Irregular facets (If) is a dominant mutation of
Drosophila that results in small eyes with fused ommatidia. Previous
results showed that the gene Krüppel (Kr), which is
best known for its early segmentation function, is expressed ectopically in
If mutant eye discs. However, it was not known whether ectopic Kr
activity is either the cause or the result of the If mutation. This study
shows that If is a gain-of-function allele of Kr. The If
mutation was used in a genetic screen to identify dominant enhancers and
suppressors of Kr activity on the third chromosome. Of 30 identified
Kr-interacting loci, two were cloned, and whether they also represent
components of a natural Kr-dependent developmental pathway of the embryo
was tested. The two genes, eyelid (eld) and
extramacrochaetae (emc), which encode a Bright family-type DNA
binding protein and a helix-loop-helix factor, respectively, are necessary to
achieve the singling-out of a unique Kr-expressing cell during the
development of the Malpighian tubules, the excretory organs of the fly. The
results indicate that the Kr gain-of-function mutation If provides
a tool to identify genes that are active during eye development and that a
number of them function also in the control of Kr-dependent developmental
processes (Carrera, 1998).
Kr expression defines the Malpighian tubule anlage at late blastoderm
stage and becomes restricted to a ring of cells at the midgut/hindgut boundary
from where Kr-expressing Malpighian tubule precursors evert. Previous
studies have shown that the specification of Malpighian tubule fate and the
segregation of the cells depend on Kr expression in the Malpighian tubule
anlage. In Kr-deficient embryos, the respective cells become part of the
hindgut epithelium (Carrera, 1998).
Once the tubules evert, Kr expression becomes restricted to a single
cell, termed the "tip mother cell". The singling-out process of this cell from
an equivalence group of Malpighian tubule precursors involves the activated
Notch pathway, which restricts the proneural bHLH proteins encoded by the
achaete-scute-complex (ASC) genes to the tip mother cell. In this
cell, the ASC proteins act in concert with bHLH protein encoded by
daughterless (da) to maintain Kr expression. The tip mother
cell divides once, and the daughters give rise to the tip cell, which controls
proliferation during tubule elongation and differentiates neuronal
characteristics, and an excretory cell, termed "satellite cell". The satellite
cell loses Kr expression in a Notch-dependent manner, whereas
Kr expression is maintained in the tip cell until the end of
embryogenesis (Carrera, 1998).
emc expression accompanies Malpighian tubule development in a manner
similar to Kr expression. However, once the tip cell is formed, the
patterns of expression become complementary, meaning that emc expression
continues in all cells of the elongating Malpighian tubules except in the tip
cell. To test whether the complementary patterns of Kr and emc
expression reflect a regulatory effect of emc on Kr, as indicated
during eye development in the If mutant, Kr expression was
examined in the Malpighian tubules of emc mutant embryos. Multiple
Kr-expressing cells are seen in emc mutant Malpighian tubules.
This finding is consistent with the previous finding that emc mutant
embryos develop multiple tip cells and that each of them continues to express
achaete. Virtually the same observations have been made with Notch
mutants, and Notch acts toward restricting the activity of the proneural
bHLH proteins, which are required to maintain Kr expression first in the
tip mother cell and subsequently in the tip cell. However, although the
activated Notch pathway acts through transcriptional repression of the
ASC genes, emc protein antagonizes proneural bHLH activities by
sequestering the proteins as heterodimers that are incapable of binding to DNA.
The results are therefore consistent with the proposal that emc functions
in the control of Kr expression by antagonizing proneural bHLH activities
that are required to maintain Kr expression in the tip mother cell
(Carrera, 1998).
The Eld protein shows a nuclear localization, consistent with its suspected
function as a transcription factor. It appears to act in multiple signaling
pathways because it antagonizes wingless activity, suppresses Ras1
activity in the eye, and blocks Notch-dependent neuronal differentiation.
During Malpighian tubule development, eld is expressed in a restriced
area of the everting precursors that corresponds to the equivalence group of
cells expressing the proneural genes (Carrera, 1998).
eld mutant embryos exert a distinct phenotype during Malpighian tubule
development that is linked to Kr activity. Whereas the anlage and the
four tubules evert normally, each tubule develops two instead of the normal one
tip cell. Tip cell development is under the control of Kr activity, so it
was next asked whether and when Kr expression is altered in eld
mutant embryos. In correspondence with the mutant phenotype, the initial
expression of Kr, including its restriction to the tip mother cell,
appears to be normal. However, once the tip mother cell has undergone division,
two instead of only one of the daughter cells maintain Kr expression.
