2 reveals that MBF1 and MBF2
localize to the nucleus and enhance BmFTZ-F1-dependent transcription only when all three factors are present. These data
illustrate the functional regulation of MBF1 and MBF2 at the step of nuclear transport and implicate MBF2 in tissue- and
stage-specific transcription (Liu, 2000).
The most interesting finding in the present study is the
dramatic change in the subcellular localization of MBF1
and MBF2 during development. The two cofactors are found
in the nucleus only in the molting D3 stage and in the
cytoplasm in other stages. Coimmunoprecipitation experiments
suggest that MBF1 and MBF2 are present as a
complex in the cytoplasm at the molting D1 stage and then
enter the nucleus and form a ternary complex with
BmFTZ-F1 at the molting stage D3. Transient expression of
these factors in S2 cells shows that simultaneous expression
of BmFTZ-F1 is essential for the nuclear localization of
MBF1 and MBF2. It is possible that the ternary complex is
formed in the cytoplasm and transported into the nucleus
together with BmFTZ-F1. Alternatively, the MBF1-MBF2
complex may be constitutively excluded from the nucleus
through a nuclear export system but it may stay in the
nucleus once the ternary complex with BmFTZ-F1 is
formed. MBF1 contains a nuclear export signal (NES)-like
sequence in its C-terminal region, which is conserved
among eukaryotes. For example, amino acid residues 119-130 of B. mori MBF1, LGKIERAIGIKL, and the corresponding
region of human MBF1, LGIERAIGLKL, are similar to the leucine-rich NES in HIV Rev
protein, LPPLERLTL, and protein kinase inhibitor,
LALKLAGLDI. Whatever the mechanism
may be, a regulation of the action of
these coactivators has been revealed at the step of nuclear transport (Liu, 2000).
Tissue staining of embryos shows the colocalization of
BmFTZ-F1, MBF1, and MBF2 in neural cells, suggesting
that these transcription factors play a role in the embryonic
neural cells. During larval development, BmFTZ-F1, MBF1,
and MBF2 form a nuclear complex at the molting stage D3.
Transient expression of a reporter gene in S2 cells demonstrated
that MBF1 and MBF2 enhance BmFTZ-F1-dependent transcription. These observations support the
model that MBF1 and MBF2 serve as coactivators that
mediate BmFTZ-F1-dependent transcriptional activation. However, BmFTZ-F1 expression only
begins from the D3 stage and culminates later at the E1 and
E2 stages, when MBF2 has already disappeared and MBF1
resides in the cytoplasm. It is possible that
MBF1 and MBF2 are required only to initiate expression of
putative BmFTZ-F1 target genes in D3 stage. The small
amount of BmFTZ-F1 in D3 stage may be enough to play
the role because MBF1 can significantly stabilize
BmFTZ-F1 binding to DNA. When present at high levels, BmFTZ-F1 no longer
requires the aid of MBF1 and MBF2 to maintain the expression
of its target gene(s) and these cofactors may be excluded
from the system to turn off the expression rapidly
upon decline in the level of BmFTZ-F1. This may allow
immediate turn on and off of the BmFTZ-F1-dependent
gene(s) (Liu, 2000).
Why is FTZ-F1 function under precise temporal control?
FTZ-F1 appears to regulate its target genes, which should be
expressed only within a limited time. For example, Drosophila
betaFTZ-F1 governs stage-specific expression of the
EDG84A gene, which encodes a cuticle protein. Manduca sexta FTZ-F1 is likely to regulate genes
acting in cuticle formation, including the dopadecarboxylase
gene, which is responsible for melanization of cuticle. Insect cuticle consists of layers of
film. Different layers contain different kinds of proteins.
These proteins are expressed in a stage-specific manner and
are deposited systematically starting from epicuticle to
endocuticle. Any disturbance
in the order of protein deposition and melanization would
be detrimental to the cuticle formation. Indeed, disruption of the cuticle structure takes place upon
ectopic expression of FTZ-F1 when endogenous ßFTZ-F1 is
absent. These observations support the notion that temporally
restricted action of FTZ-F1 is critical for development) (Liu, 2000).
