engrailed
Regulation of engrailed by Trithorax and Trithorax-like
Maintenance but not initiation of engrailed expression
requires trithorax (trx), which is also required to maintain stable long-term expression of
the homeotic genes throughout development. en expression is
dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells
in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in
the dorsal fat body is correlated with the sterility of en females that are also mutant in trx . In
addition, trx is required for normal en expression in the posterior compartment of the developing
wing, as reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx
appears to be dispensable for maintenance of en expression in other embryonic cells (Breen, 1995).
The expression of engrailed and other homeotic selector genes in imaginal discs was experimentally manipulated using mutations in trithorax group genes ash1 and
ash2. Variegated expression of engrailed as well as Antennapedia, Sex combs reduced, and Ultrabithoraxis caused by hypomorphic ash1 mutations.
These
experiments demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic
selector gene regulation
(LaJeunesse, 1995).
The proximal promoter of engrailed does not direct expression in a tissue or stage-specific manner, but contains promoter activity which can be activated by nearby genomic enhancers. Three so-called pairing sensitive sites (PS) demonstrate sensitivity to the actions of Polycomb and trithorax group genes (Kassis, 1994).
moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic
genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal
tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes: The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter.
moira is also required for transcription of the engrailed segmentation gene in the
imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ
cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).
Drosophila Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF,
CHRAC, and ACF. To clarify the biological functions of Iswi, null and dominant-negative Iswi mutations were generated and characterized. Iswi
mutations affect both cell viability and gene expression during Drosophila development. Iswi mutations also cause striking alterations in the structure of the male X
chromosome. The Iswi protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for Iswi in transcriptional
repression. These findings reveal novel functions for the Iswi ATPase and underscore its importance in chromatin remodeling in vivo (Deuring, 2000).
To determine when Iswi is required during development, the lethal phase and phenotype of Iswi null mutants were examined. Individuals heterozygous for ISWI1 or ISWI2 are viable and phenotypically normal. ISWI1/Df(2R)vg-C individuals die during late larval or early pupal development and display no obvious homeotic transformations or other pattern defects. Similar results were obtained for both ISWI2/Df(2R)vg-C and ISWI1/ISWI2 individuals (Deuring, 2000).
In vitro studies have suggested that Iswi plays an important role in transcription by facilitating the interaction of transcription factors with chromatin. One of the best candidates for a transcription factor that requires Iswi for its activity is the GAGA factor. GAGA factor binds to GA-rich sequences near the promoters of a wide variety of Drosophila genes and is thought to activate transcription by altering local chromatin structure. As the ATPase subunit of NURF, Iswi assists the GAGA factor to remodel chromatin in vitro, suggesting that the two proteins may act in concert to modulate chromatin structure in vivo as well. To examine possible interactions between Iswi and GAGA factor in vivo, the phenotypes of mutations in the two genes have been compared. GAGA factor is encoded by Trithorax-like (Trl), a member of the trithorax group of homeotic gene activators. Trl mutations enhance mutations in trithorax and cause homeotic transformations resulting from the decreased transcription of homeotic genes. Trl mutations also enhance position effect variegation, suggesting that GAGA factor antagonizes the assembly or function of heterochromatin. Unlike Trl mutations, Iswi mutations fail to enhance or suppress position effect variegation. No dominant interactions could be detected between mutations in Iswi and other genes, including Trl, other trithorax group genes (trithorax and brm), and Polycomb, a repressor of homeotic genes that is thought to act at the level of chromatin structure. These data suggest that Iswi and GAGA factor play distinct roles in chromatin remodeling in vivo (Deuring, 2000).
To investigate the role of Iswi in transcriptional activation in vivo, the effect of Iswi mutations on the expression of two targets of the GAGA factor were examined: the segmentation gene engrailed (en) and the homeotic gene Ultrabithorax (Ubx). The expression of En protein is reduced dramatically in imaginal discs of ISWI1/ISWI2 mutant larvae. Similar results are observed for Ubx. These data suggest that Iswi is essential for the expression of both en and Ubx in imaginal discs, although the possibility that this interaction is indirect cannot be ruled out (Deuring, 2000).