This indicates that eld activity is necessary to prevent Kr
expression in the sibling of the tip cell and allows for its differentiation
into a satellite cell. Thus, although emc is necessary for the
restriction of Kr to the tip mother cell, eld functions
specifically at the subsequent step during Malpighian tubule development where
an alternative and Kr-dependent cell fate decision is taken between the
daughters of the tip mother cell (Carrera, 1998).
Notch signaling is required first for the selection of the tip mother
cell and subsequently for the distinction between its daughters to either
develop a tip cell or a satellite cell. Consistently, in Notch mutant
embryos, all cells of the proneural equivalence group develop first into tip
mother cells; these cells divide and subsequently develop into the multiple tip
cells that continue Kr expression. In contrast, only two tip cells were
found in eld mutants. This finding implies that, if eld acts in a
Notch-dependent manner and/or mediates Notch signaling, its
activity is required only for the second of the two Notch-dependent
differentiation steps during Malpighian tubule development. Thus, eld
participates as an optional component in the Notch-signaling pathway and
is needed to prevent, directly or indirectly, the maintenance of Kr
expression in the satellite cell that would otherwise develop into a second tip
cell (Carrera, 1998).
The results of this study demonstrate that gene activities that were
identified via an artificial experimental situation, namely the ectopic
expression of Kr in the developing eye disc, can lead to the
identification of integral components of a Kr-dependent developmental
pathway during embryogenesis. In the eye imaginal disc, emc suppresses
Kr activity whereas eld has an opposite effect, but both act
during embryonic Malpighian tubule development as negative regulators of
Kr. No explanation is available for this phenomenon. It could mean, in
negative terms, that the Kr misexpression screen turned up
dosage-sensitive genes affecting cell fate that were several steps downstream
from Kr activity and thus have no direct interaction with Kr.
Thus, each gene identified in the modifier screen represents a candidate gene
that needs to be evaluated critically through additional criteria as outlined
here for eld and emc. The additional screening is essential to
distinguish between direct Kr interactors and genes that mediate
different read-outs of the Kr pathway in cells that have a different
organ or tissue competence. However, in view of the fragmentary information
concerning the spatial and temporal control of postblastodermal Kr
expression and in view of the fact that the few Kr target genes of
Kr were identified by molecular approaches, this experimental strategy to
assess components of a Kr-dependent regulatory circuitry seems a valid
one (Carrera, 1998).
The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific
downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the
absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from
the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream
of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).
Loss of osa can induce phenotypes similar to those caused by ectopic wg expression. Conversely, overexpression of full-length,
wild-type Osa (UAS-Osa) results in dominant, gain-of-function
phenotypes that often resemble those caused by loss of wg
function. However, osa appears to be epistatic to wg, and loss of osa function does not induce ectopic expression of wg. Therefore, the wg gain-of-function phenotypes caused by osa loss of function are likely to result from
de-repression of downstream target genes of Wg. To investigate this, the regulation of nubbin (nub) was examined. nub
encodes a POU domain protein that is required for the growth and
patterning of the wing and is expressed throughout the wing primordium
(or wing pouch) in third-instar wing discs. wg signaling is both necessary and
sufficient for the expression of nub, since ectopic expression of
wg or ectopic activation of the wg pathway can induce ectopic expression of nub, whereas blocking transmission of the wg signal in the wing pouch represses the endogenous expression of nub (Collins, 2000).
nub is ectopically expressed in wing discs that are transheterozygous for null and hypomorphic alleles of osa. An activated form of Armadillo causes similar ectopic nub expression by ectopic activation of the wg pathway. Conversely, the endogenous expression of nub is reduced along the anterior/posterior (A/P) boundary when UAS-Osa is expressed there with a decapentaplegic (dpp)-Gal4 driver. A similar loss of nub expression is caused by the expression of a dominant negative form of Pangolin (Pan) that can no longer bind Arm to activate gene expression (DN-Pan). When Osa and DN-Pan are coexpressed with dpp-Gal4, they acted synergistically to cause a severe reduction in nub expression (Collins, 2000).