During the last larval molt in Manduca sexta, a number of transcription factors are sequentially expressed. MHR4 (Drosphila homolog: Hr4) is a transcription factor that belongs to the nuclear receptor superfamily and is a homolog of germ cell nuclear factor (GCNF)-related factors (GRFs) of Bombyx mori and Tenebrio molitor and is similar to a sequence found in the Drosophila genome. Unlike E75A and
MHR3, whose mRNAs are induced when the ecdysteroid titer increases, the expression of MHR4 mRNA occurs transiently at the onset of the decline of ecdysteroid titer followed by ßFTZ-F1 mRNA expression when the ecdysteroid titer becomes
low. When day 2 fourth epidermis is exposed to 20-hydroxyecdysone (20E) in vitro, MHR4 mRNA appears between 12 and 21 h, peaks at 24 h, and then declines. Using the protein synthesis inhibitors cycloheximide and anisomycin both in
vivo and in vitro, it has been found that the MHR4 transcript is directly induced by 20E and requires the presence of 20E for its expression. The accumulation of MHR4 mRNA, however, does not occur until a 20E-induced inhibitory protein(s)
disappears. This control of MHR4 expression is unique among the ecdysone-induced transcription factors. When the epidermis is cultured with 20E, ßFTZ-F1 mRNA is not induced until after the removal of 20E as previously found for
Drosophila and the silkworm Bombyx mori. The presence of juvenile hormone had no effect on accumulation of either transcript (Hiruma, 2001).
In Drosophila, DHR3 activates ßFTZ-F1 mRNA expression and represses ecdysteroid-induced early gene expression such as that of E74A, E75A, and BRC. Yet in Manduca, relatively little MHR3
is present between 10 and 19 h after HCS when ßFTZ-F1 mRNA is increasing. Possibly MHR3 is initially activating the ßFTZ-F1 gene, but the presence of
MHR4 suppresses this expression. Then when the 20E level
declines below the threshold for MHR4 expression, ßFTZ-F1 mRNA can appear. This scenario would be similar to the complex control of MHR4 mRNA expression that has been shown in this study (Hiruma, 2001).
BmFTZ-F1 is a sequence-specific DNA-binding factor in the silkworm Bombyx mori sharing similar
biochemical characteristics with Drosophila FTZ-F1.
Amino acid sequences in the zinc finger DNA-binding region
and the putative ligand-binding domain of BmFTZ-F1 showed strong similarity to not only FTZ-F1 but also
its mammalian homologs, LRH-1, ELP, and Ad4BP, suggesting the importance of each region for the
function of these proteins. Northern blot analyses of RNA isolated from the middle and posterior silk glands
and fat bodies show the presence of a 6.1-kb BmFTZ-F1 mRNA. Expression of
BmFTZ-F1 mRNA is intermittent, being high during larval molting and both the larval-pupal and the
pupal-adult transformations. Injection of 20-hydroxyecdysone at the third day of the 5th instar larvae
induces BmFTZ-F1 mRNA in the posterior silk gland after 24 hr. When 5th instar silk glands are cultured in
vitro, BmFTZ-F1 mRNA is induced by a 6-hr exposure to 20-hydroxyecdysone followed by 6 hr in
hormone-free medium. This suggests that BmFTZ-F1 is inducible by decline in the ecdysteroid titer
and may play an important role in the development of the silkworm as a transcription factor (Sun, 1994).
Transcriptional activation by many eukaryotic sequence-specific regulators appears to be mediated through
transcription factors that do not directly bind to DNA. MBF1 and MBF2 are two polypeptides that form a heterodimer and mediate
activation of in vitro transcription from the fushi tarazu promoter by BmFTZ-F1. Neither MBF1, MBF2, nor a
combination of them binds to DNA. MBF1 interacts with BmFTZ-F1 and stabilizes the BmFTZ-F1-DNA
complex. MBF1 also makes direct contact with TATA-binding protein (TBP). Both MBF1 and MBF2 are
necessary to form a complex between BmFTZ-F1 and TBP. A model has been proposed in which MBF1 and MBF2
form a bridge between BmFTZ-F1 and TBP and mediate transactivation by stabilizing the protein-DNA
interactions (Li, 1994).
