Medea
Because both Mad and Medea are required for Dpp signaling, an attempt was made to determine whether the two proteins might physically interact. These experiments were performed
in mammalian tissue culture cells so that epitope tagged proteins could be used. To test for physical interactions between Medea and Mad, cell lysates from radioactively labeled COS-1 cells, transiently transfected with various combinations of epitope-tagged Mad (Flag-Mad) and epitope-tagged Medea, were subjected to immunoprecipitation. In cells expressing Flag-Mad, only a single specific band corresponding to transfected Mad was detected; whereas, in cells cotransfected with both Mad and Medea, an additional protein corresponding to Medea is found coprecipitating with Mad in alpha-Flag immunoprecipitates. These data indicate that Mad and Medea can physically interact. Mad(3SA) was constructed, in which the C-terminal serine residues are mutated to alanine residues. Comparison of the phosphorylation level of wild type Mad and Mad(3SA) reveals that Mad(3SA) phosphorylation levels are reduced, consistent with the notion that Mad is constitutively phosphorylated at the C terminus in COS-1 cells. The most striking effect of the mutated serines is that in co-transfected cells, Medea is unable to associate with Mad(3SA), even though wild-type Mad efficiently forms complexes with Medea (Wisotzkey, 1998).
Thus phosphorylation of Mad at the C terminus is required for association with Medea. In contrast, to Mad, phosphorylated Medea is almost undetectable; it occurs at similar levels in complexes with Mad and is unaffected by DPP signaling. These observations are similar to the known properties of mammalian Smad4. Mutation of a glycine residue within the highly conserved GWG motif (G727D) located in the MH2
domain, abolishes interaction of Medea with Mad. Interestingly, the MH1 domain mutant, Medea(C99S)
appears to interact normally with Mad, suggesting that this missense mutation does not significantly disrupt the ability of Medea to associate with Mad (Wisotzkey, 1998).
In cells transfected with Medea, the protein is predominantly localized to the
cytoplasm; this localization was not altered by coexpression of
activated TKV type I receptor. Thus, activation of DPP type I
receptor alone is not sufficient to cause accumulation of
Medea in the nucleus. In contrast, Mad protein is detected
throughout the cell in the absence of signal, and coexpression
with activated TKV receptor results in accumulation of Mad
in the nucleus. Mutation of the putative phosphorylation sites in Mad, Mad(3SA), prevents nuclear
accumulation in the presence of activated TKV receptor, indicating that phosphorylation of Mad is essential for accumulation in the nucleus. Since Mad is expressed at
extremely high levels in these cells, it is unlikely that
stoichiometric levels of a Smad4-like protein are required for Mad to accumulate in the nucleus.
To determine whether entrance of Medea into the nucleus might require physical association with Mad, the subcellular localization of both proteins was examined when coexpressed
in the same cell. Although most cells show Medea localized
to the cytoplasm when it is expressed alone,
coexpression of this protein with Mad increases the number
of cells with nuclear Medea staining. Coexpression with both
Mad and activated TKV results in strong Medea nuclear
staining. In contrast, when Medea is coexpressed
with Mad(3SA), which does not accumulate in the nucleus and does not interact with Medea, no
shift of Medea from the cytoplasm to the nucleus is
detected. Thus, the entrance of Medea into the
nucleus requires physical association with phosphorylated Mad (Wisotzkey, 1998).
The subcellular localization of mutant forms of Medea was examined in cells coexpressing Mad and Medea. Similar to wild type Medea, Medea(G727D) is
predominantly localized to the cytoplasm. However, unlike wild-type Medea, coexpression of Medea(G727D) with Mad and activated TKV does not lead to nuclear
accumulation of the mutant, consistent with the inability of this mutant to
interact with Mad. These data indicate that certain mutations in the MH2 domain block the
interaction of Medea with Mad and thus prevent nuclear accumulation of Medea. The subcellular
localization of the MH1 domain was examined in mutant Medea(C99S), which is biologically inactive but associates normally with Mad. In cells coexpressing Mad, Medea(C99S), and activated TKV receptor, nuclear
accumulation of both Mad and the mutant Medea(C99S) is blocked and the proteins
remained predominantly in the cytoplasm (Wisotzkey, 1998).
Under conditions that result in Dpp receptor activation, Mad is able to translocate to the nucleus,
while Medea remains cytoplasmic. In the presence of activated
Mad, however, Medea translocates to the nucleus. These
observations suggest that Mad, but not Medea, is a direct target
of the receptor signal, and that the signal from the activated receptor
complex to Medea is mediated by Mad. Thus it is likely that
Medea, unlike Mad, does not interact with the type I receptor.
The distinct responses of these two closely related proteins to
stimulation in cell culture, provide a biochemical explanation
for the genetic requirement for Mad and Medea in dpp
signaling. The basis of this difference in response to receptor activation
may lie in the major sites of phosphorylation for the Smads. The
class I Smads have been shown to be phosphorylated in
response to stimulus at C-terminal serines (consisting of the SSXS motif),
an event that is important for signaling. This motif is absent in Medea
and the other class II Smads, as well as in the class III Smads. From these observations, it is possible to draw a model whereby the activation of Mad occurs before the activation of Medea during Dpp signal transduction. The levels of Mad that become activated (Mad*) determine the potential of the next, equally important step, which is its hetero-oligomerization with Medea. Thus, the higher levels of signaling achieved by the Dpp/Punt/Tkv activation system in cell culture, yield higher levels of Mad*, and cause high levels of nuclear Medea, while the lower Tkv* stimulus yields low levels of Mad*, and hence undetectable levels of nuclear Medea. Since the formation of the Mad*-Medea complex is important for signaling, from this model it is also conceivable that a quantitative increase in the levels of Medea protein can compensate for a reduction of Mad, by increasing the likelihood of the hetero-oligomerization of Mad* with Medea, thereby explaining the ability of Ubi-Medea to rescue the maternal effect lethality of Mad 12 /+ flies with dpp hr27(Das, 1998).
Mad and Medea are separately mutable yet closely related genes required for Dpp signaling. To gain insight into the functional relationship between these Smads, the
subcellular localization of Mad and Medea proteins was examined in
Drosophila Schneider 2 (S2) cells, in the presence or absence
of stimulation of the Dpp pathway. To activate the pathway, constructs of dpp and its receptors, punt and thick veins were co-transfected. This strategy provides a more powerful stimulus than transfection of activated Dpp type I receptors. In the absence of signaling, Flag-Mad shows predominantly cytoplasmic staining. This is consistent with what has been reported for Mad and, further, for the vertebrate class I Smads: Smad1 and Smad2. The same
cytoplasmic staining is also observed for Medea-HA in the absence of stimulus. Only a small number of cells (6% for Mad, and 1.5% for Medea) showed predominantly
nuclear staining. However, when co-expressed with the ligand and receptors, they reveal an important difference. The localization of Medea, in the presence of Punt, Tkv and Dpp, remains cytoplasmic in the majority of cells. However, Mad, in the presence of stimulus, is localized to the nucleus in about 95% of transfected cells. A similar, but lower, response is observed when activated Tkv* is used to stimulate the pathway. In this case, Mad is seen localizing to the nucleus in about 40% of cells. Hence, when expressed alone, and in the presence of stimulus, Mad is able
to translocate to the nucleus, while Medea is not. The response was assayed when Mad and Medea are expressed together, with or without stimulus. When co-expressed,
and in the absence of stimulus, both Smads are seen to be predominantly cytoplasmic, consistent with their responses when expressed alone. However, in the
presence of stimulus, both Mad and Medea localize to the nucleus in about 40% of cells. In some cells, Mad was nuclear, and Medea was both cytoplasmic and nuclear, while in other cells, both localized primarily to the nucleus. Hence, the co-expression of Mad is required for Medea to change its subcellular localization in response to stimulus. Full-length Mad and Medea have been shown to associate directly in two-hybrid assays, and co-immunoprecipitation (IP) experiments. Vertebrate Smads have also been shown to physically associate in two-hybrid and IP experiments. Taken together, these data suggest a model whereby activated Mad interacts directly with Medea to actively translocate Medea to the nucleus (Das, 1998).