In addition to its role in transmitting the wg signal, Arm
binds directly to cadherins and is required for the formation of adherens junctions. Over-expression of Drosophila E-Cadherin (DE-Cadherin) can sequester Arm at the plasma membrane and prevent it from participating in Wg signaling; this
results in the induction of wg-like phenotypes. When DE-Cadherin (UAS-Cad) is overexpressed in the dorsal
compartment of the wing disc with an apterous (ap)-Gal4
driver, dorsal expression of nub is lost and the growth of
the wing pouch is reduced. Reduction of osa
function in discs expressing UAS-Cad restores more normal nub
expression and growth. Furthermore, the ectopic nub expression normally seen in osaeld308/osa4H discs is
suppressed by the expression of UAS-Cad in the dorsal compartment. Thus, the level of nub expression is determined by the
relative levels of Arm and Osa when either of these levels is reduced.
To increase the levels, UAS-Osa is expressed with ap-Gal4,
causing a strong reduction of nub expression in the dorsal
wing pouch. Expression of DeltaArm with the same Gal4 driver
causes nub to be expressed in almost the entire wing disc. The normal domain of nub expression is restored when UAS-Osa and DeltaArm are coexpressed.
Taken together, these data demonstrate that Osa is required for the
repression of a wg-dependent gene in vivo. Alterations in the
dosage of osa can modulate the expression of
wg-dependent genes even in the presence of an activated form
of Arm or a dominant negative form of Pan, suggesting that Osa does not
act upstream of Arm. Alterations in the level of active
Pan/Arm complexes can also modulate nub
expression in osa mutants; thus, lack of osa does not
make Wg target genes entirely independent of Arm (Collins, 2000).
The ARID DNA-binding domain of Osa fused
to the repressor domain of Engrailed (UAS-OsaRD) or the activation
domain of VP-16 (UAS-OsaAD) can target these domains to genes normally
regulated by osa in vivo. The ectopic expression of nub in
osaeld308/osa4H wing discs
can be prevented by expression of either UAS-Osa or UAS-OsaRD with
ap-GAL4. This suggests that Osa functions as a repressor of transcription
in the regulation of Wg target genes (Collins, 2000).
To test whether Osa is acting directly on Wg target genes or
regulating the expression of some other gene that interacts with the
wg pathway, attempts have been made to determine at what level in the wg pathway Osa acts. In third-instar wing discs, wg
is expressed in a narrow stripe of cells that straddles the
dorsal/ventral (DV) boundary of the wing pouch and
directs growth and patterning of the wing blade with respect to the DV
axis. Cells adjacent to the DV boundary respond to the wg signal by posttranscriptionally up-regulating cytosolic Arm. Arm then translocates to
the nucleus and binds to Pan to activate the expression of
downstream target genes such as Distal-less (Dll). When an activated form of the protein kinase Sgg, which constitutively
targets Arm for degradation (UAS-Sgg*) is
expressed in the dorsal compartment using the ap-Gal4 driver,
Arm is not up-regulated. Expression of UAS-Osa in the dorsal compartment
similarly prevents the expression of Dll on the dorsal side
of the DV boundary. However, these cells still respond to
the Wg signal by up-regulating cytosolic Arm. Therefore,
Osa represses Wg target genes without affecting the up-regulation of
Arm. This places the activity of Osa in the nucleus and argues that Osa may directly repress the expression of Wg target genes (Collins, 2000).
To test the requirements for Osa to repress the expression of Wg
target genes, the expression of a lacZ reporter gene driven by a well-characterized wg-responsive enhancer was examined.
The midgut enhancer (UbxB) of the Ultrabithorax (Ubx)
promoter drives lacZ expression in the embryonic midgut in a
pattern that is dependent on both wg and
decapentaplegic (dpp). In wild-type embryos UbxB-lacZ is expressed primarily in parasegment (ps) 6, 7, and 8, with weaker expression in ps 3. This expression is de-repressed in embryos lacking the maternal contribution of osa, such that the expression of lacZ expands anteriorly as far as ps 3. Similarly expanded expression is induced by ectopic expression of wg in the mesoderm using 24B-Gal4. Conversely, expression of
UAS-Osa or UAS-DN-Pan in the mesoderm represses the
expression of UbxB-lacZ. However, neither wg nor
dpp is ectopically expressed in the midgut in embryos lacking
maternal osa (Collins, 2000).