To study the role of ecdysone and the ecdysone inducible gene in the regulation of molting and development in crustaceans, a cDNA encoding an orphan nuclear receptor family member was cloned from the eyestalk of the shrimp Metapenaeus ensis. The size of the cDNA is 4.3 kb with the longest open reading frame encoding a protein of 545 amino acid residues. The deduced amino acid sequence of the shrimp cDNA consists of regions that are characteristic of those of the nuclear hormone receptors. It shows a high degree of amino acid sequence identity in the DNA binding domain, ligand binding domain and the FTZ box, as compared to those of other invertebrates and vertebrates. Unlike the insects Drosophila melanogaster and Bombyx mori, an AF2 transactivation domain is present in the shrimp FTZ-F1. Northern blot analysis using total RNA indicates that the FTZ-F1 mRNA can also be detected in the mature ovary. Northern blot analysis and RT-PCR analysis shows that the shrimp FTZ-F1 transcripts can be detected in the ovary, newly hatched nauplius, testis, eyestalk and epidermis of the adult shrimp. Although the cDNA clone was isolated from the eyestalk library, the shrimp FTZ-F1 appears to express most abundantly in the mature oocytes. The presence of abundant FTZ-F1 specific maternal message in the late vitellogenic ovary and early nauplius indicates that it may be important for the early embryonic and larval development of the shrimp. Interestingly, shrimp FTZ-F1 can also be found in testis of the male shrimp. The presence of FTZ-F1 in other tissues such as epidermis suggests that it may also be involved in other physiological processes such as molting (Chan, 1999).
The expression and function of the Caenorhabditis elegans gene nhr-25, a member of the widely
conserved FTZ-F1 family of nuclear receptors, has been analyzed. The gene encodes two protein isoforms, only one of which has a DNA binding
domain. nhr-25 is transcribed during embryonic and larval development. A nhr-25::GFP fusion gene is expressed in the
epidermis, the developing somatic gonad, and a subset of other epithelial cells. RNA-mediated interference indicates a
requirement for nhr-25 function during development: disruption of nhr-25 function leads to embryonic arrest due to failure
of the epidermally mediated process of embryo elongation. Animals that survive to hatching arrest as misshapen larvae that
occasionally exhibit defects in shedding molted cuticle. In addition, somatic gonad development is defective in these larvae.
These results further establish the importance of FTZ-F1 nuclear receptors in molting and developmental control across
evolutionarily distant phyla (Gissendanner, 2000).
The NHR-25 LBD has an apparent AF-2 core
motif, a conserved sequence element that contributes
to both ligand binding and NR interactions with
coactivators. This
motif is absent from the known insect FTZ-F1 family
members, suggesting that the function of the NHR-25 LBD
may be more similar to those of the vertebrate FTZ-F1
family members than to that of insect FTZ-F1.
The cuticle shedding defect exhibited by some nhr-25(RNAi) larvae is similar to that resulting from disruption
of nhr-23, another NR gene that also functions in
hypodermal development.
The requirement of both nhr-23 and nhr-25 for proper
execution of the molt is particularly intriguing since the
Drosophila orthologs of both genes (DHR3 and FTZ-F1,
respectively) have been shown to function in the metamorphic
response to the molting hormone 20-hydroxyecdysone. It is speculated that nhr-23 and nhr-25 likewise
function in a regulatory cascade for direct regulation of
nematode molting. Conservation of such a regulatory
cascade would be consistent with the recent proposal of
molting as a defining evolutionary trait for a phylogenetic
grouping of nematodes, insects, and other molting animals. The molting defects observed
in RNAi animals could result from a direct disruption
of this proposed regulatory cascade or indirectly
from defects in epidermal differentiation or function.
