bicoid
Bicoid distinguishes among related DNA-binding sites in vivo by a
specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain)
and bp 7 of the site. Bicoid binds directly to the promoters of genes whose
transcription it regulates. The amino acid 9 contact is necessary for Bicoid to direct anterior
pattern formation. Bicoid requires multiple binding
sites to activate transcription of target genes. The distance between binding sites is
critical for Bicoid activation but, unexpectedly, this critical distance differs between Drosophila
and promoter activity in S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary
protein(s) not present in S. cerevisiae (Hanes, 1994).
Comparison of the DNA targets of Bicoid, Fushi tarazu and Orthodenticle reveal the importance of the amino acid at position 50 of the homeodomain in discriminating between bases that lie adjacent to the TAAT core of homeodomain binding sites. FTZ has a preference for TAATG due to the presence of glutamine at position 50, while Bicoid prefers the consensus sequence TAATCC, specified by lysine at position 50. OTD also has a lysine at position 50 and the consensus sequence recognized is similar to that of BCD. Structural studies suggest that water-mediated hydrogen bonds and van der Walls contacts underlie the preferences for bases adjacent to the TAAT core (Wilson, 1996).
The template and coactivator requirements for synergistic transcription directed by a single
activator bound to multiple sites has been determined in the case of Bicoid. Mutagenesis studies in
combination with protein binding experiments and reconstituted transcription reactions have identified
two independent activation domains of BCD that target different coactivator subunits (TAFII110
and TAFII60) of TFIID, a major component of the transcriptional apparatus. The presence of both coactivators is
required for BCD to recruit the TATA binding protein (TBP)-TAF complex to the promoter and
direct synergistic activation of transcription (Sauer, 1995).
A
quadruple complex containing the TATA binding protein, TBP, as well as TAFII250, TAFII110, and TAFII60,
mediates transcriptional synergism by BCD and HB, whereas triple TBP-TAFII complexes lacking
one or the other target coactivator fail to support synergistic activation. Thus, the concerted action of multiple regulators with
different coactivators helps to establish the pattern and level of segmentation gene transcription
during Drosophila development (Sauer, 1996).
The graded distribution of Bicoid protein which activates subordinate genes in distinct anterior domains, specifies anterior body pattern in Drosophila.
Subsequently, transcription of
these target genes is repressed at the anterior pole owing to the activity of the receptor tyrosine
kinase torso (tor). Both activation by BCD and repression by TOR can be reproduced
by a minimal promoter containing only BCD-binding sites upstream of a naive transcriptional start
site. Repression requires the D-raf kinase and is associated with phosphorylation of BCD protein.
Repression does not require either tailless or huckebein, which were previously thought to
constitute the sole zygotic output of the TOR signaling system. Addition of a heterologous
transcriptional activation domain to bcd renders the protein insensitive to tor-mediated repression.
Thus phosphorylation of BCD, resulting from the activity of the TOR signal transduction cascade,
down-regulates transcriptional activation by the BCD morphogen (Ronchi, 1993).
The
Torso receptor tyrosine kinase cascade represses expression of Bicoid targets at the
most anterior tip of the embryo. Neither the homeodomain (HD) nor
the activation domain of BCD is targeted by Torso in the anterior repression of BCD targets. When a BCD mutant protein
whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a
reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then
repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function
but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of
its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein,
whose activation domain has been replaced by a generic activation domain, indicates that the
activation domain of BCD is also not specifically required for its down-regulation by Torso. It is
propose that Torso does not affect the ability of BCD to bind DNA, but instead directs modification
of BCD or of a potential BCD co-factor, which renders the BCD protein unable to activate
transcription (Bellaiche, 1996).
Bicoid protein
binds cooperatively to its sites within a hunchback enhancer element. A less than
4-fold increase in Bicoid concentration is sufficient to achieve an unbound/bound
transition in DNA binding. Bicoid molecules can interact with each other. One
mechanism to achieve a sharp on/off switch of gene expression in response to a
morphogenetic gradient is cooperative DNA binding facilitated by protein-protein
interaction (Ma, 1996).
Four maternal systems are known to pattern the early Drosophila embryo. The key component of the anterior system is the homeodomain protein Bicoid
(Bcd). Bcd needs the contribution of another anterior morphogen, Hunchback (Hb), to function properly: Bcd and Hb synergize to organize anterior
development. A molecular mechanism for this synergy has been proposed to involve specific interactions of Bcd and Hb with TATA-binding
protein-associated factors (TAFIIs) that are components of the general transcription machinery. Bcd contains three putative activation domains: a
glutamine-rich region, which interacts in vitro with TAFII110; an alanine-rich domain, which targets TAFII60, and a C-terminal acidic region, which has an
unknown role (Sauer, 1996a and b). Hb interacts only with TAFII60. Transcriptional activation of
a bcd target, the hb promoter, is synergistically enhanced in vitro by Bcd and Hb. However, this effect is observed only when both TAFII60 and TAFII110 are
present. It has been suggested that the synergy observed in vivo is because of the corecruitment of TAFII110 and TAFII60 by Bcd and Hb, respectively.
Flies were generated carrying bcd transgenes lacking one or several of these domains to test their function in vivo. Surprisingly, a bcd
transgene that lacks all three putative activation domains is able to rescue the bcdE1 null phenotype to viability. Moreover, the development of these embryos
is not affected by the presence of dominant negative mutations in TAFII110 or TAFII60. This means that the interactions observed in vitro between Bcd and
TAFII60 or TAFII110 aid transcriptional activation but are dispensable for normal development (Schaeffer, 1999).
Bcd contains three putative activation domains: a glutamine-rich
(Q) domain, an alanine-rich (A) domain, and a C-terminal acidic region (C). The domains of interaction of Bcd with the
TAFIIs have been mapped in vitro: TAFII110 binds to the Q domain, whereas TAFII60 binds to the A domain (Sauer, 1995b). The
C-terminal region has been shown to be dispensable for bcd function in vivo, but it contributes to increase activation in yeast
(Dreiver, 1989). Interestingly, the entire activation domain of Bcd can be functionally replaced by a generic activation domain (random acidic
amphipathic helix B6) from Escherichia coli (Schaeffer, 1999 and references).
Because the corecruitment of TAFIIs could provide
a powerful explanation to the synergistic effect observed between Bcd
and Hb during early development, bcd rescue
constructs were assembled encoding proteins lacking one or several domains of
interaction with the TAFIIs. These
constructs contain DNA encoding the wild-type or deletion variants of
the Bcd protein in the context of the genomic bcd locus. Their ability to rescue the bcdE1
mutant phenotype was examined. bcdE1 is a null allele
that is truncated after the homeodomain and does not appear to produce
a stable protein. Embryos from bcdE1
mothers lack all head and thorax segments and some abdominal segments.