To directly observe interactions between Iswi and chromatin in vivo, the distribution of Iswi protein on salivary gland polytene chromosomes in third instar larvae was examined by immunofluorescence microscopy. Consistent with a fairly general role in transcription or other processes, Iswi protein is present at a large number of euchromatic sites in the polytene chromosomes. The same pattern was observed using whole sera and affinity-purified antibodies. The chromosomal distribution of Iswi protein is not appreciably altered following heat shock (Deuring, 2000).
Iswi protein is also associated with a subset of heterochromatin, as evidenced by punctate staining at the chromocenter. It is difficult to analyze the distribution of heterochromatic proteins on salivary gland chromosomes, since heterochromatic sequences are underreplicated in polytene tissues. To more accurately map the regions of heterochromatin with which Iswi interacts, the distribution of Iswi protein on mitotic chromosomes from larval neuroblasts was examined. On mitotic chromosomes, Iswi protein is abundantly present on the euchromatic arms of all chromosomes and is concentrated in regions of heterochromatin enriched with middle-repetitive sequences. For example, on the heterochromatic Y chromosome, Iswi is concentrated in the h11–13 region, which is composed almost entirely of middle repetitive DNA families. By contrast, little Iswi protein is detected in regions containing predominantly satellite DNA. The distributions of Iswi and GAGA factor on polytene and mitotic chromosomes were determined by double-label immunofluorescence microscopy. Both GAGA factor and Iswi are associated with hundreds of sites in the euchromatin of polytene chromosomes, but the distributions of the two proteins do not overlap extensively. Even greater differences in the distributions of the two proteins were observed in mitotic chromosomes where the GAGA factor, but not Iswi, is associated with GAGA-satellite sequences. The lack of extensive colocalization does not rule out an interaction between Iswi and GAGA at specific loci, but it does suggest that Iswi and GAGA are not obligatory partners (Deuring, 2000).
The decrease in en and Ubx expression in Iswi mutant larvae is consistent with reports that Iswi is involved in transcriptional activation in vitro. Consequently, it was not anticipated that the distributions of Iswi and RNA Pol II on salivary gland polytene chromosomes would be mutually exclusive. The preferential association of Iswi with transcriptionally inactive regions suggests that Iswi may create changes in chromatin structure that are not conducive to RNA Pol II transcription in vivo. Although there is no direct evidence that Iswi represses transcription, such a function would be consistent with the proposal that Iswi acts antagonistically toward histone acetyltransferases to compact chromatin structure. Based on these observations, further investigation of the role of Iswi in transcriptional repression is clearly warranted (Deuring, 2000).
How can the distributions of Iswi and RNA Pol II on polytene chromosomes be reconciled with the effect of Iswi mutations on gene expression in imaginal discs and the ability of Iswi complexes to activate transcription in vitro? One possibility is that Iswi has roles in both transcriptional repression and activation. NURF, ACF, and CHRAC were purified from Drosophila embryo extracts, and nothing is known about the nature or relative abundance of Iswi complexes in larvae. Perhaps only one Iswi complex is associated with transcriptionally inactive chromatin in the larval salivary gland, while others are either less abundant or transiently interact with chromatin to activate transcription. It is also possible that the interaction of Iswi with en and Ubx is indirect. For instance, the decreased expression of the two genes may be a secondary consequence of reduced cell viability in Iswi mutant larvae (Deuring, 2000).
Regulation of engrailed Polyhomeotic
Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many
developmental pathways. These include limb morphogenesis in Drosophila, since PcG genes interact with identity and pattern
specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated
with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. The
results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the
level of engrailed expression in posterior ones. It is suggested that PcG targets are not exclusively regulated by an on/off mechanism, but that
the PcG also exerts negative transcriptional control on active genes (Randsholt, 2000).
polyhomeotic is expressed in all imaginal
disc territories. ph is required for limb patterning. Induction of ph null clones in larvae, by irradiating ph heterozygotes,
leads to appendage pattern defects resembling those caused
by ectopic expression of the hedgehog pathway. Adults that are heterozygous for ph null (pho) mutations and that have been irradiated as larvae, show only small ph null clones, no larger than 16
cells, that are not homeotically transformed. Irradiation of such animals performed during late
second or early third larval instars (L2 or L3) causes the
appearance of small vesicles of tissue trapped inside the
body cavity and easily visualized in the wing.