Intracellular signaling of the TGF-beta
superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in
Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory
Smad genetically shown to antagonize Dpp signaling. A common mediator Smad, Medea, is described, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Medea
upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp (Inoue, 1998).
Dad stably associates with Tkv and thereby inhibits
Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. The effect of Dad on Mad phosphorylation by
Tkv was studied. Various combinations of Mad, Dad, and constitutively active Tkv
were introduced into COS cells. In the first experiment, cells were labeled with [32P]orthophosphate in
vivo, and incorporation of radioactivity into Mad was detected. Dad inhibits
phosphorylation of Mad by constitutively active Tkv. Next, anti-phosphoserine antibody was used. As in the
orthophosphate labeling, phosphorylation of Mad diminishes in the presence of Dad.
In vertebrates, inhibitory Smads such as Smad6 and Smad7 have been shown to stably associate with
type I receptors. The interaction of Mad or Dad with Tkv was studied: cells were transfected with an
appropriate combination of expression plasmids, affinity labeled with iodinated BMP-2, and subjected
to immunoprecipitation with antibodies against Mad or Dad. Pathway-specific Smads are known to associate with type I receptors upon ligand stimulation, but this
interaction is too brief to detect under natural conditions. The interaction
can be observed when the type I kinases are rendered inactive or when the C-terminal phosphorylation
sites of the Smads are modified to be resistant to phosphorylation. Mad interacts with the
kinase-defective form of Tkv, whereas the interaction of Mad with wild type Tkv is also detectable. The interaction of Mad with Tkv might thus be more stable than that of mammalian
Smads with receptors. Dad interacts with wild-type Tkv as efficiently as with the kinase-defective
form of Tkv. Notably, almost the same amount of Tkv is immunoprecipitated with Mad and Dad,
although the expression level of Mad is much higher than that of Dad. Thus the affinity of Dad with
Tkv seems to be higher than that of Mad. It was found that the interaction
of Mad with Tkv was hampered by expression of Dad. Dad thus inhibits
the Tkv phosphorylation of Mad by competing with Mad in association with the receptor.
Constitutively active Tkv causes hetero-oligomerization of Mad with Medea. The effect of Dad on
the hetero-oligomerization was examined: could Dad inhibit the constitutively active Tkv-induced
complex formation of Mad and human Smad4? The hetero-oligomerization of
Mad with Smad4 was shown to be efficiently blocked. Dad thus blocked a critical step in the activation of Mad (Inoue, 1998).
The following model is suggested for Dpp signaling by Mad, Medea, and Dad: Dpp
induces phosphorylation of Mad through Tkv and Punt. Mad then forms homo-oligomeric complexes
and/or hetero-dimerizes with Medea. Oligomers of Mad and Medea translocate into the nucleus where
they transactivate target genes, such as vestigial. Dad is one such target, and its expression is induced
by Dpp. Dad stably binds to Tkv and interrupts phosphorylation of Mad by Tkv (Inoue, 1998).
Baboon has been shown to function through the newly identified dSmad2 (Smad on X), a Drosophila homolog of mammalian Smad2 and Smad3, which function in Activin signaling. Activated Babo induces
phosphorylation on the last two serines of dSmad2. These data suggest that dSmad2 is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus. Babo-dependent
phosphorylation of dSmad2 also induces association with Medea, a homolog of mammalian Smad4. Phosphorylation of dSmad2 on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of
dSmad2 and Medea. Mammalian Activin receptors require dimerization with a type II receptor for their function. Punt, the Drosophila type II receptor was shown to function to activate
Babo. dSmad2 interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these
functional and biochemical analyses strongly suggest that dSmad2 is a Drosophila homolog of Smad2/Smad3 and functions as a downstream
signaling component that directly interacts with Babo (Brummel, 1999).
Interactions between the various components of the putative activin pathway of Drosophila were characterized. In vertebrates, R-Smads have been shown to associate with, and be phosphorylated by, specific type I receptors. Baboon, along with thick veins (tkv) and saxophone (sax) have been cloned in searches for Drosophila TGFbeta-like receptors. Since Sax and Tkv participate in the Dpp pathway, and biochemical studies have shown that Tkv activates Mad, it was possible that Baboon could be the receptor responsible for activating Smox. In addition, a previous study had shown that Baboon could bind human Activin, supporting the view that Smox, along with Baboon, may comprise part of a Drosophila activin pathway (Das, 1999).
Co-Smads have been identified in C. elegans, Drosophila and vertebrates. They differ from the R-Smads in that they are not phosphorylated on the C terminal serine residues in the'SSXS' motif. However, they form complexes with R-Smads and translocate to the nucleus, where they have been shown to bind DNA and stimulate transcription. In vertebrates, the Co-Smad Smad4, has been shown to participate as a partner in the TGFbeta, activin and BMP signaling pathways. Medea is the only Co-Smad known in Drosophila, and hence a possible association with Smox was tested. FLAG-Smox and myc-Medea were transfected into COS cells in the presence or absence of Baboon and Punt. Cells were lysed and immunoprecipitated with anti-FLAG antibodies. The association of Smox and Medea could only be observed in the presence of the receptors. These data show that Smox and Medea form a complex in the presence of the receptors and indicate that the ability of Co-Smads to function via multiple pathways is an ancient and conserved property (Das, 1999).
R-Smads translocate into the nucleus following phosphorylation and oligomerization. The nuclear translocation of Smox was examined using transfected COS cells. COS cells were transfected with expression plasmids for Smox, Baboon and punt, and the subcellular localization of Smox was determined by immunofluorescent staining. In unstimulated cells, Smox is distributed throughout the cell, but after activation by Baboon and Punt, Smox accumulates in the nucleus (Das, 1999).
The ability of Smox to activate transcription was studied using a heterologous promoter reporter construct (CAGA)-MLP-Luc. In vertebrates, Smad3 and Smad4, but not Smad1 or Smad2, bind directly to the sequence 'AG(C/A)CAGACA' (CAGA boxes), both in vitro and in vivo; furthermore, stimulation by TGFbeta and Activin, but not by BMPs, induces the transcriptional activation of this reporter construct. Failure of Smad2 to bind the CAGA box is due to the presence of a 30-amino acid insert next to the DNA binding domain in the MH1 domain of Smad2, which is not found in Smad3 nor, significantly, in Smox. Smox does however, have a putatively inactive DNA binding domain, and in addition, preliminary data reveal that Smox does not bind to the CAGA DNA sequence, suggesting that it may behave much like the vertebrate Smad2 in its DNA binding characteristics. Smox or Medea alone weakly induces transcriptional activation, but the co-transfection of Smox and Medea cause a dramatic increase in the luciferase activity. This observation suggests that Smox regulates the expression of target genes activated by activin/TGFbeta-like ligand(s) in Drosophila (Das, 1999).
dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).
dpp expression in posterior
tracheal branch anlagen appears to be initiated by prior episodes of wg
and dpp expression in the undifferentiated dorsal ectoderm.