Instead, they have a second telson at the anterior end. A construct
encoding a wild-type Bcd protein completely rescues the phenotype and
behaves like the endogenous gene.
Surprisingly, deletion of the A or Q (BcdDeltaA or BcdDeltaQ, respectively) domains, or of both domains (BcdDeltaQA) does not abolish rescue activity. The
BcdDeltaC transgene displays a hypomorphic bcd phenotype with
only a very partial head but normal thorax and abdominal segments. The BcdDeltaAC construct shows an almost wild-type
cuticular phenotype with only small head defects.
The lines bearing the BcdDeltaQC deletion exhibit very low rescue, with only the lack of a duplicated telson at the
anterior. Although the lines lacking the C domain have a lower potential for rescue than the BcdDeltaQ, BcdDeltaA, and BcdDeltaQA lines, this is likely because of lower levels of expression rather than weaker
activator proteins. Consistent with this interpretation, the domain of
the hb staining is moved anteriorly in these lines. It has been argued that the position of the
posterior border of hb indicates the amount of Bcd protein produced, whereas the intensity of staining within this domain reflects
the activation potential of the Bcd deletion variant. In fact, two
lines expressing a truncation construct lacking the C domain as well as
both the Q and A domains (BcdDeltaQAC) have strong rescue activity: these
lines exhibit rescue of the thorax and abdominal segments, loss of
duplication of the telson at the anterior, and development of head
structures. One of these two BcdDeltaQAC lines
completely rescues the bcdE1 phenotype. Seventy-five percent of the embryos exhibit wild-type cuticles, and 30% of those are viable. This rescue is probably because of high-level expression of the transgene. Consistent with this, the posterior border of bcd target genes and the
position of the cephalic furrow (both strong indicators of the slope of the Bcd protein gradient) are more posterior in this line than in
wild-type embryos.
Because the phenotypic rescue of BcdDeltaQAC is not fully penetrant, the
Q, A, and C domains do provide some contribution to the activity of Bcd
but are not essential (Schaeffer, 1999).
In a wild-type embryo, hb is transcribed in a broad anterior
domain as well as in a more restricted posterior domain. Transcription
of the anterior domain of hb is under the control of Bcd
and, therefore, is lost in embryos from
bcdE1 mutant mothers. Whenever Bcd
activity is altered, hb expression is also changed. The posterior border of the anterior hb expression
domain is, like the cephalic furrow, positioned in response to the
amount of Bcd, whereas the level of expression within this domain
reflects the activation potential of the respective Bcd variant.
In embryos laid by females homozygous for the
bcdE1 mutation and carrying two copies of
the transgene BcdDeltaC, BcdDeltaAC, or
BcdDeltaQC, the posterior boundary of hb expression
is significantly moved toward the anterior. This is consistent with the fact that
these lines only show partial rescue of the bcdE1 phenotype, which appears to be
because of a lower level of expression of the transgenes. However,
within the hb domain, hb is transcribed at normal
levels, which indicates normal activity of these Bcd deletions. The
BcdDeltaQ and BcdDeltaQA lines
that fully rescue the bcdE1 phenotype show
normal amounts of hb expression within a slightly enlarged
domain. One of the BcdDeltaA lines exhibits a widely enlarged domain of hb transcription. This is most likely because of the
very high level of transgene expression observed by immunostaining. Another BcdDeltaA line shows a normal rescue pattern and exhibits a normal position of the cephalic furrow.
Finally, the domain of expression mediated by the BcdDeltaQAC line that is able to rescue the
bcdE1 phenotype to viability appears
slightly enlarged compared to wild-type Bcd, indicating a high level of
transgene expression. Thus, Bcd is able to activate its target gene
hb, even in the absence of the protein domains that have
been shown to interact in vitro with
TAFII60 and TAFII110 (Schaeffer, 1999).
TAFII110 and TAFII60 have
been shown to mediate transcriptional activation in vitro, and mutations in the respective genes have been isolated in a
dominant modifier screen in Drosophila (Sauer, 1996). Mutations in
TAFII60 and
TAFII110 are homozygous embryonic lethal and cell lethal during eye development and oogenesis. However, viable dominant negative alleles of these two genes have allowed Sauer (1996) to investigate their effects on transcription. These mutations do not appear to cause a general reduction of transcription, but instead, they affect the expression of only a subset
of genes. The
TAFII60YY
allele encodes a protein that contains two tyrosines residues inserted
at amino acid 207. The
TAFII110DeltaC allele
lacks the C-terminal 126 amino acids of the protein. Although TAFII60 and TAFII110 have
been shown to bind to both Bcd and TAFII250, the
mutant proteins are defective for their interaction with
TAFII250 but still retain their in
vitro interaction with Bcd. Because these mutant proteins can
still interact with the transcription activators but cannot mediate
interaction with the transcription machinery, they can be considered
dominant negative alleles (Schaeffer, 1999).
Although the BcdDeltaQAC truncation construct lacks the identified
TAFII60 and TAFII110
interaction domains, it is still able to strongly activate the
bcd target genes. It is possible that the BcdDeltaQAC
truncation does not affect Bcd function because Bcd can still interact
with Hb, its partner involved in synergistic activation. Hb might still
recruit TAFII60 to the promoter, even if Bcd
cannot do it directly. If this is the case, the genetic rescue of
bcdE1 by BcdDeltaQAC should be highly
dependent on the interaction between Hb and
TAFII60. To test this possibility,
the ability of BcdDeltaQAC to rescue a bcd mutant phenotype was examined in
the presence of either the dominant negative
TAFII60YY,
which is expected to interfere with activation by BcdDeltaQAC, or TAFII110DeltaC, which
should not interfere with the rescue (Schaeffer, 1999).
Typically, a single copy of the
BcdDeltaQAC transgene is able to almost completely rescue the progeny of
females homozygous mutant for bcdE1. Neither of the
TAFII60YY nor
TAFII110DeltaC
dominant negative mutations affect the rescue ability of the BcdDeltaQAC
construct: the bcdE1 phenotype in the
progeny of double mutant females
TAFII60YY,
bcdE1/bcdE1 or
TAFII110DeltaC,
bcdE1/bcdE1 is
rescued by the BcdDeltaQAC as well as that of the single
bcdE1/bcdE1 mutant. Rare rescue to wild type can
be observed at the same frequency, independently of the presence or absence of dominant negative
TAFII mutations (Schaeffer, 1999).