Animals irradiated earlier during larval development can
show pattern abnormalities of all their appendages: this is particularly striking in the anterior wing
compartment. Wing phenotypes range from slight margin
or vein deformations to blisters and symmetrical mirror
duplications of anterior compartment elements (Randsholt, 2000).
One copy of P[phd+] rescues lethality and homeotic
transformations of pho. Furthermore, in the presence of a P[phd+] transgene, pho/1 irradiated flies never exhibit limb pattern defects,
indicating that these are indeed induced by the ph null
clones. To understand the origin of these abnormalities,
the expression patterns of a series of lacZ
reporters were examined in wing discs where ph null clones had been induced. The anterior-specific pattern disruptions in irradiated pho/1 animals mimic exactly those caused by ectopic expression of either
engrailed, hedgehog or decapentaplegic, all genes that
control A/P identity specification and limb morphogenesis.
These developmental processes depend on interactions
between posterior cells (which express en, the en-related
gene invected and hh), and anterior cells, which respond to
the Hedgehog signal by activating dpp through Ci in a stripe
of cells at the A/P compartment border (Randsholt, 2000).
Starting with en-lacZ, ectopic en-lacZ expression is rapidly detected (three to
four cell divisions after irradiation) in the shape of a spotted pattern in anterior disc
compartments. Larger spherical anterior en-lacZ expressing
domains are seen in late L3 discs that had been irradiated
during L1. The shape of these ectopic en-lacZ
domains suggests that they correspond to cells that are minimizing their contact surface with the rest of the disc. When
ph null cells are induced in a hh-lacZ background, ectopic
hh-lacZ expression is detected with the same time-lag and
in a similar pattern in the anterior compartment. A
ptc-lacZ reporter, whose peak expression is normally
restricted to anterior cells along the compartment boundary,
reveals ectopic ptc-lacZ expression in irradiated pho/1
discs in both compartments. A similar experiment
performed in a ci-lacZ background detected ectopic ci-lacZ
expression in the posterior compartment, despite
the presence of Engrailed in these cells. Activation of dpp
along the A/P boundary is the normal result of hh signaling,
so dpp expression was monitored with a dpp-lacZ reporter.
Ectopic dpp-lacZ expression is consistently seen in the
anterior wing compartment of irradiated pho/1;dpp-lacZ/1 discs. dpp-lacZ expression
was never detected in the posterior compartment. This anterior-specific induction of dpp-lacZ
makes dpp a likely candidate for causing the anterior appendage defects of irradiated pho/1 animals. From these data, it has been concluded that loss of ph product leads to
misregulation of hh pathway genes in both compartments. Additional experiments have shown that maintenance of A/P cellular identities in the developing appendages requires not only ph, but also several PcG products (Randsholt, 2000).
It was important to determine whether anterior activation of hh is a
consequence of anterior engrailed activation or reflects
an independent effect of loss of ph on the hh gene. The latter
situation would agree with the fact that hh is upregulated in
a trans-heterozygous mutant context for both ph and en. The data collectively suggest that anterior deregulation of hh in a ph mutant
background can be independent of engrailed. It is
suggested that Ci expression is increased when the ph level
decreases (Randsholt, 2000).
Double-immunostaining of irradiated discs from different
genetic backgrounds allows a precise determination of which
genes are deregulated by the absence of functional Ph. pho cells were followed by Myc expression after irradiation
of pho/Myc larvae. Staining of discs from pho/Myc;en-lacZ/1 larvae show that the anterior ph null cells form
bubble-like shapes that are in a different plane from the rest
of the disc. This shows that the pho cells are
sorting-out from the disc surface and explains the origin
of the vesicles of tissue trapped between the wing surfaces
or in the body cavities of irradiated pho/1 adults.