The maintenance of dpp expression in posterior tracheal
branches appears to require continuous input from wg and
from a dpp feedback loop. The initiation and maintenance
of dpp expression in posterior tracheal branches also requires
continuous nej activity. Overall, the data are consistent
with the following combinatorial signaling model. The transcriptional activator Medea (Med, signaling for the Dpp
pathway) interacts with the transcriptional activator Arm
(signaling for the Wg pathway) via the transcriptional coactivator
Nej. This multimeric complex initiates and, with
continuous signaling, maintains dpp expression in posterior
tracheal branches with the help of Zw3. These data extend
previous studies of dpp expression and Dpp signaling in
several ways. nej has been reported to participate in
Dpp signaling. Expression from Dpp responsive
enhancers is reduced in nej zygotic mutant
embryos. While they show that nej3 enhances dpp wing
phenotypes, this study shows that Mad1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch
forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in
Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement
of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain
of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain
about the regulation of dpp expression by Wg, Dpp, and Nej.
Two questions arise about the mechanism of signal integration:
how is zw3 involved and how is Nej recruited to bridge
the two pathways? It is tempting to speculate that, in
response to a Wg or a Dpp signal, Zw3 (a serine-threonine
kinase) is involved in Nej recruitment. Numerous studies
have shown that p300/CBP transcriptional coactivation
functions are stimulated by its phosphorylation, but the
site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the
posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of conserved, overlapping consensus binding sites for dTCF and Mad/Med. Analyses of DNA-protein interactions predicted by the data involving this candidate combinatorial enhancer have begun (Takaesu, 2002).
Highwire, a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses (McCabe, 2004).
To search for proteins that interact with Hiw, a yeast two-hybrid screen of a Drosophila cDNA library was carried out using two regions of the Hiw protein as bait (HWB1: aa 2063–3461; and HWB2: aa 4082–5233). From a screen of 4 × 107 transformants, three candidate proteins were isolated that interacted positively with HWB1 and one candidate protein that showed positive interaction with HWB2. One of the three candidate proteins that interacted with HWB1 was identified as the C-terminal region of the second mad homology domain (MH2) of Med (aa 616-745). The Med clone did not self-activate, and when positive interaction was tested by cotransforming the Med clone together with either HWB1 or HWB2, a strong interaction was found with HWB1 but no interaction was seen with HWB2. Furthermore, GST-fused Med protein is able to bind to in vitro translated preparations of Hiw. The binding region for Med was between amino acids 2063 and 3461 of Hiw (included in HWB1), while binding was not observed with the region of Hiw found in HWB2. Interestingly, the Med binding domain of Hiw includes the region used to generate the partial Hiw transgene, Hiw-DN, that produces a dominant-negative hiw phenotype when expressed in the nervous system. The results of yeast two-hybrid experiments together with in vitro binding results show that Med and Hiw proteins can interact and that the MH2 region of Med is sufficient for this interaction (McCabe, 2004).
This study demonstrates that Hiw negatively regulates the BMP signaling cascade, required for the normal growth and function of neuromuscular synapses in Drosophila. hiw mutants have a dramatic overexpansion of synaptic structures, with many more synaptic boutons than wild-type. In contrast to hiw mutants, mutants of Med have smaller synapses and many fewer synaptic boutons compared to wild-type. Med is part of a presynaptic signaling cascade that includes three cell surface receptors (Wit, Tkv, and Sax) and two intracellular transcription factors (Med and Mad) that transmits a BMP signal from the NMJ to the motoneuron nucleus. Genetic removal of either Med or Wit can completely suppress the synaptic overgrowth in hiw mutants, while activation of BMP signaling in hiw mutants produces even more synaptic growth. These findings provide evidence for a functional link between the action of Hiw and BMP signaling to control synaptic growth at the Drosophila NMJ (McCabe, 2004).
The type II BMP receptor Wit and the BMP ligand Gbb are required for synaptic growth at the neuromuscular junction. While Wit is required in presynaptic neurons for normal NMJ growth, the requirement for Gbb in postsynaptic muscles is consistent with a retrograde BMP signal. Yet it
was not clear from these studies how Wit carried out this function, whether by a local signaling mechanism within the synapse or via a signaling cascade. Evidence is provided that strongly suggests BMP signaling through Wit regulates synaptic growth primarily by altering transcription. Two lines of evidence support this conclusion. (1) Mutants of both of the Smad transcription factors Med and Mad have similar defects in synaptic growth, presynaptic ultrastructure, and function to mutants of the cell surface receptors wit, tkv, and sax. (2) Phosphorylated Mad is absent from the nucleus of motoneurons in wit, sax, and tkv mutants. The absence of P-Mad in the nuclei of motoneurons in BMP receptor mutants combined with the similarity of synaptic phenotypes between mutants in receptors and mutants of intracellular Smads argues that BMPs exert their influence on synapses primarily by signaling to the nucleus rather than having a local synaptic activity (McCabe, 2004).
The specificity of synaptic BMP signaling seems to be maintained by Wit, which is expressed only in the nervous system, while Med, Mad, Tkv, and Sax are found in many tissues. This conclusion is supported by the finding of developmental defects in many tissues such as the fat body in Med, Mad, tkv, and sax mutants that are not found in wit mutants. Recently, an independent study found synaptic defects in sax and Mad mutants (Rawson, 2003). These studies have been extended, demonstrating that Med and Tkv, in addition to Mad and Sax, are required in presynaptic neurons for normal NMJ growth, and mutants of all these molecules have similar characteristic defects in presynaptic active zone ultrastructure. This study further shows, by rescuing wit mutants with a pair of Tkv::Wit chimeric receptors, that Tkv and Wit function together in vivo, and that Tkv is localized at the NMJ. All these data therefore suggest that Wit, Tkv, and Sax receive a retrograde BMP signal from muscles by Gbb (and possibly other BMP ligands) at the NMJ and transmit this signal to the nucleus via Mad and Med to induce transcriptional change. This neurotrophic signal is essential for the coordination of presynaptic NMJ expansion with postsynaptic muscle growth (McCabe, 2004).
While BMP signaling plays an essential role in neuromuscular junction expansion, BMP signaling mutants also have dramatic reductions in the levels of neurotransmitter release and aberrant presynaptic ultrastructure at active zones. Several pieces of data suggest the role of BMP signaling in the regulation of neurotransmitter release may be separable from its role in synaptic growth. Restoration of Gbb in the nervous system of gbb mutants can rescue neurotransmitter release to wild-type levels while not restoring normal synaptic size. This result is reminiscent of Fasciclin II mutations, which also have reduced synaptic size but normal neurotransmitter release. Furthermore, Wit is necessary for the homeostatic regulation of neurotransmitter release. It may be that the involvement of BMP signaling in this process is independent of, but complementary to, its role in regulating synaptic structural growth (McCabe, 2004).
Despite the central requirement for BMP signaling in synaptic growth, when attempts were made to increase BMP signaling in motoneurons, no synaptic overgrowth beyond wild-type levels was seen. These observations are explained by proposing the presence of a negative regulatory process that tightly controls the levels of synaptic BMP signaling. Hiw is a key and necessary component of this regulatory process (McCabe, 2004).