The ability of BcdDeltaQAC to fully rescue the
bcdE1 phenotype is surprising with respect to
previous reports by using injection of mRNA encoding truncation
constructs of bcd (Dreiver, 1989). mRNA injections of a truncated
construct similar to BcdDeltaQAC show a low rescue potential, even at
high concentrations: the construct only suppresses the formation of
posterior structures at the anterior. It induces with high frequency
thoracic structures and, more rarely, structures of the gnathal region
of the head. The difference between the results obtained by mRNA
injection or by using transgenic flies could be because of the lack of
anterior localization of the injected mRNA. To carry out its
morphogenetic function, a gradient of Bcd activity is established by
the tight localization of the bcd mRNA to the anterior pole
of the egg. This localization depends on the maternal genes
exuperentia (exu), swallow
(swa), and staufen (stau). Embryos
from females homozygous mutant for exu, swa, or
stau show head defects similar to those of embryos
from females mutant for weak bcd alleles. The labrum is
absent and the pharyngeal head skeleton is reduced. Strikingly, the
swa, exu, and stau mutants, the weak
bcd alleles, and the embryos rescued by injection of the
bcd (1-264) mRNA, are all reminiscent of the
torso phenotype: lack of dorsal bridge and labrum. This suggests that Tor has a enhancing effect on Bcd activity, as has
been reported for bcd target gene expression. The tight localization of the BCD mRNA might, thus, be required
for Tor to enhance the function of the Bcd protein before it migrates (Schaeffer, 1999 and references).
It has been shown that
Bcd is phosphorylated in response to Tor activity (Ronchi, 1993). The strong
remaining activity in the BcdDeltaQAC protein that presents such an
extensive deletion might be because of the presence of the S/T rich
region located between the homeodomain and the Q, A, and C activation
domains. The S/T-rich sequence is the target of
phosphorylations induced by the activity of the terminal Tor pathway:
in embryos lacking Tor activity, Bcd phosphorylation is greatly
diminished. Activation of the Tor signal transduction pathway
leads to activation of a mitogen-activated protein kinase, a nuclear
kinase that has been implicated in the phosphorylation of transcription
factors. Most mitogen-activated protein phosphorylation sites of Bcd
have been mapped in vitro to the S/T rich region.
Site-directed mutagenesis indicates that most of the Bcd
phosphorylations occur on these sites in vivo (F. y, R. Sturny, F. Catala, and N. Dostatni, unpublished work, cited in Schaeffer, 1999). The ability of
the Tor pathway to create negative charges in this region might allow
the generation of an acidic activation domain that compensates for the
loss of the other Q, A, and C activation domains (Schaeffer, 1999).
It has been shown that the Bcd phosphorylations mediate part of the Tor
enhancing effect on Bcd activity (F. Janody, R. Sturny, F. Catala, and N. Dostatni, unpublished work cited in Schaeffer, 1999). Hence, the rescue by BcdDeltaQAC could be
explained by the fact that the S/T rich region is phosphorylated
in vivo by the Tor cascade, thus creating an acidic
activation domain on Bcd before it forms its concentration gradient. As
long as the homeodomain and the S/T rich region are intact, good rescue
is achieved, especially if the transgene is expressed at high levels
and mRNA translation occurs in the region of Tor activity (Schaeffer, 1999).
The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its
mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however,
are required for segmentation, nor for expression of most genes known to be regulated by Chip. Chip also interacts
with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and Chip potentiates activity of
one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in
segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers (Torigoi, 2000).
Full-length Chip interacts with the HD proteins Bicoid (Bcd) and Ftz,
and with a fragment of the Su(Hw) insulator protein. The HD protein Otd binds almost as efficiently as does Bcd and Ftz to Chip, but the Eve HD protein binds poorly, a result possibly attributable to improper folding of the in
vitro-translated protein. The domains of Chip involved in homotypic and heterotypic interactions include the LIM interaction domain (LID) and the self-interaction domain (SID). Deletion of the LID reduces interaction with
Apterous. That deletion,
however, has no effect on interaction with Bcd, Ftz, Su(Hw)DeltaCTD, or
Chip. In contrast, two other deletion
mutants, ChipDelta404-465 and ChipDelta441-454, reduce binding to Bcd,
Ftz, Su(Hw)DeltaCTD, and Chip but have little effect on binding to
Apterous. On the basis of this and
additional deletion mutants, Chip residues 439-456 are identified as the region that interacts with
the HD proteins, Su(Hw), and with Chip itself. This region is termed
the other interaction domain (OID) (Torigoi, 2000).
The domains of Bcd and Su(Hw) that interact with Chip were mapped to
determine if the OID recognizes a common motif in its diverse
interaction partners. The N-terminal half of Bcd (residues 1-255)
contains the HD and everything needed to rescue bcd mutants in vivo. The N-terminal half of Bcd interacts with Gst-Chip, whereas the C-terminal half (residues 246-489) does not. Smaller Bcd fragments containing the HD (residues 1-190, 1-166 or 57-255) bind more weakly than does the 1-255 fragment, and a fragment (residues 57-166) consisting mostly of the HD (residues 92-151) does not bind. Thus, residues on both sides of the HD are required for strong binding. Similar results were obtained with the Otd HD protein. The region of Su(Hw) that contains 12 zinc fingers (residues 204-672) interacts with Gst-Chip, whereas the N-terminal region (residues 1-190) and the C-terminal region (residues 706-944) do not. Mutation of any one of the 12 zinc fingers does not significantly affect binding to Chip. The regions of Bcd, Su(Hw), and Chip that interact with the Chip OID are not homologous at the primary sequence level (Torigoi, 2000).
The interactions between Chip and HD proteins in vitro raise the question of whether Chip affects the activities of HD proteins in vivo. The effect of Chip on Bcd activity in embryos was tested because
both Chip and Bcd are provided maternally and do not regulate each
other's expression. Thus, any effect of Chip on Bcd is likely to be
direct. The design of the experiment that shows that in embryos reducing Chip
activity decreases the activity of a partially defective Bcd protein,
was guided by the following considerations. To demonstrate a helping effect of Chip on Bcd activity, maternal Chip could not be simply eliminated because that manipulation results in a more severe segmentation defect than does elimination of Bcd itself. Nor could the dosage of maternal Chip be halved because that
change has no effect, even if the maternal Bcd level is also reduced by
one-half. Moreover, zygotic Bcd makes no contribution to
segmentation; heretofore, no effect has been seen on
segmentation by changing the level or nature of zygotically expressed
Chip. To detect an effect of Chip on Bcd activity, therefore, the activities of both Bcd and Chip were reduced to less than that provided
by a single maternal dose of each. This was accomplished by producing
doubly mutant mothers: these mothers were homozygous for the
bcdE3 allele, which encodes a mutant with
reduced DNA-binding activity, and were also heterozygous for the
Chipg96.1 allele. This latter mutant allele
encodes the SID fragment, which acts as a
dominant negative, inhibiting, but not eliminating, maternal Chip
activity. It was deduced that the SID fragment inhibits maternal Chip
activity from the observations that
Chipg96.1/Chipg96.1
embryos produced by Chipg96.1/+
mothers die before reaching the larval stage (some display a mild
segmentation defect), whereas all
Chip-/Chip-
embryos produced by Chip-/+
mothers segment normally and die as larvae. It was further deduced that at
least one maternal and two zygotic doses of the SID fragment are
required to cause embryonic lethality from the fact that
Chipg96.1/+ embryos from
Chipg96.1/+ mothers segment
normally and survive to adulthood. Presumably the SID fragment,
produced in this experiment both maternally and zygotically, forms
nonfunctional multimers with maternal wild-type Chip. On average, embryos from Chipg96.1/+; bcdE3/bcdE3 mothers (and wild-type fathers) produce nearly one segment less than do embryos from bcdE3/bcdE3 mothers (Torigoi, 2000).