Dissection of the exact roles of nhr-23 and nhr-25 in the
regulation of nematode molting must await a more
detailed genetic analysis. A full comparison of the functions
of the C. elegans genes with those of their apparent
Drosophila orthologs will also require a more comprehensive
analysis of the early zygotic and larval phenotypes
of the corresponding Drosophila mutants. Nevertheless, the results
described here establish an essential function for nhr-25
in nematodes, adding another phylum to those in which
members of the ancient FTZ-F1 NR family are known to
perform key developmental roles (Gissendanner, 2000).
Steroidogenic factor 1, a member of the Fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Both zFF1A and B transcripts are present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA is absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members shows a close structural relationship between the mouse liver receptor homolog, which activates the alpha1-fetoprotein gene in rodent liver. Similar to mouse steroidogenic factor 1, zFF1A regulates chinook salmon gonadotropin IIbeta subunit gene expression. zFF1B, which can bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacks both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells show that zFF1B is a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. This investigation suggests that (1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; (2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary, and (3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface (Liu, 1997).
SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homolog of Drosophila Ftz-F1. Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. Frog (Rana rugosa) SF-1/Ad4BP cDNA has been isolated from a testis lambdagt10 cDNA library. It encodes a protein of 468 amino acids, and its open reading frame shares 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homolog) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA is expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, the 5'-untranslated region (4.6kb) of the SF-1/Ad4BP gene was cloned with exons I and II. Genomic structure analysis has shown that frog SF-1/Ad4BP is also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. The use of SF-1/Ad4BP instead of Ftz-F1 is proposed for the gene that encodes SF-1/Ad4BP in vertebrates (Kawano, 1998).
A homolog (rrFTZ-F1alpha) of the FTZ-F1 gene of Drosophila has been cloned from the frog Rana rugosa. The frog gene is expressed at high levels in the testis. The FTZ-F1alpha mRNA level
in adult frogs does not change throughout the year, even during hibernation.
However, when immunohistological studies using the anti-rrFTZ-F1alpha antibody
were employed to examine which testicular cells expressed this gene, Sertoli
cells were found to produce rrFTZ-F1alpha in two seasons: the breeding
season (from March through May) and the pre-hibernating season (from October
through November). Interstitial cells, however, expressed the gene only in the breeding season (from April through May). Taken together, the results suggest that the rrFTZ-F1alpha expression is regulated at the post-transcriptional step, and that the rrFTZ-F1alpha may play an important role(s) in the seasonal activities of
Sertoli and interstitial cells in the frog testis (Takase, 2001).
The human homolog of the Drosophila melanogaster orphan nuclear receptor Fushi tarazu factor 1 (Ftz-F1), NR5A2 (hB1F), was initially identified as a regulatory factor that binds and activates enhancer II of hepatitis B virus. NR5A2 (hB1F) is expressed specifically in pancreas and liver, playing important roles in the regulation of several liver-specific genes. A detailed analysis on the genomic
structure and promoter activity will greatly promote future studies on the
function of the NR5A2 (hB1F) gene. In this report, a bacterial artificial
chromosome clone and several phage clones covering the NR5A2 (hB1F) gene were
isolated and the complete genomic sequence was obtained. Alignment of different
cDNAs of the NR5A2 (hB1F) gene with the genomic sequence facilitated the
delineation of its structural organization, which spans over 150 kb and consists
of eight exons interrupted by seven introns. RT-PCR and 3'-RACE reveals that
utilization of two polyadenylation signals results in 3.8 and 5.2 kb
transcripts. The transcription start site of the NR5A2 (hB1F) gene maps downstream of a canonical TATA box. An upstream fragment containing binding sites for several liver-specific and ubiquitous transcription factors exhibits hepatocyte-specific promoter activity. Transient transfections indicated that hepatocyte nuclear factors HNF1 and HNF3beta can activate NR5A2 (hB1F) promoter (Zhang, 2001).
Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action,
its domain function was investigated by mutagenesis and functional analyses. The putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation (Li, 1999).