Hiw is an extremely large protein, making in vitro confirmation of its ubiquitination activity difficult. Despite the absence of direct biochemical data, several lines of evidence suggest that Hiw does function as an E3 ubiquitin ligase. Hiw has a signature RING-H2 finger, a domain that has a general function in ubiquitin-mediated protein degradation. RING fingers can function as modules that interact with E2 ubiquitin-conjugating enzymes to catalyze ubiquitination. Futhermore, hiw mutants have a strong genetic interaction with the deubiquitinating enzyme Fat Facets. Overexpression of either Fat Facets or the yeast deubiquitinating protease UBP2 in presynaptic neurons produces a synaptic overgrowth phenotype very similar to the hiw mutant phenotype. Given this evidence, a model is proposed whereby Hiw negatively regulates BMP signaling at the NMJ by a ubiquitination-dependant mechanism, antagonizing BMP signaling and controlling synaptic growth (McCabe, 2004).
In support of this model, Hiw has been shown to specifically binds Med protein in both yeast two-hybrid and in vitro binding assays. This is consistent with a function for Hiw as an E3 ubiquitin ligase, since these proteins specifically bind to their substrates before targeting them for proteolysis. Unfortunately, several antibodies against mammalian Smad4 failed to detect Med, precluding assays of Med's ubiquitination status. Interestingly, however, the region in Hiw that interacts with Med is included in the sequence of a partial Hiw transgene, Hiw-DN, that causes synaptic overgrowth when expressed in the nervous system. Since this transgene does not include the RING finger domain, its dominant-negative effect could be mediated by its ability to inhibit the binding of Med by endogenous Hiw. In addition to physical interaction between Hiw and Med, genetic removal of Med has been demonstrated to suppress the increase in the number of synaptic boutons in hiw mutants to Med mutant levels. This implies that the dramatic increase in the number of synaptic boutons in hiw mutants is completely dependant upon the presence of Med. This increase is also suppressed by wit mutants, showing that it is Med's role as part of a BMP signaling cascade that mediates its suppression of hiw. Furthermore, it was shown that synaptic overgrowth due to the overexpression of the deubiquitinating enzymes Faf or UBP2 is also suppressed by disrupting BMP signaling. These results together support the model whereby Hiw regulates BMP signaling via a ubiquitin-dependent mechanism (McCabe, 2004).
Overexpression of a constitutively active Tkv type I receptor transgene in neurons does not cause any overgrowth at the NMJ. Similarly, loss-of-function mutants of the inhibitory Smad, Dad, does not show any synaptic overgrowth. Consistent with the model that Hiw regulates BMP signaling, in hiw mutants, activation of BMP signaling can now lead to further synaptic overgrowth. This suggests that while hiw mutants may have elevated levels of BMP signaling, further activation of the BMP pathway can induce yet more synaptic growth. By activating BMP signaling using transgenic constitutively active type I receptors, other factors that could conceivably limit the signal can presumably be bypassed, such as the availability of Wit or Gbb (McCabe, 2004).
In contrast to the current findings, dad mutants have previously been reported to produce large numbers of extra synaptic boutons. While the current study examined only one homoallelic mutant combination of dad, several homo and heteroallelic mutant combinations were examined, to eliminate the possibility of second site mutations, and this prior result was not confirmed. Thus the discrepancy remains unresolved. Consistent with the current data, it has been shown that overexpression of Wit cannot induce synapse overgrowth, despite the ability of overexpressed type II receptors to activate signaling in the absence of ligand. The current results are also supported by findings of Rawson (2003) (McCabe, 2004).
While the current data indicate that synaptic structural growth can be controlled by Hiw regulating BMP signaling, neurotransmitter release at the NMJ does not seem to be governed by an identical mechanism. BMP mutants have decreased neurotransmitter release, in addition to reduced numbers of synaptic boutons, when compared to wild-type. In contrast, hiw mutants have many more synaptic boutons than wild-type, but despite this, neurotransmitter release in hiw mutants is also reduced to levels similar to that of BMP mutants. Interestingly, double mutants of hiw;wit or hiw;Med have levels of neurotransmitter release similar to those of wit or Med mutants alone. This result indicates that the role of Hiw in controlling neurotransmitter release is distinct from its role as a negative regulator of synaptic structural growth. Previous results support this idea; the unknown retrograde signal that controls the homeostasis of neurotransmission at the NMJ is disrupted in wit mutants but remains functional in hiw mutants. Other aspects of the hiw mutant phenotype also appear to be independent of BMP signaling. Individual synaptic bouton size is reduced in hiw mutants, a phenotype not observed in BMP mutants and not suppressed by the inhibition of BMP signaling in hiw mutants. Also, the excessive degree of synaptic branching and arborization observed in hiw mutants is only partially suppressed by the disruption of BMP signaling. It is likely therefore that Hiw regulates other molecules responsible for these aspects of synaptic development (McCabe, 2004).
How is synaptic growth maintained? A model is proposed whereby a positive BMP signaling cascade is negatively regulated by the ubiquitin-protein ligase action of Hiw on the Smad Med. This model, however, leaves an important question unanswered: how is the balance between these two opposing forces maintained? This question cannot be answered with current knowledge, but some scenarios can be suggested. One possibility is that the level of phosphorylated Mad competes for binding of Med with Hiw. Once phosphorylated by type I receptors, Mad forms a complex with Med, and the formation of this complex is required for efficient signaling to the nucleus. It is conceivable that an equilibrium exists between the binding of phosphorylated Mad to Med and the binding of Med to Hiw. This equilibrium could potentially act to set a consistent level of BMP signaling and thus normal synaptic growth at the NMJ. Another alternative is that the ability of Hiw to block BMP signaling could be regulated by a third protein that is itself under the transcriptional control of BMP signaling. In this scenario, activation of BMP signaling leads to increased levels of this third protein that in turn activate Hiw's ability to target Med for ubiquitination, completing a negative feedback loop. Future studies will allow these and other possibilities to be tested to further dissect the opposing molecular forces that govern synaptic growth and function (McCabe, 2004).
The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. A simple mechanism has been uncovered through which Dpp, a TGFß/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. These findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation (Pyrowolakis, 2004).
One of the primary events controlled by the Dpp morphogen gradient during growth and patterning of imaginal discs is the establishment of an inverse gradient of brk expression. brk expression is controlled by two opposing activities, a ubiquitous enhancer and a Dpp-dependent silencer. The minimal requirements for a functional silencer complex, both at the DNA and at the protein level, have been determined. Importantly, it has been demonstrated that the minimal element functions in vivo when assayed in the vicinity of a strong enhancer (the brk enhancer) or when present in a single copy in chimeric transgenes (brk enhancer-bamSE fusions) or from within an endogenous gene (gsb-enhancer lacZ fusions). The minimal functional silencer contains a distinct, single binding site for each of the two signal mediators, Mad and Med. Med binds to a GTCTG site, previously recognized as a high-affinity site for Smad binding. Mad binds to a different, GC-rich sequence. Upon binding of Mad and Med, the zinc finger protein Shn is recruited to the protein-DNA complex, bringing along a highly effective repression domain. Although ShnCT contains three essential zinc fingers, it does not bind the silencer element in the absence of Mad and Med. These data suggest that even in the triple protein complex, Shn might bind DNA with moderate sequence specificity, since only a single nucleotide position was identified that is essential for Shn recruitment. However, a number of other cis-regulatory elements that bind Mad and Med (derived from the vestigial, labial tinman, and ubx genes failed to recruit Shn, demonstrating the exquisite selectivity of the element defined in this study (Pyrowolakis, 2004).