These results suggest that Chip increases interactions between Bcd
molecules. Thus, in yeast with nonsaturating levels of Bcd,
Chip increases activation by Bcd from two strong binding sites
separated by a weak site or by a nonbinding spacer, but not from one or
three contiguous strong sites. Moreover, Chip does not increase
activation above levels that are achieved with high concentrations of
Bcd itself. Bcd binds DNA cooperatively, mediated by interactions of
regions overlapping those that interact with Chip, and it is suggested that Chip interacts with Bcd to amplify that
cooperativity. It is unlikely that Chip itself is a
transcriptional activator. Previous experiments have shown that Chip
does not activate when tethered upstream of yeast promoters but it
can induce activation by recruiting an activation domain fused to LIM
domains (Torigoi, 2000).
Chip potentiates Bcd activity in the Drosophila embryo when the Bcd activity is low. This effect
is consistent with previous studies on the expression of segmentation
genes in embryos lacking maternal Chip activity. Embryos contain a
gradient of Bcd protein, with a high concentration at the anterior end
and a low concentration at the posterior end. Loss of maternal
Chip strongly reduces all seven blastoderm stripes of Eve protein
produced by the eve pair-rule gene. Many, if not all of
these stripes are also regulated by Bcd, even though most occur in
regions with low to intermediate Bcd concentrations. The
eve stripes are activated by several remote enhancers located ~1.5-9 kb from the promoter, and Bcd-binding sites
are critical for activation by at least the stripe 2 enhancer.
It is likely, therefore, that Chip increases eve expression
at least in part by increasing binding of Bcd to the enhancers. Accumulation of the Hb protein is not substantially affected by loss of
maternal Chip even though hb expression is dependent on
Bcd and several Bcd-binding sites just upstream of the promoter. This lack of an effect of Chip is not unexpected, however,
because hb is expressed in the anterior end where the Bcd
concentration is the highest (Torigoi, 2000 and references therein).
It is suggested that
Chip plays two roles in the regulation of gene expression: (1) Chip is likely to aid binding of proteins to
enhancers, and (2) Chip is also likely to
function between enhancers and promoters to support enhancer-promoter
communication. The in vitro interaction between Chip
and the Su(Hw) insulator protein shown here is consistent with the
notion that Su(Hw) is directly antagonistic to Chip activity as
previously demonstrated genetically at the cut locus. It remains to be seen how, if these speculations are correct, Chip facilitates enhancer-promoter communication and how that communication is disrupted by Su(Hw). It is believed that Su(Hw) blocks activation not by reducing the binding of proteins to enhancers, but rather by hindering enhancer-promoter communication. For instance, an enhancer blocked in its interaction with one
promoter by Su(Hw) can nevertheless activate a second promoter located
on the opposite side of the enhancer from Su(Hw). Thus,
although Su(Hw) is antagonistic to Chip, it is unlikely to affect
binding of proteins to enhancers. It is also unlikely that Chip
functions merely by preventing binding of Su(Hw) to gypsy because Chip
is also important for the expression of several genes, e.g.,
cut and eve, in the absence of gypsy and Su(Hw) (Torigoi, 2000).
Bicoid directs anterior development in Drosophila
embryos by activating different genes along the
anterior-posterior axis. However, its activity is down-regulated
at the anterior tip of the embryo, in a process
known as retraction. Retraction is under the control of
the terminal polarity system, and results in localized
repression of Bicoid target genes. A
Drosophila homolog of human SAP18 (Sin3A-associated polypeptide p18), a member of the
Sin3A/Rpd3 histone deacetylase complex (HDAC), is described. Termed Bicoid interacting protein 1 (Bip1), the SAP18 homolog interacts
with Bicoid in yeast and in vitro, and is expressed
early in development coincident with Bicoid. In tissue
culture cells, Bip1 inhibits the ability of Bicoid to
activate reporter genes. These results suggest a model in
which Bip1 interacts with Bicoid to silence expression
of Bicoid target genes in the anterior tip of the embryo (Zhu, 2001).
A cDNA encoding Bin1 was identified using a custom two-hybrid selection in
which Bicoid was bound to DNA via its homeodomain. The 5' end of the bin1 cDNA was cloned by RACE and a full-length cDNA sequence was
assembled. The bin1 cDNA encodes a 150-amino-acid protein with a
predicted molecular weight of 17.3 kDa. The protein is
58% identical to the human and murine SAP18 proteins and 42% identical
to a C. elegans ORF. The Drosophila
genome sequence does not predict any other homologs. A search of
the Berkeley Drosophila Genome Project database revealed an EP insertion
line EP(3)3462 in which an EP-transposon
is inserted 259 bp upstream of the bin1 start
codon, and 151 bp upstream of the transcription initiation
site. This insertion is within the 5' UTR of
nebula, an ORF oriented opposite to that of bin1 (Zhu, 2001).
LexA-Bicoid fusion proteins were used to map the regions within Bicoid that are important
for the Bin1-Bicoid interaction. In these experiments, Bin1 was
fused to the B42 activation domain. Results from these
assays show that interaction with Bin1 does
not require the Bicoid acidic activation domain (AD), or
the polyglutamine (Q) or polyalanine (A) domains. The
homeodomain is not sufficient for interaction, but seems
to be required along with flanking regions, each of which
contributes modestly to the interaction. Thus, the interaction
requires two distinct regions of Bicoid, aa 1-95 and
aa 163-246. To test whether Bin1 interacts directly with
Bicoid in vitro, the full-length Bin1 was
expressed as a GST fusion protein in Escherichia coli.
GST-Bin1 was attached to glutathione beads and used
in pull-down experiments with 35 S-methionine-labeled,
full-length Bicoid generated by in vitro translation. GST-Bin1
interacts with Bicoid in this system. Thus, Bin1
interacts directly with Bicoid in vitro (Zhu, 2001).