When the dpp response element in UbxB is mutated (UbxBC), the expression of the lacZ reporter is severely reduced; only weak levels of lacZ expression are detectable in ps 8. Expression of UbxBC-lacZ
is unchanged in the absence of maternal osa, suggesting that
the dpp response element is still required for the expression
of the reporter construct in the absence of Osa. When one of the two wg response elements in UbxB is mutated (UbxB4), the expression of lacZ is reduced in wild-type embryos. However, removal of
maternal osa allows an expansion of UbxB4-lacZ
expression. This suggests that lack of osa can
compensate for a reduction in the responsiveness of the promoter to Wg
but not to Dpp. Furthermore, the expression of wild-type
UbxB-lacZ is also de-repressed in embryos lacking maternal osa even in the presence of DN-Pan. These data argue that Osa functions specifically to repress the activation of the UbxB enhancer by the Wg pathway (Collins, 2000).
Osa functions as a component of Brm chromatin-remodeling complexes and might be acting through the Brm complex to repress Wg target genes. Other components of the
Brm complex were therefore tested for genetic interactions with the wg pathway. Blocking Wg signaling at the wing margin by expressing UAS-Sgg* with
vg-Gal4 causes a reduction in wing growth and a loss of the
wing margin. These phenotypes are strongly enhanced in flies heterozygous for wg
or in those that coexpress UAS-Osa; they are suppressed in flies
heterozygous for axin (a negative regulator of Wg signaling or osa. The effects of UAS-Sgg* expression are also suppressed by the loss of one copy of
brm or moira (mor), which encodes an
essential component of the Brm complex, or by
coexpression of a dominant negative form of Brm (DN-Brm). In contrast, two other
trithorax group genes [trithorax (trx) and
absent, small, or homeotic discs 2 (ash2)] that
encode components of other nuclear complexes thought to regulate
chromatin structure, failed to modify the UAS-Sgg* phenotype (Collins, 2000).
This demonstrates that there is a specific genetic interaction between
the wg pathway and components of Brm complexes and suggests
that these complexes are required for the repression of Wg target
genes. Indeed, the wg-dependent gene nub is ectopically expressed in wing discs that contain large clones of cells mutant for brm or mor or that expressed DN-Brm in the dorsal compartment.
Furthermore, the loss of nub expression caused by expression
of UAS-Osa with ap-GAL4 is rescued by coexpression of DN-Brm, indicating that Brm activity is required for the repression of Wg target genes by Osa. The Wg-dependent
UbxB-lacZ reporter is also de-repressed in embryos that
express DN-Brm, and coexpression of DN-Brm can rescue the
loss of UbxB-lacZ expression caused by DN-Pan.
These results suggest that Osa acts through the Brm
chromatin-remodeling complex to prevent the expression of Wg target genes (Collins, 2000).
In addition to transducing the wg signal in a complex with
Arm, Pan is also required for the active repression of Wg target genes in the absence of the Wg signal. This repression requires the
association of Pan with the corepressor Groucho (Gro). Gro functionally interacts with the histone deacetylase Rpd3, and this interaction is important for at least some of the repressive activity of Gro. Thus, both Osa-containing Brm complexes and Pan/Gro/Rpd3 complexes repress the expression of Wg target genes and probably mediate this repression by altering the local chromatin architecture at the
promoters of these genes. Consistent with this, reduction of gro or rpd3 dosage reduces the ability of Osa to
repress nub. The loss of nub expression caused by
expression of UAS-Osa with ap-Gal4 is significantly rescued in wing discs
homozygous for a hypomorphic allele of rpd3. Also, larvae transheterozygous
for osaeld308 and groE48 often ectopically express nub in the wing disc, and 40% of transheterozygous adults have notum-to-wing transformations. These phenotypes are not seen when osa or gro single mutants are crossed to wild-type flies (Collins, 2000).