Furthermore, all cells that do not express Myc in the anterior compartment, and thus are ph null, express beta-
Galactosidase, hence ectopic Engrailed.
This shows that ph negatively regulates engrailed in the
anterior compartment and that the induction of En is cell
autonomous in all anterior compartment ph null clones.
Ectopic engrailed expression allowed for the identification of the
ph null cells in the anterior compartment (Randsholt, 2000).
Similar results were obtained when clones were induced
in a hh-lacZ or ptc-lacZ background, showing that both hh and ptc are autonomously
induced in anterior compartment ph null cells. In discs
from pho/1 larvae stained with anti-En and anti-Ci antibodies, ci expression is present in some but absent in other
anterior compartment ph clones. The
presence of En in pho cells might secondarily lead
to repression of ci when sufficient En has accumulated.
Finally, in discs from pho/1;dpp-lacZ/1 larvae, all the ph null clones in the anterior compartment are
associated with non-autonomous dpp-lacZ expression in the
surrounding cells. Furthermore, similar to what has been observed for ci, ectopic dpp-lacZ expression is detected in
some of the anterior ph null clones. These results suggest
that the effect of ph on dpp expression is indirect, and that
the relative levels of En and Hh within the clones are likely
responsible for the expression or non-expression of dpp.
Indeed, En is a strong repressor of dpp, whereas Hh stabilizes the activator form of Ci, which promotes dpp expression (Randsholt, 2000).
To conclude, loss of ph in the anterior compartment leads
to sorting-out of the ph null cells and to misregulation of en
and hh. This changes the identity of the cells toward cells
that are not quite posterior either since they strongly express
ptc, and sometimes even ci and dpp (Randsholt, 2000).
Discs from irradiated pho/phlac+2
larvae stained with antibodies against Ci and beta-Galactosidase exhibit cell autonomous expression of Ci in all posterior ph null cells,
recognized by the fact that they do not express beta-Galactosidase. ph null cells also
sort-out in the posterior compartment. Similarly, posterior
ph null clones are found to be associated with ectopic ptc
expression. Irradiated discs from pho/phlac+2
larvae labeled with Ptc and beta-Galactosidase antibodies exhibit Ptc in all
the ph null cells. Ectopic expression of either ci or
ptc allows the posterior ph null clones to be identifed by the presence of these markers.
Discs from pho/1 larvae stained with antibodies against
En and Ci suggest that loss of ph (detected in this case by
ectopic Ci expression) is likely to have an effect on engrailed
expression in posterior clones. Indeed, the posterior ci-expressing cells apparently express en at a
higher level than the surrounding wild-type cells. The clonal cells sort-out, and they are not on the same focal plan as surrounding cells (Randsholt, 2000).
Enhanced En expression is also revealed in clones
induced in pho/1 larvae and labeled with antibodies directed against Ptc and En. It is noteworthy that discs from irradiated pho/1;dpp-lacZ/1 larvae labeled with anti-beta-Galactosidase antibody
never show dpp-lacZ expression in the posterior compartment. Indeed, pattern defaults of irradiated pho/1 flies occur
anteriorly, which is in agreement with the presence of ectopic dpp expression associated only with anterior pho clones.
Together these analyses reveal that posterior pho clones
also sort-out. Loss of ph in posterior cells induces misregulation of ci and ptc, which are anterior specific genes, suggesting that ph is involved in maintenance of posterior cell
identity. Several sets of data indicate that engrailed expression is affected by loss of ph in both compartments, suggesting that ph participates in the repression of engrailed in the
anterior compartment, but also in the maintenance of a
certain level of engrailed expression in posterior cells (Randsholt, 2000).
Negative regulation of engrailed in the anterior compartment is complex. Indeed, engrailed does not depend on
transcriptional repression by the PcG alone; the groucho
gene product, for one, also participates in silencing of en
in anterior cells. The data presented here
indicate that posterior ph null cells that have lost all Ph
product express engrailed more strongly than their wild-type neighbors, suggesting that posterior compartment regulation of engrailed also involves more than a simple on/off
mechanism. ph could intervene in this regulation either
through direct regulation of en or the loss of ph could deregulate
other genes that in turn control the expression level of en.