If Bin1 is required for Bicoid function, then its
protein expression pattern should overlap with that of
Bicoid temporally and spatially. Bicoid is translated
from maternally deposited mRNA shortly after egg
laying. After 3 h of development, the protein level begins
to diminish, and after 4 h, Bicoid is undetectable. To determine
when the Bin1 gene is expressed, Northern
analysis was carried out using mRNA isolated from
unfertilized eggs and from 0- to 2-h, 2- to 4-h, and 4- to
24-h developing embryos. A Bin1 mRNA of about 500 nt is detected in
unfertilized eggs and in early embryos. The mRNA levels peak around the cellularization to early gastrulation stages (2-4 h). These results indicate that Bin1 is transcribed both maternally and zygotically, and the Bin1 mRNA is present at the time that Bicoid is present (Zhu, 2001).
To determine the spatial distribution of Bin1
mRNA within the early embryo, whole-mount in situ
hybridization was carried out using anti-sense. In contrast to the highly localized BCD mRNA, Bin1 mRNAs are distributed throughout
the early embryo and in the unfertilized egg. Thus, the expression
pattern of Bin1 overlaps spatially with that of
Bicoid protein, which is detectable over the anterior two-thirds
of the embryo. The temporal and spatial expression pattern of
Bin1 mRNA suggests that Bin1 protein
is present throughout the embryo, although proof of
protein localization will require anti-Bin1 immunostaining (Zhu, 2001).
Based on the role of human SAP18 in transcription
repression by HDAC complexes, tests were performed to see whether over-expression
of Bin1 inhibits Bicoid-dependent
transcription in Drosophila S2 cells. In this assay, plasmids
expressing Bicoid and Bin1 were co-transfected
along with a Bicoid binding site-CAT reporter
construct. The results indicate a dose-sensitive inhibition of
Bicoid-dependent transcription by Bin1.
The effect is greater at lower Bicoid concentrations,
suggesting that the ratio of Bin1 to Bicoid is
important for the effect (Zhu, 2001).
The Sin3A Rpd3 histone deacetylase complex is conserved in Drosophila.
Both Sin3 and an Rpd3 homolog (HDAC1)
have been identified in Drosophila and are required for
embryogenesis. By analogy with mammalian systems,
Bin1 is likely to function in co-repression as
part of a Drosophila Sin3/HDAC1 complex.
It is proposed that interaction with Bin1
recruits the HDAC complex to DNA, converting Bicoid
from an activator into a repressor, or at least neutralizing
its ability to stimulate expression of its target genes. In this model, interaction of Bicoid with Bin1 would be stimulated by the action of the
terminal polarity-system kinases. For example, phosphorylation
of either Bicoid or Bin1 might trigger a
conformational change that strengthens their interaction.
The Bin1-Bicoid complex would then recruit
Sin3/HDAC1 to down-regulate Bicoid's transcription
activity beginning at late cellularization stages. In this
way, Bicoid-dependent gene expression could be down-regulated
exclusively at the anterior tip of the embryo,
where the Bicoid concentration is high and the terminal
system is active, resulting in the observed retraction (Zhu, 2001).
Human SAP18 has been found to interact with a cAMP-GEF
protein. cAMP-GEF proteins function in MAPK signal transduction pathways
to activate the GTPases Rap1 and Ras, which in turn
leads to activation of Raf kinases (MAPKKK). Members of this pathway are present
in Drosophila, including two putative proteins similar to
human cAMP-GEFs, CG3427, located at 42C4-5, and
CG9494, located at 26C3, as well as dRap1 (Roughened) and Raf kinase (Pole hole
protein), which is the kinase downstream of the Torso
receptor in the terminal system. By
analogy with human SAP18, Drosophila Bin1 might
interact with a cAMP-GEF, and thereby be linked directly
to the terminal system MAP-kinase pathway. For example,
interaction of Bin1 with cAMP-GEF might
result in phosphorylation of Bin1 by Raf upon
stimulation of the Torso receptor tyrosine kinase. This,
in turn, might stimulate Bin1 to interact with
Bicoid and trigger recruitment of the HDAC complex to
Bicoid-regulated promoters (Zhu, 2001).
Bin1 has also been identified as a protein
that interacts with Enhancer of Zeste, E(z), (L. Ding and
R. Jones, personal communication to Zhu, 2001), which is a Polycomb
group protein important for maintenance of repression of
homeotic genes, and with GAGA factor, the trithorax-like
gene product required for activation of homeotic genes. These and other examples suggest that control of expression of homeobox genes by histone
deacetylases is important for embryogenesis. Histone deacetylases may also alter homeodomain protein activity by direct interaction. Mobilization
of the EP-transposon insertion near Bin1 should
make it possible to generate mutant alleles, which will
be important for studying the role of Bin1 in development (Zhu, 2001).
To decipher the mechanistic roles of Mediator proteins in regulating
developmental specific gene expression and compare them
to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing
Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal
transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement
for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by
promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates
promoter-specific activation signals to the basal transcription machinery (Park, 2001).
Previous studies in yeast and human cells have suggested
that transcriptional activator proteins interact with Mediator
complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as
TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been
suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes
with the VP16 protein were examined. After incubation of nuclear extracts with an
excess of GST fusion protein beads containing either wild-type or
mutant (Delta456FP442) VP16 activation domain, the supernatants were
analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating
with GST-VP16 but not with GST-VP16Delta456FP442. However, the amounts
of dGCN5, dTAFII40, dTAFII250, and dTBP in the
extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID
and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the
amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most
strongly bound to and most readily recruited to the activation domain (Park, 2001).
In addition to the model VP16 activator derived from herpesvirus,
dMediator interacts with Drosophila transcriptional
activators Dorsal and heat shock factor (dHSF). When dMediator complex
was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads
even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein
G-agarose beads through anti-dSOH1 Ab and the binding of
diverse 35S-labeled Drosophila transcription
factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained
specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).
To evaluate the requirement of dMediator for activated transcription in
response to the Drosophila activator proteins that interact
with dMediator, the ability of dMediator-deficient nuclear
extracts to support transcriptional activation by the Dorsal and
Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to
mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level
of transcriptional activation is reduced significantly (five- and
three-fold activations, respectively) in nuclear extract that has been
depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by
the sequence-specific transcription factor Even-skipped (Park, 2001).
dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer
elements. However, the requirement of TAFs, or at least
dTAFII250, in activated transcription appears to be
redundant in the in vitro transcription system used and affected by
such factors as the core promoter organization or nucleosomal structure
of transcriptional templates. Several TAF components in the TFIID
complex indeed have biochemical activities and structural motifs
adequate for the recognition of specialized settings of transcription
templates. For example, certain TAFs recognize the Inr and DPE
sequences located in many Drosophila core promoters and
increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAFII250, which has a HAT catalytic activity and also possesses a
histone octamer-like module comprising the histone H2B-, H3-, and
H4-like TAFs. Although not experimentally
demonstrated, these TAFs may have some roles in the transcriptional
regulation of nucleosomal templates (Park, 2001).