Whereas many of the genes regulated by the wg pathway
require wg for their expression, several genes appear to be
repressed by high levels of wg signaling. To determine the effect of Osa on the expression of genes that are normally repressed by wg, the expression of dpp in leg discs was examined with altered
dosage of osa. In third-instar leg discs, wg and dpp are expressed along the A/P boundary in the ventral and dorsal
compartment, respectively, and mutually antagonize each other's
expression. dpp expression is repressed when UAS-Osa is expressed in a broad central domain of the leg disc with a Dll-Gal4 driver and
dpp is ectopically expressed in the ventral compartment
in osaeld308/osa4H leg discs. Clones of cells mutant for osa can also induce
leg duplications in the ventral compartment of the leg,
consistent with the ectopic expression of dpp. Thus, in addition to repressing the expression of genes that are normally activated by the wg signal, Osa is also required for the repression of at least one of the genes that are repressed by wg (Collins, 2000).
The most likely explanation of these data is that Osa functions to
directly repress Wg target gene expression, with such target genes
being defined by their inclusion of a Pan-binding site. Osa function is
not exclusive to the Wg signaling pathway; Osa also functions as a
promoter specific activator of Antennapedia expression and as
a coactivator for Zeste and likely represses E2F-mediated gene
expression. Furthermore, the expression of even-skipped is
perturbed in embryos lacking maternal osa, a phenotype that
precedes the expression of wg in the embryo. However, the strong correlation of the expression of Wg target
genes with the level of Osa suggests that counteracting Osa activity is
an important function of the Wg pathway (Collins, 2000).
Osa functions as a component of Brm chromatin-remodeling complexes. These complexes and closely related complexes in
other species such as the yeast SWI/SNF complex catalyze an ATP-dependent alteration in the structure of nucleosomal DNA that
can run in either direction to render the DNA either more or less
accessible to binding by transcription factors. Whereas chromatin-remodeling complexes are generally
thought to promote gene expression, recent reports have demonstrated
that they are also required for the repression of some genes.
Genome-wide analysis shows that more genes have elevated than reduced
expression in a swi2 mutant yeast strain, and some of these
genes are directly repressed by SWI/SNF. The hBRM complex in humans has been
shown to cooperate with the retinoblastoma protein (Rb) to repress
E2F-1-mediated activation. Furthermore, brm, mor, and osa have been identified as
enhancers of an E2F gain-of-function phenotype, suggesting that Brm
complexes also repress E2F activation in Drosophila (Collins, 2000 and references therein).
Because Osa can antagonize Brm complex function in
some tissues, it has been thought possible that Brm complex
activity could be required for the expression of Wg target genes and
that Osa might be a negative regulator of Brm complex function.
However, the findings that the effects of blocking the wg
pathway at the wing margin can be suppressed by reducing the dosage of
brm or mor and that nub and
UbxB-lacZ are ectopically expressed when brm or
mor function is lost suggest that Brm complexes are required
for the repression, rather than the activation, of Wg target genes.
Furthermore, expression of a form of Brm that has a mutation in its
ATP-binding site also induces ectopic expression of nub and
UbxB-lacZ and can rescue the loss of nub expression caused by overexpression of Osa. Because the ATPase activity of Brm is
required for the chromatin-remodeling activity of the Brm complex, this suggests that chromatin remodeling by the
Brm complex is necessary for Osa to repress the expression of Wg target genes (Collins, 2000 and references therein).
In addition to activating gene expression by recruiting Arm to the
promoters of wg-responsive genes, Pan also represses these same genes by recruiting the
transcriptional corepressor Gro. Interestingly, Gro has been shown to interact with the N-terminal tail of histone H3
and with the histone deacetylase Rpd3, and it has therefore been
proposed that Gro mediates repression by altering chromatin structure. Consistent with this, a strong genetic interaction exists between osa and gro that suggests that their activities in repressing Wg
target genes are closely related. Although it has not previously been reported that Rpd3 functions in the repression of wg target
genes, reducing the function of rpd3 can
partly rescue the loss of nub expression caused by the
overexpression of Osa. Rpd3 is therefore important for the repression
of Wg target genes; testing whether it is essential awaits the
isolation of null alleles (Collins, 2000).