Part of this selectivity is accounted for by the specific spacing and orientation of the Mad and Med binding sites in the silencer. Deletion and insertion of single base pairs between the two sites abolish Shn recruitment in vitro and Dpp-dependent repression in vivo, although such alterations still allow the efficient formation of a Mad/Med complex. These findings suggest that Shn recruitment requires a specific steric positioning of amino acid residues in the Smad signal mediators. Strikingly, GTCTG- and GC-rich elements were also found to be crucial for the activation of the Id gene by BMP signaling, but in this case the spacing between the GTCTG- and the GC-rich sites is much larger, and additional factors might be involved in the signal-dependent activation of the Id gene. A more recent study also links these two elements to transcriptional activation of the BMP4 synexpression group in Xenopus. It is tempting to speculate that simple sequence elements similar to the one identified here in several Drosophila genes might be involved in the repression of genes by BMP signaling. Interestingly, human Smad1/5 and Smad4 do form a complex with ShnCT on the Drosophila silencer element from brk; however, a mammalian protein sharing clear homology with Shn in the C-terminal three zinc fingers has not been identified (Pyrowolakis, 2004).
The Dpp-dependent SE allows cells in the developing organism to read out the state of the Dpp signaling pathway. This readout is relatively straightforward because the SE participates in a single switch decision, that is, either to repress (bind Mad/Med and recruit Shn along with its repression domain) or not to repress (not bind Mad/Med, thus failing to recruit Shn). This decision is critically dependent upon one major parameter: the amount of available nuclear Smad complex. For the SE to be functional in vivo, it only needs to interact with a Mad/Med heteromer in those regions of the genome that are actively transcribed; genes that are not active in a given tissue do not need to be repressed by Dpp signaling. This might be one of the main characteristics explaining why such a simple sequence element can have operator-like function in vivo; the element only needs to be recognized by the relevant trans-acting factors in open and active chromatin regions (Pyrowolakis, 2004).
A minimal Dpp-dependent silencer element derived from the brk gene has been identified and demonstrated to function in vivo in a single copy. Its interaction with relevant trans-acting factors have been identified. Based on the results of this analysis, it was possible to derive a consensus sequence, GRCGNCN(5)GTCTG, which allowed scanning of the entire Drosophila genome for potential additional elements. Approximately 350 sites were identified, that, when assayed using transgenic approaches in vivo or in cell culture, should function in a manner analogous to the SEs isolated from the brk regulatory region. Strikingly, and likely significantly, in silico search revealed that the brk gene contains a total of ten SEs, three of them in regions that have been shown to respond to Dpp-dependent repression. Since brk transcription responds to (or can respond to) Dpp signaling in all tissues examined so far, brk might require a SE in the vicinity of each of the different enhancers driving expression in distinct tissues. Alternatively, the readout of the Dpp morphogen gradient might require several SEs, each contributing to the graded repression by Dpp signaling (Pyrowolakis, 2004).
Interestingly, subsequent analysis of two genes containing such Dpp-dependent SEs has demonstrated that these elements function in these transcription units the same way as they do in the brk regulatory region. Therefore, the same molecular principle underlies morphogen readout (brk repression), germline stem cell maintenance (bam repression), and restriction of gene expression to the ventral side of the developing embryo (gsb repression). When the SEs from these three genes are aligned, all the parameters determined to be important for complex formation and for repression are conserved; at all other positions, different base pairs were found in different SEs. In addition, several genes harboring silencer elements are expressed in the wing imaginal disc in a pattern similar to brk or are known to be repressed by Dpp signaling. In contrast, SEs were not found in the vicinity of enhancers known to be activated by Dpp signaling (Pyrowolakis, 2004).
Clearly, these findings implicate that Dpp-induced, Shn-dependent repression via SE elements is a key aspect of development. The readout of the brk gradient contributes to growth and patterning of appendages, and the repression of bam in the germline is essential for the maintenance of germline stem cells. To what extent the repression of gsb contributes to proper cell fate determination along the dorsoventral axis will have to be determined by rescuing the gsb phenotype with a transgene lacking the gsbSE. However, it has been observed that wingless (wg) expression expands from ventral positions to the dorsal side in shn mutant embryos. Since gsb activates wg transcription, the expansion of gsb (in the absence of the gsbSE) possibly leads to the expansion of wg and subsequently to the alteration of dorsoventral cell fate assignments (Pyrowolakis, 2004).
It is important to note that genes repressed by a signaling pathway will not easily be identified in genetic screens because the loss-of-signaling phenotype does not correspond to the loss-of-function phenotype of a repressed gene; in the absence of the signal, such genes are ectopically expressed, leading to a locally restricted gain-of-function phenotype of the corresponding gene. Moreover, since these specific, local patterns of misexpression are likely to result in different phenotypes than widespread overexpression would, simple gain-of-function screens for candidate targets of signal-mediated repression are unlikely to offer straightforward results. Since the target sequence of Dpp/Shn-mediated repression have been identified, the genome can now be scanned and potential target genes can be identified by expression studies and enhancer dissection. It is likely that additional Dpp-repressed genes will be identified using this approach, and this will allow the painting of a much clearer picture of the gene network controlled by Dpp signaling (Pyrowolakis, 2004).
Only a few cases of signal-induced repression have been studied at the molecular level. In most of these cases, repression relies on cooperative action of cell type-specific transcription factors with nuclear signal mediators. The DNA elements that have been demonstrated to mediate repression of particular genes have not been demonstrated to be important for the regulation of other genes, and genome-wide identification of potential target genes using a bioinformatic approach might therefore be difficult, if not impossible (Pyrowolakis, 2004).
The Dpp-dependent repression system identified in this study relies on the organization of Smad binding motifs into Smad/Shn complex-recruiting SEs. The simplicity of these SEs and their capacity to repress transcription in different tissues argues that they function in the absence of tissue-restricted factors. The simple consensus sequence of the SE provides a signature for Dpp-dependent repression, allowing for a genome-wide analysis of potential target genes. Confirmed Dpp-repressed target genes can then be expressed ectopically under the control of the appropriate SE-mutated enhancers to assess the biological importance of repression in a given tissue (Pyrowolakis, 2004).
Substrate-specific degradation of proteins by the ubiquitin-proteasome
pathway is a precise mechanism that controls the abundance of key cell
regulators. SCF complexes are a family of E3 ubiquitin ligases that target
specific proteins for destruction at the 26S-proteasome. These complexes are
composed of three constant polypeptides -- Skp1, Cullin1/3 and Roc1/Rbx1
-- and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a
Drosophila F-Box protein that fulfills several roles in development
and cell physiology. Slmb participation in egg chamber development was analyzed
and slmb was found to be required in both the follicle cells and the
germline at different stages of oogenesis. In slmb
somatic clones, morphogenesis of the germarium and encapsulation of the cyst
are altered, giving rise to egg chambers with extra germline cells and two
oocytes. Furthermore, in slmb somatic clones, ectopic
Fasciclin 3 expression was observed, suggesting a delay in follicle cell differentiation,
which correlates with the occurrence of ectopic polar cells, lack of
interfollicular stalks and mislocalization of the oocyte. Later in oogenesis,
Slmb is required in somatic cells to specify the position, size and
morphology of dorsal appendages. Mild overactivation of the Dpp pathway causes
similar phenotypes that are antagonized by simultaneous overexpression of
Slmb, suggesting that Slmb normally downregulates the Dpp pathway in
follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer
trap reveals that the Dpp pathway is upregulated in slmb somatic
clones and, consistent with this, ectopic accumulation of the co-Smad protein,
Medea, occurs. By analyzing slmb germline clones, it was found that
loss of Slmb provokes a reduction in E2f2 and Dp levels, which correlate with
misregulation of mitotic cycles during cyst formation, abnormal nurse cell
endoreplication and impairment of dumping of the nurse cell content into the
oocyte (Muzzopappa, 2005).