The sequence-specific transcription factors which interact physically with
dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and
Fushi-tarazu. These factors contain different types of
activation domains (acidic and glutamine-rich domains). Most of these
transcription factors have been shown to activate transcription either
constitutively or inducibly. It is noteworthy that dHSF interacts with
and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170),
suggests that some function of Mediator is required for HSF-mediated
transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is
conserved between yeast and Drosophila,
transcriptional activation by HSF might utilize the conserved Rgr1
components of the Mediator complexes (Park, 2001).
Although some human Mediator complexes appear to have a negative effect
on activated transcription, dMediator does not
exhibit such an activity in an in vitro transcription system
reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is
more plausible that the dMediator-Krüppel interaction observed
is a part of the mechanism for transcriptional activation rather than
transcriptional repression. Taken together with the fact that dMediator
is dispensable for basal transcription, the lack of defect of the
dMediator-depleted nuclear extracts on transcriptional repression by
Even-skipped protein suggests that dMediator is required mainly for the
mediation of transcriptional activation signals to the basal
transcription machinery. Very recently, developmental roles of certain
dMediator proteins found in the Drosophila genome database
have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).
Like yeast Mediator, dMediator bind with the CTD repeats of
Drosophila Pol II. This implies that though
dMediator was purified separately from Pol II, these two complexes
indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS.
Such interactions may be involved in the regulation of Pol II
preinitiation complex assembly. Related with this idea, it has been
reported that in yeast, recruitment of general transcription factors
such as TBP, TFIIB, and TFIIH to active promoters requires the function
of Mediator. Also, TFIIE interacts with the
Mediator protein Gal11. Further analyses will be
required to clarify whether these interactions, observed both in yeast
and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).
dMediator contains the protein kinase component Cdk8, which can
phosphorylate serine residues in the CTD. This catalytic kinase subunit
seems responsible, at least in part, for the Pol II phosphorylation by
dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II
repeats, suggesting the presence of a functional interaction between
these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH
has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).
The Drosophila mophogenetic protein Bicoid (Bcd) can activate transcription
in a concentration-dependent manner in embryos. It contains a self-inhibitory
domain that can interact with the co-repressor Sin3A. A
Bcd mutant, BcdA57-61, that has a strengthened self-inhibitory function and is unable to activate the hb-CAT reporter in Drosophila cells, has been used to analyze the role of co-factors in regulating Bcd function. Increased
concentrations of the co-activator dCBP in cells can switch this protein from
its inactive state to an active state on the hb-CAT reporter. The C-terminal
portion of BcdA57-61 is required to mediate such activity-rescuing function of dCBP. BcdA57-61 has a normal ability to bind to a single TAATCC site when analyzed in vitro. Although capable of binding to DNA in vitro, BcdA57-61 is unable to access the hb enhancer element in cells, suggesting that its DNA binding defect is only manifested in a cellular context. Increased concentrations of dCBP restore not only the ability of BcdA57-61 to access the hb enhancer element in
cells but also the occupancy of the general transcription factors TBP and TFIIB
at the reporter promoter. These and other results suggest that an activator can
undergo switches between its active and inactive states through sensing the
opposing actions of positive and negative co-factors (Fu, 2005).
As a molecular morphogen, Bcd can undergo switches, in a
concentration-dependent manner, between its active and inactive states in
activating transcription of its target genes. The experiments described in this
report suggest another mechanism that can facilitate on-off switches of Bcd
activity in a Bcd concentration-independent manner. In particular, the mutant
BcdA57-61 is incapable of activating the hb-CAT reporter gene in
S2 cells at all concentrations tested. The
inability of this mutant Bcd to activate the hb-CAT reporter reflects a
distinct functional state of this protein rather than its defects in protein
stability. In fact, this same mutant protein is only modestly weaker than the wt
protein on another reporter gene, kni-CAT, which contains the
Bcd-responsive kni enhancer element. These and
other results suggested that the A57-61 mutation may cause its functionally
inactive state on hb-CAT by more efficiently interacting with a
co-repressor protein(s), such as Sin3A and its associated complex(es).
The experiments described in this
report show that increased concentrations of dCBP can restore activity to
BcdA57-61 on the hb-CAT reporter in cells. These results suggest
that the opposing actions of positive and negative co-factors can facilitate Bcd
to switch between its active and inactive states in a manner that is Bcd
concentration-independent (Fu, 2005).
Although BcdA57-61 can bind to both a single site and natural enhancer
elements in vitro, it is unable to access the hb enhancer element
in cells. These
results suggest that the DNA binding defect of this mutant protein is only
manifested in a cellular context. This notion is consistent with the finding
that the PAH domains of Sin3A do not exhibit any increased ability to reduce DNA
binding by BcdA57-61 in vitro when compared with wt Bcd. It is proposed
that other co-repressors or those
that are associated with Sin3A, such as the HDACs, can reduce the ability of Bcd
to access a natural enhancer in cells. It is possible that the enzymatic HDAC
activity that is more stably associated with BcdA57-61 makes it unable to
negotiate with histones for accessing DNA. It is also possible that a more
stable Bcd-co-repressor complex may sterically hinder the interaction between
BcdA57-61 molecules and prevent cooperative binding to the enhancer
element in cells (Fu, 2005).
The most striking finding of this report is that high levels of dCBP can switch
BcdA57-61 from its inactive state to an active one on the hb-CAT
reporter in cells. ChIP data further show that dCBP increases both the
ability of BcdA57-61 to access the hb enhancer element in cells
and the occupancy of GTFs at the reporter promoter. How does dCBP switch the activity states of BcdA57-61 on
hb-CAT in cells? Since Bcd and dCBP can physically interact with each
other through multiple domains, it is possible that dCBP may increase the DNA binding ability of Bcd in
cells by stabilizing the interaction between Bcd molecules and thus enhancing
its cooperativity. It is also possible that dCBP may physically compete with
co-repressor complexes in interacting with Bcd. Co-IP results suggest that
dCBP may negatively affect the interaction between Bcd and Sin3A in cells.
dCBP could also play a role in facilitating the
interaction between Bcd and the transcription machinery. For all these actions,
dCBP may play a structural (rather than enzymatic) role.