The loss of either osa or gro leads to ectopic
expression of Wg target genes; thus, the activity of one is not
sufficient to repress the expression of these genes without the
activity of the other. Osa and Gro may, therefore, be mediating the
same repressive event rather than acting in parallel. Interestingly, human SWI/SNF forms a repressor complex with Rb and the histone deacetylase HDAC. This complex interacts with the cyclin E promoter through the binding of Rb to E2F-1 and represses E2F-1 activation of cyclin E
expression. This suggests the intriguing possibility that Osa and the
Brm complex function in a larger repressor complex containing Gro and
Rpd3 and that this complex is recruited to Wg target genes though the
binding of Gro to Pan. However, Gro acts as a corepressor for a large
number of transcription factors, and Osa cannot be required for all repression mediated by Gro
because loss of osa does not result in neurogenic phenotypes
like those caused by the loss of gro. Further research is needed to determine if Gro and/or Rpd3 can directly
interact with components of the Brm complex and, if so, what determines
the specificity of this interaction (Collins, 2000 and references therein).
The mechanism by which Wg signaling leads to the active repression of
genes such as dpp is not fully understood, although it is
counteracted by Sgg. However, the observation
that dpp expression is repressed by Osa suggests that other
factors may allow Wg signaling to reinforce repressive chromatin modeling by the Brahma complex on such promoters (Collins, 2000).
A model for the regulation of gene expression by components
of the Wg pathway is presented. The chromatin remodeling activity of the OsaBrm complex is required to
maintain the chromatin at the promoters of wg-responsive genes
in a repressive conformation. This would prohibit the association of
other transcription factors with their binding sites and prevent the
recruitment of components of the basal transcription machinery.
Osa/Brm complexes may be recruited to Wg-responsive genes
through an association with Pan/Gro/Rpd3
complexes. In response to the Wg signal, Arm is stabilized and
accumulates in the cytosol. This accumulation of cytosolic Arm permits
Arm to translocate to the nucleus and displace Gro from Pan and, in so
doing, relieve the repression mediated by Gro, Rpd3, and
Osa/Brm complexes. Arm may also promote a more open
chromatin conformation by recruiting the HAT activity of dCBP, thus
permitting the association of other transcription factors with their
binding sites. Also, the stimulation of the DNA-bending activity of Pan
by Arm may bring distantly spaced transcription factors into
juxtaposition to promote the activation of gene expression. In the absence of osa, the chromatin is maintained in a more
open and less repressive conformation. This would permit other transcription factors to interact with their binding sites at lower
concentrations than would otherwise be possible. Under these conditions, the low levels of Arm that are always present in the cell
may be sufficient to promote the activation of gene expression without
the Wg signal (Collins, 2000).
Protein Interactions
The Drosophila 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. Although Osa and Brm are present in similar fractions, Snr1 sediments in the bottom half of the gradient and could also
be part of another complex that does not contain Osa or Brm. Alternatively, the anti-Snr1 antibody might be much more sensitive, detecting very low levels of the
Snr1 protein. When glycerol gradient fractions are immunoprecipitated with anti-Osa antibody, Osa, Brm and Snr1 co-precipitate in the same region of the
gradient in which they co-sediment. ISWI and Ash2 both show broad sedimentation patterns, appearing in the bottom half of the gradient, but neither
protein is immunoprecipitated from the gradient fractions with anti-Osa antibody. 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 the domain 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. 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).
osa genetically interacts with trithorax group genes. Ectopic expression of a dominant-negative form of Brm with a mutation in the ATP binding site (UAS-brmK804R) disrupts many developmental processes. An optomotor-blind (omb)-GAL4 driver was used to direct expression of UAS-brmK804R in the central region of the wing disc; this results in loss of the distal wing margin, formation of ectopic campaniform sensillae and wing margin bristles, and disruptions in wing vein morphology. These phenotypes are strongly enhanced in animals heterozygous for osa. Expression of UAS-brmK804R at the wing margin using vestigial (vg)-GAL4 results in the loss of the proximal, posterior wing margin, a phenotype that is again enhanced in osa heterozygotes. The effect of increasing osa dosage was tested by co-expressing a full-length osa transcript under the control of the same vg-GAL4 driver, and this completely rescues the dominant-negative Brm phenotype. Interestingly, ectopic expression of osa alone with vg-Gal4 induces a dominant loss of proximal wing hinge structures, and this phenotype is also rescued in animals co-expressing osa and dominant-negative brm. This suggests that the functions of Osa and Brm are closely related, because a reduction in the activity of one can compensate for an excess of the other (Collins, 1999).