In limb discs, Slmb is a negative regulator of the Dpp
pathway, although the molecular mechanism involved is still unclear. Mild overexpression of Dpp causes a wide
spectrum of phenotypes that are largely coincident with those caused by
slmb loss of function in FC. Supporting the idea that loss of
slmb might cause hyperactivation of the Dpp pathway, the strongest
chorion phenotypes originated by overexpression of Dpp are completely
antagonized by simultaneous overexpression of Slmb in FC. Moreover, expansion
of dad-lacZ expression occurs in slmb mutant follicles,
further suggesting that ectopic induction of the Dpp pathway indeed occurs as
a consequence of slmb loss of function. Consistent with this,
a downstream component of the Dpp pathway, the co-Smad protein
Medea, is upregulated in slmb mutant egg chambers. Because in
mammalian cell culture it was demonstrated that Smad4 is a direct target of
the mammalian Slmb ortholog, ßTrcp1, it is believed
that Medea could be a direct target of Slmb. Further molecular work is
required to assess whether this is indeed the case or if alternatively, the
effect of Slmb on Medea is indirect (Muzzopappa, 2005).
A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno; termed snoN in FlyBase). The SnoN locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for SnoN function. This is an intriguing possibility from the perspective of developmental evolution. SnoN is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. SnoN mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and Smad2 mutants. This suggests that SnoN is a mediator of Baboon signaling. SnoN binds to Medea and Medea/SnoN complexes have enhanced affinity for Smad2. Alternatively, Medea/SnoN complexes have reduced affinity for Mad such that, in the presence of SnoN, Dpp signaling is antagonized. It is proposed that SnoN functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFß signaling and a novel function for Sno/Ski family proteins (Takaesu, 2006).
Studies in mammalian cells showed that, when overexpressed, Sno is an antagonist of TGFß/Activin signaling. Overexpression of dSno with A9.Gal4 (throughout the presumptive wing blade) resulted in small wings with multiple vein truncations at 100% penetrance. A9.Gal4:UAS.dSno pupal wing discs were examined for Drosophila serum response factor (dSRF) expression, an intervein marker repressed by Dpp signaling. In A9.Gal4:UAS.dSno pupal discs, dSRF expression is highly irregular with no obviously downregulated regions corresponding to vein primordia. These wing and disc phenotypes are strongly reminiscent of those expressing the dominant-negative allele Mad1 (DNA binding defective but competent to bind Medea) with a variety of drivers, including A9.Gal4 (100% penetrant) and 69B.Gal4. Mad1 dominant-negative effects are due to the titration of Medea into nonfunctional complexes. The similarity of dSno and Mad1 phenotypes suggests that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).
This was further tested this by coexpressing dSno with Medea or Mad or dSmad2. Coexpression of dSno with Medea or Mad rescues the dSno phenotype to nearly wild type in size and vein pattern. In dSno and Medea coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 28% penetrance. In dSno and Mad coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 19% penetrance. Alternatively, coexpression of dSno with dSmad2 has little effect on the dSno phenotype. In dSno and dSmad2 coexpressed wings, reduced size and multiple vein defects remained 100% penetrant. Coexpression of Mad1 and dSno significantly enhanced the dSno phenotype. One hundred percent of Mad1 and dSno coexpressing the wings are more abnormal than those expressing either dSno or Mad1. The coexpressing wing is very small and veinless and resembles wings expressing UAS.Dad (Dpp antagonist) or dpp class II disc mutants (e.g., dppd5). The enhanced phenotype suggests that dSno and Mad1 antagonize BMP signaling in distinct ways that have additive effects (Takaesu, 2006).
Experiments with a constitutively activated form of the Dpp type I receptor Thickveins (CA-Tkv) are also consistent with this hypothesis. One hundred percent of A9.Gal4:UAS.CA-Tkv wings are overgrown and have numerous ectopic veins as well as vein truncations. This phenotype is suppressed in 98% of the individuals when UAS.dSno is coexpressed with UAS.CA-Tkv. In fact, the coexpression phenotype is not much different from A9.Gal4:UAS.dSno alone, indicating that dSno antagonism of Dpp signaling is fully epistatic to activated Tkv. Finally, ubiquitous overexpression of dSno in the embryonic ectoderm with 32B.Gal4 resulted in discless larvae—a phenotype seen in Mad and Medea null genotypes and in dpp class V disc mutants ( e.g., dppd12. It is concluded that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).
In Drosophila, as in vertebrates, two TGFß subfamilies are present. The
bone morphogenetic protein (BMP) subfamily member Dpp signals through its type I receptor Thickveins to its dedicated transducer Mad (Smad1 homolog) and the Co-Smad Medea (Smad4 homolog). The TGFß/Activin subfamily member activin signals through its type I receptor Baboon to its dedicated transducer dSmad2 and Medea. This study shows that dSno binds Medea and then functions as a mediator of Activin signaling by enhancing the affinity of Medea for dSmad2. Antagonism for BMP signaling likely arises as a secondary consequence of dSno overexpression. Examination of dSno loss-of-function mutants shows that dSno is required in cells of the optic lobe of the brain to maintain proper rates of cell proliferation. Given that Dpp signaling is essential for neuronal differentiation in the optic lobe, these data suggest that dSno functions as a switch that shunts Medea from the Dpp pathway to the Activin pathway to ensure a proper balance between differentiation and proliferation in the brain (Takaesu, 2006).
To resolve the apparent contradiction between the effect on signaling of dSno overexpression (antagonism) and the role identified in dSno mutants (mediation), dSno was examined biochemically. Expression constructs encoding Flag or T7 epitope-tagged Medea, Mad, and dSmad2 were used and various combinations were co-expressed in COS1 cells. It was possible to clearly detect interaction of Medea with both dSmad2 and Mad by co-immunoprecipitation. A T7-tagged dSno expression construct was generated and coexpressed with Flag-dSmad2, Mad, or Medea or with a control vector. Complexes were isolated on Flag agarose and analyzed for the presence of coprecipitating dSno by T7 Western blot. T7-dSno was readily detectable in complexes isolated from cells expressing Medea, but not Mad or dSmad2 (Takaesu, 2006).
Since Medea is a shared partner for both Mad and dSmad2, whether co-complexes containing Medea and dSno together with either Mad or dSmad2 was tested. COS1 cells were transfected with T7-dSno and Flag-Mad or dSmad2 with or without T7-Medea. T7-dSno was present in a complex with Flag-dSmad2 only when T7-Medea was also present. Interestingly, approximately equal amounts of Medea and dSno appeared to coprecipitate with dSmad2, suggesting that much of the Medea that interacts with dSmad2 in this assay is also bound to dSno. In contrast, dSno was not detected in complex with Mad, even when Medea was present, even though Medea clearly interacted with Mad in this assay. These results suggest that dSno interacts specifically with Medea and that the dSno-Medea complex can interact with dSmad2 but not with Mad (Takaesu, 2006).