Finally, the fact that the HAT-defective mutant of dCBP does have a
reduced ability to restore activity to BcdA57-61 indicates that its enzymatic activity has a positive role, possibly
through modifications of histones. It is likely that dCBP can affect the
BcdA57-61 activity through multiple mechanisms that may be weak
individually but, when
combined, can lead to a dramatic switch from its inactive state to an active one
on the hb-CAT reporter in cells (Fu, 2005).
Currently, it is poorly understood how precisely Bcd activates transcription.
Previous studies suggest that much of its activation function is conferred by
the C-terminal portion of Bcd.
This portion of the protein contains several domains, including the acidic,
glutamine-rich and alanine-rich domains, that are characteristic of activation
domains capable of interacting with components of the transcription machinery.
Interestingly, the alanine-rich domain previously thought to play an activation
role was shown recently to exhibit an inhibitory function instead.
The C-terminal domain of Bcd can also interact with dCBP, and the results show that this domain is responsible
for mediating the activity-switching function of dCBP.
Although much of the activation function of Bcd is provided by its
C-terminal domain, the N-terminal portion of the protein also contains some
activation function. Studies have shown that Bcd(1-246), a derivative
lacking the entire C-terminal portion of Bcd, can rescue the
bcd- phenotype when expressed at high levels.
These results suggest that Bcd can achieve its activation
function through multiple domains presumably by interacting with different
proteins, including co-activators and components of the transcription machinery.
The results described in this report further support the importance of dCBP in
facilitating activation by Bcd (Fu, 2005).
Bcd is a morphogenetic protein whose behavior can be regulated not only by its
own concentration but also by the enhancer architecture.
On the kni and hb enhancer
elements, the N-terminal domain of Bcd is preferentially used for either
cooperative DNA binding or self-inhibition, respectively. It is proposed
that the interaction between Bcd molecules bound
to the kni enhancer element, through its N-terminal domain, can interfere
with its interaction with co-repressors, such as Sin3A.
Co-activators such as dCBP and co-repressors such as Sin3A can also
functionally antagonize each other, possibly by competing for Bcd interaction as
part of the mechanisms. Bcd is more sensitive to
the self-inhibitory function on the hb enhancer element than on the
kni enhancer element: consistent with dCBP's
antagonistic role, dCBP increases the activity of Bcd more robustly on the
hb enhancer element than on the kni enhancer element.
However, the interplay between positive and negative
activities that regulate Bcd functions is probably far more complex than the
simple physical competition: as already discussed above, dCBP can affect Bcd
activity through multiple mechanisms in both HAT-dependent and independent
manners. Moreover, in the
presence of exogenous dCBP, high levels of BcdA57-61 cause a reduction in
its activity on the hb-CAT reporter in cells, a reduction that is not observed with wt Bcd,
suggesting that the optimal concentration ratio between Bcd and dCBP may vary
depending on the strengths of the self-inhibitory function and interaction with
co-repressors. In addition, high concentrations of dCBP can rescue the inactive
derivative BcdA57-61, but not another inactive derivative lacking the
C-terminal portion, Bcd(1-246; A57-61), suggesting that the
Bcd-dCBP interaction strength can also influence the balance between
positive and negative activities that regulate Bcd function (Fu, 2005).
The experiments described in this report suggest that an activator's function is
subject to intricate controls by both positive and negative activities in cells.
A fine balance between these activities is critical for normal cellular and
developmental processes. Transgenic experiments show that both
BcdA57-61, which has a strengthened self-inhibitory function, and
BcdA52-56, which has a weakened self-inhibitory function, cause embryonic
defects. In addition, embryos
with reduced dCBP activity exhibit defects in early expression patterns of a Bcd
target gene, even-skipped. Finally, mutations affecting
SAP18, a component of the Sin3A-HDAC complex, can alter Bcd function and
anterior patterning in embryos. In addition to the
co-factors discussed in this study (Sin3A, dCBP and SAP18), Bcd likely has the ability to
interact with many other proteins, including not only regulatory proteins but
also components of the transcription machinery. Precisely how all these different proteins harmoniously
regulate and facilitate the execution of Bcd functions during development
remains to be determined. Recent studies have shown that the Bcd gradient in
embryos possesses a strikingly sophisticated ability to activate its target
genes in a precise manner.
These findings further underscore the need of intricate control mechanisms that
facilitate Bcd to switch between its active and inactive states in target gene
activation. These studies suggest that on-off switches of Bcd activity can be
achieved not only in a Bcd concentration-dependent manner but also in a Bcd
concentration-independent manner. It remains to be investigated whether and how
Bcd interacting proteins, including those yet to be identified, participate in
the precision control of target gene activation during development (Fu, 2005).
The Drosophila morphogen gradient of Bicoid (Bcd) initiates anterior-posterior (AP) patterning; however, it is poorly understood how its ability to activate a target gene may have an impact on this process. This paper reports an F-box protein, Dampened (Dmpd) as a nuclear cofactor of Bcd that can enhance its activating potency. A quantitative platform was established to specifically investigate two parameters of a Bcd target gene response, expression amplitude and boundary position. Embryos lacking Dmpd have a reduced amplitude of Bcd-activated hunchback (hb) expression at a critical time of development. This is because of a reduced Bcd-dependent transcribing probability. This defect is faithfully propagated further downstream of the AP-patterning network to alter the spatial characteristics of even-skipped (eve) stripes. Thus, unlike another Bcd-interacting F-box protein Fates-shifted (Fsd), which controls AP patterning through regulating the Bcd gradient profile, Dmpd achieves its patterning role through regulating the activating potency of Bcd (Liu, 2013).
Morphogen gradients such as those of Bcd are excellent experimental paradigms for dissecting the mechanistic operations of the regulatory networks that control patterning decisions. They provide a unique window to probing the regulatory impacts of F-box proteins both spatially and temporally at a fine resolution. Increasing evidence supports the notion that F-box proteins - there are up to 45 of them in Drosophila and 75 in humans - belong to a critical class of regulatory proteins; however, few of them have been studied in native developmental contexts. This work reports the identification of a nuclear cofactor of Bcd, Dmpd, that can enhance the potency of Bcd as an activator. Dmpd thus joins an expanding list of F-box proteins that can act as transcriptional co-activators; it remains to be determined whether the co-activator role of Dmpd for Bcd in the embryo is dependent on a functional SCF complex and the E3 ligase activity. The results show that embryos lacking Dmpd have a lower amplitude of Bcd-activated hb expression, a defect that can be passed further downstream of the network, causing an enlargement of ΔELeve3-4, the spacing between eve expression stripes that are sensitive to absolute concentrations of Hb. In embryos that lack another Bcd-interacting F-box protein Fsd, neither the hb expression amplitude nor ΔELeve3-4 is affected. This stems from the fact that, unlike Dmpd, Fsd regulates the Bcd gradient profile through a proteolytic pathway without a detectable co-activator function. The contrasting functions of these two F-box proteins thus document that a normal AP-patterning outcome is subject to regulation by two distinct mechanisms. These mechanisms control two distinct properties of Bcd that are indispensable to its morphogen action: the formation of a concentration gradient and the activation of its target genes (Liu, 2013).