Alternatively, ph could, together with other PcG products,
maintain a rate of en transcription in posterior cells, possibly
by regulating chromatin structure or accessibility. The
repressive mechanism controlling en expression in the
posterior compartment might, as in mammalian cell
systems, change the probability that a given promoter is
transcribed.
The fact that Ph and Psc can bind transcribed loci in cell
cultures suggests that control of
gene activity by PcG products could extend to the regulation
of active genes. The data from ph null clones provides further
evidence that such a regulation does indeed take place
during Drosophila development, and suggests that it plays
a crucial role in the regulation of selector genes whose wildtype function requires, like engrailed, a strict control of their
expression level (Randsholt, 2000).
To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).
For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and
Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA
polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).
Acetylation of histones H3 and H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of
amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one
that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels
in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the
inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive
promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with
results in other systems (Breiling, 2001).
The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF
(RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).
The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at
PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).
Regulation of engrailed by Eyelid and Dead ringer
In maternally mutant eyelid/osa gene embryos, although the initial engrailed expression is initiated in a relatively normal fashion, its later expression is abnormal: several stripes appear broadened, others are partially missing, and their spacing is disrupted. Nevertheless, wingless stripes are not expanded. These results are consistent with eld acting to counteract Wingless signaling in regions posterior to the engrailed stripes, in addition to responding to earlier patterning signals that affect the positioning of pair-rule gene stripes (Treisman, 1997).
It has been suggested that eld acts downstream rather than upstream of wingless for the following reasons:
The Drosophila eyelid/osa gene, like yeast SWI1, encodes
an AT-rich interaction (ARID) domain protein. Genetic and biochemical evidence is presented that Osa is a component of the Brahma
complex, the Drosophila homolog of SWI/SNF. To determine whether Osa is associated with the high molecular weight Brm complex, Schneider cell nuclear extracts were fractionated through a glycerol gradient
and immunoblotted with antibodies against the various proteins. Osa, Brm and Snr1 co-sediment in the bottom third of the gradient, suggesting
that they are part of a large protein complex. Thus, in vivo, Osa is found in a large complex with Brm and
Snr1, but does not bind to proteins in other chromatin remodeling complexes.
The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is
sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation
of some of these genes. Brm-related complexes are thought to promote transcription by altering the architecture of nucleosomal DNA, thus generating a conformation that is more favorable to binding by transcription factors and the basal transcriptional machinery. Some genes, such as even-skipped, show reduced levels of expression in osa mutant embryos, supporting the role of Osa as an activator of gene expression. However, other genes, such as engrailed, show expanded domains of expression in osa mutants. These genes could be directly activated or repressed by Osa, or their changes in expression level could be secondarily due to the regulation of other transcription factors by Osa. Some Brahma-containing complexes do not contain Osa and Osa is not required to localize Brahma to
chromatin. These data suggest that Osa modulates the function of the Brahma complex (Collins, 1999).
Regulation of engrailed by Single minded
The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in
the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the
embryonic CNS. In sim mutant embryos the
midline cells fail to differentiate properly into their mature CNS cell types and do not take their
appropriate positions within the developing CNS. sim is required
for midline expression of a group of genes, engrailed among them. Others include slit, Toll, rhomboid, and a gene at
91F. The sim mutant CNS defect may be largely due to loss of midline slit expression. The snail gene is required to repress sim and other midline genes in the presumptive mesoderm (Nambu, 1990).
Regulation of engrailed by Egfr signaling
Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in
development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the
first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (Egfr) signaling
has many functions in fly development. Antagonizing Egfr signaling during the second larval instar leads to notum to
wing transformations and wing mirror-image duplications. Egfr signaling is necessary for confining the wing subregion in the developing
wing disk and for the specification of posterior identity. To do so, Egfr signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing
discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells (Baonza, 2000).