To test whether incorporation of dSno affected formation of the Medea-dSmad2 complex, Flag-Medea and T7-dSmad2 were coexpressed with or without dSno. The amount of dSmad2 that coprecipitated with Flag-Medea was clearly increased in the presence of dSno. In the reverse of this experiment, it was also observed that an increase in T7-tagged Medea present in Flag-dSmad2 precipitates when dSno was coexpressed. These results suggest that dSno may promote the formation of Medea-dSmad2 complexes (Takaesu, 2006).
To test whether dSno has any effect on the formation of Mad-Medea complexes, similar experiments were performed in which Flag-Medea and Western blotted was isolated for coprecipitating T7-Mad or dSmad2 in the presence or absence of coexpressed dSno. The interaction of Medea with Mad was more readily detectable than with dSmad2. However, inclusion of dSno again increased the interaction between Medea and dSmad2. In contrast, no increase was seen in the Medea–Mad interaction when dSno was coexpressed and it appeared that increasing dSno expression decreased the amount of Mad that coprecipitated with Flag-Medea. Thus it appears that dSno not only may promote interaction of Medea with dSmad2, but also may compete with Mad for Medea interaction, suggesting that dSno may play a role in determining the pathway specificity of Medea (Takaesu, 2006).
Nuclear envelope proteins play important roles in chromatin organization, gene regulation, and signal transduction; however, the physiological role of these proteins remains elusive. This study found that otefin (ote), which encodes a nuclear lamin, is essential for germline stem cell (GSC) maintenance. Ote, as an intrinsic factor, is both necessary and sufficient to regulate GSC fate. Furthermore, ote is required for the Dpp/BMP signaling pathway to silence bam transcription. By structure-function analysis, it was demonstrated that the nuclear membrane localization of Ote is essential for its role in GSC maintenance. Ote physically interacts with Medea/Smad4 at the bam silencer element to regulate GSC fate. Thus, this study demonstrates that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).
In adult tissues, stem cells are characterized by their unique capacity to produce daughter stem cells for self-renewal as well as differentiated daughter cells for maintaining homeostasis. Understanding how the self-renewal and differentiation processes of stem cells are controlled will not only reveal the fundamental biological mechanisms that govern the formation and maintenance of tissues, but may also influence future stem cell-based therapies for regenerative medicine (Jiang, 2008).
The Drosophila ovarian germline stem cells (GSCs) within the germarium region provide an attractive system to study the regulatory mechanisms that determine stem cell fate. A typical Drosophila ovary is composed of 16-20 ovarioles, each consisting of an anterior functional unit called a germarium and a linear string of differentiated egg chambers posterior to the germarium. In the tip of the germarium, GSCs normally divide asymmetrically to ensure that one daughter remains attached to the stromal somatic cap cells (or niche cells) for self-renewal. The remaining daughter cell is displaced from the niche and becomes a cystoblast (CB), which initiates differentiation and sustains oogenesis. During this process, one gene, bag-of-marbles (bam), has been shown to act autonomously in the germline to play an instructive role in CB differentiation. In contrast, gene products, such as Piwi and Dpp, a homolog of BMP2/4 in mammals, are produced from niche cells; however, they function as maintenance factors for GSC self-renewal. It has been shown that Dpp signaling from stromal cells activates Smad signaling in GSCs, directly silences bam transcription, and blocks the formation of Bam:Bgcn complexes that would otherwise antagonize translational repression. However, the issue of how Dpp/Smad signaling is transduced in the nucleus and acts especially at the bam silencer element to repress bam transcription remains poorly understood (Jiang, 2008).
The nuclear envelope separates the nucleoplasm from cytoplasm and is composed of outer and inner membranes that are separated by the perinuclear space and joined at nuclear pore complexes. The nuclear lamina is a network of lamin polymers and lamin-associated proteins that are embedded in the inner membrane. Increasing evidence indicates that these nuclear membrane proteins play important roles in chromatin organization, gene regulation, and signal transduction at the cellular level. However, the physiological roles of these proteins remain elusive. Otefin (Ote) is one member of the 'LEM' family, which represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain. Previous studies have shown that Ote physically interacted with lamin B and YA proteins and localized at the nuclear envelope. Although inhibition of lamin activity by anti-lamin antibody prevented nuclear assembly in vitro, RNAi experiments demonstrated that knockdown of Ote exhibited no effect on Drosophila Kc167 cells, which suggests that Ote might not be a limiting component for the maintenance of the nuclear architecture. Thus, the function and physiological role of Ote remain elusive (Jiang, 2008).
This study shows that otefin (ote), which encodes a nuclear lamin, is essential for GSC maintenance. Ote, as an intrinsic factor, is both necessary and sufficient for GSC maintenance by silencing bam transcription via interaction with Dpp signaling. Furthermore, nuclear membrane localization of Ote is critical for its function in the maintenance of GSC. Finally, biochemical evidence is presented to support that Ote physically interacts with Medea, a Drosophila Smad4, at the bam silencer element to regulate GSC fate. Thus, these data indicate that an integral membrane protein, the nuclear lamin Ote, functions at target gene loci to mediate BMP signal-dependent repression (Jiang, 2008).
This study has identified and characterized Otefin (Ote) as a protein the plays an important role in the regulation of GSC fate via BMP/Dpp signaling. The data support the notion that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).
Observation of the abnormality and loss of germ cells in ote mutant ovaries prompted an exploration of whether ote is involved in the regulation of GSC fate. Using germline clonal analysis and rescue tests, it was demonstrated that ote plays an intrinsic role in GSC self-renewal. In addition, it was also observed that ectopic expression of ote increased the number of GSC-like cells, most likely through repression of GSC/CB differentiation. Thus, the results suggest that, like Dpp signaling, Ote is also both necessary and sufficient to regulate GSC fate. A previous study has demonstrated that knockdown of Ote by RNAi interference exhibited no effect on Drosophila Kc167 cells, suggesting that Ote might not be a limiting factor for the maintenance of the nuclear architecture in cultured cells. Consistently, clonal data showed that ote mutant GSCs could develop into normal cysts and egg chambers rather than undergo apoptosis, suggesting that Ote plays a specific role in maintaining GSC self-renewal but not germ cell viability. As supportive evidence, it was also shown that loss of function of ote did not affect the nuclear architecture and the normal expression of other nuclear lamin components in ovaries. In addition, it was found, except in germ cells, neither overexpression nor loss of function of ote exhibited obvious defects in other developmental processes. Together, these data suggest that Ote may play a role in the maintenance of GSC and germ cell development rather than performing a general cell biological function (Jiang, 2008).
Previous studies have revealed two major signaling mechanisms, dpp-dependent bam transcriptional silencing and bam-independent translational repression, that function cooperatively in the repression of GSC differentiation. In GSCs, Pum/Nos-mediated and microRNA-mediated translational control have been proposed to repress translation of the mRNA pool that promotes GSC/CB differentiation; in contrast, Dpp signaling from the niche cells is responsible for silencing bam transcription in GSCs by activating Smad complexes that physically bind the bam silencer element. Thus, the question becomes how Ote integrates into this signal network. Several lines of genetic evidence strongly suggest that Ote acts through the Dpp signaling pathway rather than through a parallel (Dpp-independent) pathway. (1) The removal of Ote activity not only results in the loss of GSCs, but also replicates the mad or med mutant phenotypes. (2) ote suppressed the TKVca-overexpression phenotype, suggesting that the function of Dpp signaling required Ote activity in order to repress germ cell differentiation. (3) Genetic analysis showed that the ote and dpp pathway are functionally dependent on each other. Thus, these results strongly suggest that Ote serves as a positive component in the Dpp signaling pathway rather than acting through a parallel (Dpp-independent) pathway to regulate GSC fate (Jiang, 2008).