The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Mullerian inhibiting substance, and gonadotropin genes. Attempts have been made to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, all of which share the DNA-binding domain. Analyses of the transcriptional activity of these variants reveal that the activity of ELP-2 and SF-1 is much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins results in a complete loss of transactivation. Several lines of evidence demonstrate that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhances GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibits the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirms the interaction between SRC-1 and GAL4-SF-1 and demonstrates that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhance transcriptional activity of GAL4-SF-1. It is concluded that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1 (Ito, 1998)
Multiprotein bridging factor 1 (MBF1) is a coactivator that mediates transcriptional activation by interconnecting the general
transcription factor TATA element-binding protein and gene-specific activators such as the Drosophila nuclear receptor FTZ-F1
or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function,
especially in transcription, remains unclear. Reported here is the cDNA cloning and the functional analysis of hMBF1. Two isoforms,
termed hMBF1alpha and hMBF1beta, have been identified. hMBF1alpha mRNA is
detected in a number of tissues, whereas hMBF1beta exhibits tissue-specific expression. Both isoforms bind to TBP and Ad4BP/SF-1, a mammalian
counterpart of FTZ-F1, and mediate Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 is detected in the cytoplasm by immunostaining,
coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induces accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus
through its binding to Ad4BP/SF-1. hMBF1 also binds to ATF1, a member of the basic leucine zipper protein family, and mediates its activity as a transcriptional
activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes (Kabe, 1990).
PNRC (proline-rich nuclear receptor coregulatory protein) was identified using
bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening
of a human mammary gland cDNA expression library. PNRC is unique in that it has
a molecular mass of 35 kDa, significantly smaller than most of the coregulatory
proteins reported so far, and it is proline-rich. PNRC's nuclear localization
has been demonstrated. In the yeast
two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1
in a ligand-independent manner. PNRC was also found to interact with the
ligand-binding domains of all the nuclear receptors tested in a ligand-dependent manner, including estrogen
receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR),
progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid
receptor (RAR), and retinoid X receptor (RXR).
Functional AF2 domain is required for nuclear receptors to bind to PNRC.
Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to
demonstrate a direct contact between PNRC and nuclear receptors such as SF1.
A coimmunoprecipitation experiment using Hela cells that express PNRC and ER was
performed to confirm the interaction of PNRC and nuclear receptors in vivo in a
ligand-dependent manner. PNRC functions as a coactivator to enhance
the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor
alpha-1), PR, and TR. A 23-amino acid sequence in the
carboxy-terminal region, amino acids 278-300, is critical and sufficient for
the interaction with nuclear receptors. This region is proline rich and contains
a SH3-binding motif, S-D-P-P-S-P-S. The two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast
two-hybrid screening study. These two proteins belong to a new family of nuclear
receptor coregulatory proteins (Zhou, 2000).
PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified
using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening
of a human mammary gland cDNA expression library. PNRC2 is an unusual
coactivator in that it is the smallest coactivator identified so far, with a
molecular weight of 16 kDa, and interacts with nuclear receptors using a
proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan
receptors SF1 and estrogen receptor-related receptor alpha1 in a
ligand-independent manner. PNRC2 was also found to interact in a ligand-dependent manner with the
ligand-binding domains of estrogen receptor, glucocorticoid receptor,
progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X
receptor. A functional activation function 2 domain
is required for nuclear receptors to interact with PNRC2. Using the yeast
two-hybrid assay, the region amino acids 85-139 were found to be responsible for
the interaction with nuclear receptors. This region contains an SH3
domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A
mutagenesis study has shown that the SH3 domain-binding motif is important for
PNRC2 to interact with all the nuclear receptors tested. These results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins (Zhou, 2001).