To study Egfr function during early wing development, Egfr signaling was reduced at different times by using thermosensitive alleles of Egfr or by overexpression of dominant negative Raf (DNRaf). Hypomorphic vein (vn) and connector enhancer of ksr (cnk) [a regulatory
member of the Ras signaling cascade] alleles were also analyzed. Under these
conditions, posterior to anterior
transformations, proximal
(notum) to distal (wing) transformations, and a
reduction (or absence) of the notum region were observed with high frequency. When DNRaf is
expressed in clones induced during the second instar,
different kinds of phenotypes are found. Large clones in the posterior
notum/hinge anlage lead to notum to wing transformations, whereas large clones covering the posterior of the wing
give rise to posterior to anterior transformations. These phenotypes were found only after inducing a large
amount of confluent clones.
Clones of cells overexpressing DNRaf in other regions at this age, or
anywhere at later stages, give rise to different defects, such as those previously described on cell proliferation and vein cell fates (Baonza, 2000).
Posterior to anterior transformations are associated with
mirror-image duplications that are reminiscent of those observed after
reducing the expression of engrailed, a gene that confers posterior identity in posterior wing cells. En represses
ci and limits the expression of dpp to
anterior compartment cells adjacent to En-expressing cells. Dpp acts as
a long-range morphogen emanating from the compartment border and it
directs the growth and patterning of the wing. Local loss of en function is sufficient to generate a complete
transformation of posterior cells to anterior, and as a consequence, to
induce the ectopic expression of dpp and an ectopic anterior
compartment (Baonza, 2000).
The mirror-image wing duplication resulting from the reduction of Egfr
signaling in the posterior compartment correlates with a
down-regulation of En protein expression and the up-regulation
of Ci. Accordingly, ectopic dpp expression is activated and a new A/P border is implemented. It should be mentioned that dpp is
activated within the posterior compartment,
suggesting that the posterior En-nonexpressing cells are not recruited
from anterior regions of the wing disk (Baonza, 2000).
Cells that neighbor those that express DNRaf in clones are recruited to
generate the new A/P border. This is a nonautonomous effect
that also has been described for en clones, leading to mirror-image duplications. Moreover, when DNRaf is expressed in
clones, not all cells within the clone down-regulate En. Egfr signaling
is therefore important for the maintenance of En expression in the
posterior cells of the wing pouch and the enactment of posterior cell
fates, although its effects appear to be nonautonomous (Baonza, 2000).
Badenhorst, P., Voas, M., Rebay, I. and Wu, C. (2002). Biological functions of the ISWI chromatin remodeling complex NURF. Genes Dev. 16: 3186-3198. 12502740
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).
ISWI, the catalytic subunit of NURF, is required for expression of the homeotic gene engrailed (en). However, ISWI is also a component of two other chromatin remodeling complexes, ACF and
CHRAC. To resolve which ISWI-containing complex is required for homeotic gene expression, expression of Ultrabithorax (Ubx) and engrailed (en) were examined in nurf301 mutant animals. When both copies of nurf301 are mutated, in homozygous mutant
nurf3011 larvae, expression of the Ubx protein becomes undetectable. The normal expression of Ubx in the haltere and third leg discs of wild-type third instar larvae is absent in nurf301 mutant animals. Expression of the homeotic gene en requires nurf301. The normal expression of En in the posterior compartment of imaginal discs is abolished in nurf3012 mutants. Semiquantitative RT-PCR analysis confirms that Ubx and en transcript levels are reduced in nurf301 mutant animals. These results confirm that the defects in homeotic transcription seen in iswi mutants are caused by abrogated NURF function (Badenhorst, 2002).