A critical feature of morphogen gradients is their ability to induce downstream responses in a concentration-dependent manner. This particular feature has been subjected to extensive investigations, in part because of a significant interest in the question of what morphogen gradients do. It has been proposed recently that the concentration-dependent input-output relationship between Bcd and hb also contributes directly to the formation of AP patterns that are scaled with the length of the embryo. By contrast, the regulation of the amplitude of a response to the morphogen input has been relatively underexplored. Its importance may be better appreciated from the perspective of regulatory networks that control the patterning outcome. In the case of hb as a direct target gene of Bcd, its encoded protein Hb acts as an input, in a concentration-dependent manner, for genes (such as eve) that are further downstream of the AP-patterning network. The regulation of the expression of these downstream genes allows the regulatory network to refine and evolve towards the desired final outcome of patterning. Since these downstream genes respond to absolute concentrations of Hb, the boundary position for hb expression in response to the Bcd gradient input has become no longer directly relevant to the decision-making processes of these genes. However, as shown by this study, another feature of the hb response to the Bcd gradient input, namely its expression amplitude, remains directly relevant to the continued operation of the AP-patterning network (Liu, 2013).
Analysis of the impact of the dmpd mutation on active hb transcription reveals important mechanistic insights into the regulation of the transcription process in a native developmental context. As documented recentl, Bcd-activated hb transcription becomes detectable immediately upon entering the nc 14 interphase; however, it is shut off within a few minutes. Intron-staining results show that, at time classes t1 and t2, the hb-transcribing probability at the plateau region is largely unaffected by the dmpd mutation. They suggest that the onset of active hb transcription upon entering the nc 14 interphase is largely insensitive to dmpd mutation. The reduction in ρplat (the plateau region of the hb expression domain) at t3 and subsequent time classes is thus consistent with the possibility that dmpd embryos might have a hastened shutdown of active hb transcription at the nc 14 interphase. To test this possibility, quantitative hb mRNA FISH was performed in wt and dmpd embryos with an exclusive focus on nc 13. Embryos at this stage already have a significant accumulation of hb mRNA suitable for quantitative measurements that are necessary for effective comparisons between wt and dmpd embryos. In addition and importantly, active hb transcription is known to span the entire interphases prior to nc 14. Thus, if the defect of dmpd embryos is specific to hb shutdown at the nc 14 interphase, hb mRNA level should remain unchanged prior to this shutdown-that is, at nc 13. This prediction is supported the results. An implication of these findings is that altering the hb expression amplitude at, and only at, the last interphase (nc 14) prior to cellularization and gastrulation can still have an impact on the AP-patterning outcome, suggesting that this interphase represents a critical time period in making patterning decisions forward (Liu, 2013).
An important feature of developmental systems is that their desired spatial properties must be attained within the allotted periods of time when an entire system is progressing along the irreversible temporal axis. This interconnection between the spatial and temporal aspects of the developmental systems poses significant constraints on their operation. In Drosophila, the early embryo undergoes rapid cycles of nuclear division. This poses a constraint on the transcription process itself and the decoding of maternal gradient inputs, since mitosis is known to abort transcription. It has been documented that active hb transcription can resume almost immediately upon entering the interphase in the blastoderm embryo; however, it remains unknown precisely how this can be achieve. For developmental systems evolving rapidly along the temporal axis such as the early Drosophila embryo, patterning decisions may need to be made before true steady states could be achieved. In a recent study, it was shown that how quickly a gene can resume efficient transcription upon entering the nc 14 interphase can affect the amount (the amplitude) of the gene products at a later time when such products are needed for action. Thus, it was shown that a slowed onset of snail transcription led to gastrulation defects. In the case of dmpd mutation investigated in this report, while the onset of active hb transcription upon entering the nc 14 interphase is unaffected, a hastened shutdown reduces the amplitude of hb expression products, a defect that alters the spatial characteristics of the patterning outcome. Together, these two latest examples of dynamic regulation of transcription illustrate the importance of understanding the actual transcriptional decisions in a developmental system through the prism of time, an area of research that has only begun to be explored (Liu, 2013).
Three recent studies have reported investigations of the dynamics of transcription in early embryos. Two of the studies were based on a live-imaging technique using the MS2. For evaluating the onset of transcription upon entering an interphase, the use of this system requires an adjustment by the delay between transcription initiation and detection of fluorescent signals at the reporter locus, as dictated by the time necessary for RNA polymerase to transcribe through the MS2 stem loop repeats and for the MS2 coat protein-green fluorescent protein to bind to these RNA repeats. With this and detection limit-imposed delay adjustments, both of the live-imaging studies are consistent with a quick onset of Bcd-dependent transcription initiation upon entering an interphase as documented in the current and previous studies. A live-imaging study also supports a role of Bcd in directly lengthening the time period of active transcription during an interphase. At the nc 14 interphase, two studies support a shutdown of hb. Importantly, an observation that a reporter gene driven by the ~250-bp Bcd-responsive hb enhancer element is shut down at nc 14 in a manner that is broadly similar to the endogenous hb shutdown is consistent with the documented role of Dmpd and the activating potency of Bcd in influencing this shutdown process (Liu, 2013).
A contribution of the current work is the establishment of a quantitative platform for specifically (and simultaneously) analysing the amplitude and expression boundary of a target response to the Bcd gradient input. Under the current experimental framework, these two parameters are primarily subjected to regulation by two distinct mechanisms. The results document that Dmpd has a role in enhancing the activating potency of Bcd as an activator and in regulating the AP-patterning outcome. Interestingly, a detectable, although small, posterior shift in the hb expression boundary (xhb) in dmpd embryos, suggests that Dmpd may have regulatory roles beyond its primary role of regulating the amplitude of hb expression. This posterior shift in xhb cannot be simply explained by the Bcd gradient profile properties because a smaller B0 (Bcd concentration at the anterior) in dmpd embryos, if biologically meaningful, would have predicted a small shift in xhb towards the anterior. This small shift is detectable in embryos not yet exhibiting a significant sign of PS4 expression, suggesting that it is related to Bcd-dependent hb transcription. It remains to be determined mechanistically whether Dmpd may have a meaningful role in regulating the affinity of Bcd for the hb enhancer during development. It is anticipated that, as more experimental tools are developed and more regulatory players are identified, it will be able to further improve mechanistic knowledge about how the AP-patterning network operates in space and time at an even finer resolution and precision (Liu, 2013).
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