Ectopic expression of Osa in eye imaginal discs using eyeless (ey)-GAL4 results in a variable reduction in eye size. Rather than the expected suppression, an enhancement of this phenotype has been observed in flies that either co-express dominant-negative Brm or are heterozygous for brm. The eye phenotype is also enhanced by mor and SNF5-related 1 (Snr1), both of which encode components of the Brm complex. However, reducing the dosage of the trithorax group genes trx, ash1 or ash2 does not enhance the Osa overexpression phenotype. As expected, a reduction in osa dosage suppresses the small eye phenotype. Clones of mor mutant cells in the eye disc exhibit a severe reduction in growth, which is partially rescued if the cells are also mutant for osa. Taken together, these data demonstrate that osa shows strong and specific genetic interactions with components of the Brm complex. However, in the wing, osa appears to act in concert with brm, whereas in the eye, osa opposes the functions of brm, snr1 and mor (Collins, 1999).
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. The lack of specificity of DNA binding by Osa in vitro prevents the demonstration of direct action of Osa by altering Osa binding sites in the promoters of potential target genes. As an alternative approach, attempts were made to preserve Osa's target specificity in vivo and to determine the effect of making it an obligate activator or repressor of transcription. Either the exogenous activator domain of VP16 or the repressor domain of Engrailed was fused to the DNA-binding domain of Osa. The effects of misexpressing these activator (UAS-osaAD) and repressor (UAS-osaRD) forms of Osa under the control of the GAL4-responsive UAS sequences were compared with those caused by misexpressing the full-length wild-type Osa protein. The Osa DNA-binding domain appears to be sufficient for chromosomal localization of these fusion proteins, because an antibody to VP16 detects the OsaAD protein along the length of polytene chromosomes (Collins, 1999).
The notum of the adult fly contains a regular pattern of small (microchaetae) and large (macrochaetae) bristles. Expression of the osa transgenes in the developing notum using a GAL4 insertion in the pannier (pnr) gene results in a dominant alteration of bristle formation. Ectopic expression of osa causes the loss of both micro- and macro-chaetae, and defects in the midline of the notum, scutellum and abdomen. Expression of UAS-osaAD with the same GAL4 driver leads to a very similar phenotype, and co-expression of UAS-osa and UAS-osaAD induces a stronger, apparently additive phenotype. Expression of UAS-osaRD with pnr-GAL4 has the opposite effect, inducing the formation of ectopic macrochaetae on the notum. Co-expression of UAS-osa with UAS-osaRD rescues the bristle loss phenotype caused by the expression of UAS-osa alone. Thus, targeting an activation domain to Osa-regulated genes has an effect similar to overexpression of the full-length protein, while a repressor domain has the opposite effect (Collins, 1999).
In the wing, expression of UAS-osaRD with omb-GAL4 produces ectopic campaniform sensillae and wing margin bristles. This phenotype is enhanced in flies heterozygous for osa, suggesting that it results from interference with wild-type osa function. It is also very similar to the effect of expression of dominant-negative brm. Expression of UAS-osaAD causes the opposite phenotype, loss of campaniform sensillae. Expression of full-length osa with this driver results in dominant pupal lethality; although a small number of flies expressing osa eclose, their wings are deformed, making a phenotypic comparison difficult. The observation that UAS-osaAD and UAS-osaRD cause specific phenotypes in the developing wing disc, related to those caused by full-length Osa, implies that the DNA-binding domain of Osa has functional specificity in spite of its lack of DNA sequence specificity in vitro. Binding to other proteins could contribute to its ability to act on specific promoters. Expressing the DNA-binding domain alone has no effect, suggesting that its promoter interactions are not strong enough to compete significantly with endogenous Osa. The similar effects of UAS-osa and UAS-osaAD and opposite effects of UAS-osaRD also indicate that, in the wing imaginal disc, Osa functions as an activator of gene expression (Collins, 1999).
The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. This study reports the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (1) the BAP complex, defined by the presence of Osa and the absence of Polybromo and BAP170, and (2) the PBAP complex, containing Polybromo and BAP170 but lacking Osa. Determination of the genome-wide distributions of Osa and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. It is concluded that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. These results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes (Mohrmann, 2004).