A positive regulator of the Bithorax-Complex, E(bx), has
been localized genetically to 61A, the same cytological interval as nurf301. However, unlike numerous regulators of the
BX-C, E(bx) had not been cloned. Since NURF is required for
expression of Ubx, whether nurf301
corresponds to E(bx) was tested. Both alleles of E(bx) were no
longer extant, so whether the mutations that were isolated in
nurf301 recapitulated the published morphological properties
of E(bx) mutants was tested (Badenhorst, 2002).
nurf301 mutants, like E(bx), increase
the severity of bithorax (bx) mutant phenotypes.
bx is a DNA regulatory element required for correct expression
of Ubx in regions that give rise to the third (T3), but not
second thoracic segment (T2) of the adult fly. This expression distinguishes T3 from T2 identity. Loss or reduction of Ubx levels in bx mutant animals (Ubx6.28/bx34e and
Ubx6.28/bx8 mutant combinations causes a homeotic transformation of the third thoracic segment to the anterior second thoracic segment. Thus, the third thoracic segment, which is normally vestigial and naked, is transformed into the second thoracic segment, increasing its size and causing sensory bristles to develop. Moreover, the
haltere (T3) is transformed toward wing fate (T2), manifested by
increases in size and the development of bristles. The strength of
these transformations is increased when one copy of E(bx) also is removed. Mutation of one copy of nurf301 similarly enhances bx phenotypes. With one
copy of either the nurf3011, nurf3012 or a
deficiency that removes nurf301 -- Df(3L)3643 -- the
strength of the transformation is enhanced. nurf301 enhances both bx34e and bx8 mutations (Badenhorst, 2002).
Although NURF is required for expression of the homeotic genes in
imaginal discs, neither E(bx) nor nurf301 homozygous
mutant larvae display obvious homeotic transformations of the larval cuticle. The absence of mutant larval cuticle phenotypes is likely due
to the large maternal dowry of nurf301 transcript contributed to embryos. Larval cuticular patterning is established before these transcripts have dissipated. Attempts were made to generate embryos lacking the maternal nurf301 contribution through use of the dominant female sterile technique. Although germ-line clones were produced using the parental chromosome, it was not possible to recover germ-line clones using nurf3011. Like ISWI, NURF301 is required for ovary development (Badenhorst, 2002).
An important question is how NURF is recruited to target sites in vivo.
Four genes were shown in this study to be dependent on nurf301 for expression: Ubx, en, hsp26, and
hsp70. All contain multiple binding sites for the GAGA factor,
which is genetically required for their correct expression. On the Drosophila hsp70 and hsp26 promoters, (GA.CT)n cognate elements (to which the GAGA factor binds) are required for HSF-binding.
When these sequences are deleted, HSF-binding to transgenes in polytene
chromosomes is impaired, consistent with the defects seen in nurf301 mutant animals. It is therefore compelling that recent biochemical studies show that NURF and the GAGA factor bind to each other in crude extracts, and that purified NURF301 and GAGA factor interact directly in vitro. The principal interacting domains map to an N-terminal
region of NURF301 and a stretch flanking the Zn finger DNA-binding
motif of GAGA factor. These data suggest that NURF
is recruited by the GAGA factor through specific, direct interactions
with the NURF301 subunit, to catalyze local sliding of nucleosomes at
bx, en, hsp26, and hsp70 promoters,
increasing accessibility to sequence-specific transcription factors and
RNA polymerase II. Curiously, though, reduction of nurf301
levels fails to enhance phenotypes of mutations in Trithorax-like, the gene that encodes the GAGA factor (Badenhorst, 2002).
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moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).
Eld meets the criteria for a direct nuclear effector: its expression is ubiquitous in the early embryo and imaginal discs, and therefore cannot be dependent on localized Wingless signaling. Eld appears to function as a repressor of engrailed expression although this effect need not be direct. Intriguingly, a homolog of Eld, Drosophila Dead ringer, binds to target sequences of Engrailed protein, which are derived from possible autoregulatory sites within the engrailed genomic region (Gregory, 1996). If Eld also binds to these sites, it might compete with En to prevent the establishment of autoregulation. This would imply that Eld acts as a repressor; several active repression domains have been shown to have a similarly high proline content (Treisman, 1997).
engrailed:
Biological Overview
| Evolutionary Homologs
| Targets of activity
| Protein Interactions
| Developmental Biology
| Effects of mutation
| References
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