The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a critical
developmental regulator in the urogenital ridge, because mice targeted for
disruption of the SF-1 gene lack adrenal glands and gonads. SF-1 was recently
shown to interact with DAX-1, another orphan receptor whose tissue distribution
overlaps that of SF-1. Naturally occurring loss-of-function mutations of the
DAX-1 gene cause the human disorder X-linked adrenal hypoplasia congenita (AHC),
which resembles the phenotype of SF-1-deficient mice. Paradoxically, however,
DAX-1 represses the transcriptional activity of SF-1, and AHC mutants of DAX-1
have lost repression function. To further investigate these findings, the interaction between SF-1 and DAX-1 was characterized and it was found that their
interaction indeed occurs through a repressive domain within the carboxy
terminus of SF-1. Furthermore, DAX-1 recruits the nuclear
receptor corepressor N-CoR to SF-1, whereas naturally occurring AHC mutations of
DAX-1 permit the SF-1-DAX-1 interaction, but markedly diminish corepressor
recruitment. Finally, the interaction between DAX-1 and N-CoR shares
similarities with the interaction between the nuclear receptor RevErb and N-CoR, because the
related corepressor SMRT is not efficiently recruited by DAX-1. Therefore,
DAX-1 can serve as an adapter molecule that recruits nuclear receptor
corepressors to DNA-bound nuclear receptors like SF-1, thereby extending the
range of corepressor action (Crawford, 1998).
Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1) genes are
essential for mammalian gonadogenesis prior to sexual differentiation. In males,
SF-1 participates in sexual development by regulating expression of the
polypeptide hormone Mullerian inhibiting substance (MIS). WT1-KTS isoforms [the KTS isoform is an alternative splice variant that has an insertion of three amino acids (KTS) between the third and fourth zinc fingers] associate and synergize with SF-1 to promote MIS expression. In
contrast, WT1 missense mutations, associated with male pseudohermaphroditism in
Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked,
candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between
SF-1 and WT1, most likely through a direct interaction with SF-1. It is proposed
that WT1 and Dax-1 functionally oppose each other in testis development by
modulating SF-1-mediated transactivation (Nachtigal, 1998).
Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription
factor that is also essential for adrenal and gonadal development. Two
mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene
encoding Ad4BP in adrenocortical cells have been proposed: (1) the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and (2) a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. The
transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells has been investigated from several angles, including the above two mechanisms. Using a
series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and
site-directed mutagenesis for transient transfection studies, an E box element,
CACGTG at -87/-82 from the transcriptional start site, was found to be
essential for the transcription of the hFTZ-F1 gene in mouse or human
adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite
the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first
intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was
found to be unlikely in the hFTZ-F1 gene mainly due to site-directed
mutagenesis. In addition, the forced expression of Ad4BP has little effect on
hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such
Ad4BP-independent regulation of the hFTZ-F1 gene is in striking contrast to the
regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which
the proximal promoter activity is Ad4BP-dependent and the transactivation by
Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional
regulation of the DAX-1 gene has been reported, DAX-1 did not affect the
transcriptional activity of the hFTZ-F1 gene in this study. Taken together, these
observations suggest that the E box is indeed required for the expression of the
FTZ-F1 gene, at least in mammalian species, but may not determine the
tissue-specific expression of the hFTZ-F1 gene, and that, unlike the
steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be
independent of both Ad4BP and DAX-1 (Oba, 2000).
The nuclear receptor steroidogenic factor-1 (SF-1) is essential for development of the gonads, adrenal gland, and the ventromedial hypothalamic nucleus. It also regulates the expression of pivotal steroidogenic enzymes and other important proteins in the reproductive system. A domain located C-terminal to the DNA binding domain of SF-1, exhibits transcriptional repression function. Point mutations in this domain markedly potentiate the transcriptional activity of native SF-1. Using an SF-1 region that spans this proximal repression domain as bait in a yeast two-hybrid system, an SF-1 interacting protein has been cloned that is homologous to human DP103, a member of the DEAD box family of putative RNA helicases. DP103 directly interacts with the proximal repression domain of SF-1, and mutations in this domain abrogate its interaction with DP103. DP103 is expressed predominantly in the testis and is also expressed at a lower level in other steroidogenic and nonsteroidogenic tissues. Functionally, DP103 exhibits a
native transcriptional repression function that localizes to the C-terminal
region of the protein and represses the activity of wild-type, but not mutant,
SF-1. Together, the physical and functional interaction of DP103 with a
previously unrecognized repression domain within SF-1 represents a novel
mechanism for regulation of SF-1 activity (Ou, 2001).
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ftz-f1
continued:
Biological Overview
| Regulation
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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