The loss of ote results in a female sterile phenotype but does not affect Dpp signaling in other developmental stages, implying that Ote regulates Dpp signaling only in the ovary. It is possible that ote plays a specific role in regulation of the Dpp pathway in ovary, but is dispensable for dpp pathway regulation in other tissues. A similar example is brinker (brk), which also functions in a tissue-specific manner. It has been shown that brk acts as a negative regulator of the dpp pathway in wing growth control; however, it is dispensable for the dpp pathway in the regulation of GSC fate. Another possibility is that ote could have a redundant function with other nuclear membrane protein(s) in the regulation of the dpp pathway in other tissues (Jiang, 2008).
Structure-function analysis revealed that nuclear membrane localization is essential for Ote function in the regulation GSC fate, the co-IP and FRET assays showed a direct interaction between Ote and Med at the nuclear membrane, and the ChIP assay verified that Ote associated with the bam silencer element in a Med-dependent manner, indicating that Ote/Med interaction might be important for recruiting the bam locus to the nuclear envelope. Combined with the data that Ote is necessary and sufficient for bam silencing in vivo, the results further suggest that Ote/Med-mediated relocalization of the bam locus to the nuclear periphery might be important for bam silencing in the regulation of GSC fate. It has been proposed that subnuclear environments at the nuclear periphery promote gene silencing and activation. Silenced regions of the genome, such as centromeres and telomeres, are statically tethered to the nuclear envelope. Thus, Ote/Med interaction recruiting the bam locus to the nuclear periphery that results in bam silencing may provide an interesting example to support the role of the nuclear periphery in target-gene silencing at the transcriptional level to maintain the identity of the specific type cells (Jiang, 2008).
It has been shown that Schnurri (Shn), a negatively acting Mad cofactor, is genetically required for GSC maintenance. The biochemical evidence showed that the bam silencer element could also form a ShnCT-containing protein-DNA complex with high affinity when Dpp signaling was activated. Thus, studies have proposed that Shn probably serves as a component in the bam silencing complexes/Smad complexes required for bam silencing, and germline stem cells are maintained by Shn recruitment to the bam silencer element. However, so far, the direct experimental evidence that loss of shn results in derepression of bam in GSCs is still lacking. Since Shn, like Ote, has tissue-specific functions mediated by its ability to confer repressive activity on Smad complexes, it will be interesting to test whether Shn acts together with Ote at the bam silencer element in GSCs (Jiang, 2008).
The LEM family represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain. A number of studies have focused on the potential biochemical properties of these proteins and their relationship with nuclear assembly and cell division at the cellular level. Recently, several studies revealed that certain nuclear envelope components are involved in signal transductions, such as MAN1, a nuclear membrane protein that binds Smad2 and Smad3 and antagonizes TGF-β signaling in vertebrates. These findings are in contrast with the current results indicating that Ote functions positively to regulate Dpp signaling transduction in the regulation of GSC. It has been reported that a Drosophila LEM domain protein encoded by the annotated gene CG3167, named dman1, is the putative ortholog to vertebrate MAN1. Similar to Ote, downregulation of dMAN1 by RNAi has no obvious effect on Kc167 cells, suggesting that the dMAN1 protein is also not a limiting component of the nuclear architecture either. Since ote and dman1 possess opposite roles in the regulation of TGF-β/BMP signaling, and dMan1 potentially interacts with Mad in yeast two-hybrid assays and co-IP assays in S2 cells, it would be interesting to determine whether Ote and dMan1 collaborate together to balance the self-renewal and differentiation of GSCs by controlling the proper induction of Dpp pathway activity. There is no known counterpart to Ote in mammals; however, Emerin has a domain arrangement similar to Ote, since it also contains a LEM at its N terminus and a single TM at its C terminus. It has been reported that mutations in emerin cause Emery-Dreifuss muscular dystrophy in humans; however, the molecular mechanism of these mutations and their phenotypes remain poorly understood. This study has characterized a new role in the regulation of stem cells for the nuclear lamin Otefin. It will also be interesting to determine whether nuclear lamina components in mammals, including humans, are also involved in fate determination of stem cells, as well as in mediating signal-dependent gene silencing related to human diseases (Jiang, 2008).
Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. This study demonstrates that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos (see SUMO). Med is identified as the primary SUMOylation target in the Dpp pathway; failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and evidence is provided that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, these results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer (Miles, 2008).
Together, these data suggest a model whereby Med enters the nucleus either by shuttling in a signal-independent manner or through pathway activation, leading to its SUMOylation. Since less SUMOylated Med is detected in the presence of signal, it is proposed that pMad slows the rate of Med SUMOylation, possibly via an effect on Ubc9 recruitment [Ubc9 is encoded by the lesswright (lwr, also called semushi) gene in flies]. FRAP data and imaging of Med in lwr mutant embryos suggest that SUMO modification of Med acts as a trigger to promote its mobility and nuclear export. This finding could explain the necessity for pMad to delay SUMO modification of Med, in order that active Smad complexes have sufficient time to activate transcription. It has been reported previously that TGF-β signaling decreases the nuclear mobility of vertebrate Smad4. It is proposed that this decrease may reflect a slower rate of Smad4 SUMOylation in the presence of phosphorylated R-Smad, which in turn retains Smad4 in an unmodified immobile form (Miles, 2008).
Like Med, the pMad domains are also expanded in lwr mutant embryos and those with non-SUMOylatable Med. More pMad was associated with the non-SUMOylatable MedABC mutant than with wild-type Med. Therefore, the loss of nuclear Med upon SUMOylation appears to promote loss of pMad, even though pMad can accumulate in the nucleus without Med interaction. Recently, pyruvate dehydrogenase phosphatase (PDP) has been shown to terminate Dpp signaling through dephosphorylation of pMad. Although it is presently unclear if PDP dephosphorylates pMad in the nucleus or cytoplasm, the Smad2/3 phosphatase PPM1A acts in the nucleus, resulting in Smad2/3 nuclear export. Therefore, it is possible that SUMO and PDP function together in the nucleus to terminate Dpp signaling. The expanded pMad domains observed when Med SUMOylation is prevented suggest a model in which Med SUMO modification in a wild-type embryo precedes pMad dephosphorylation. This model is consistent with the evidence that dephosphorylation of the receptor-activated Smad promotes complex dissociation and export (Miles, 2008).
SUMO-dependent export of Med from the nucleus following signal activation provides a mechanism to ensure that cells activate Dpp-dependent transcription only in response to the continual receipt of an extracellular Dpp signal. Removal of this sensing mechanism in lwr mutant embryos leads to an inappropriate signaling duration as detected by prolonged zen expression and the cuticle phenotypes (Miles, 2008).
The fate of SUMOylated Med is currently unknown. However, as Ulp1, one of the major SUMO deconjugating enzymes in Drosophila, is localized to the nuclear pore complex (Smith, 2004), it is likely that Med is deSUMOylated upon export. It is suggested that ultimately SUMOylated Med is either recycled following deSUMOylation or degraded. Despite the apparently large cytoplasmic pool of Med, overexpression of wild-type Med expands Dpp target gene expression and the number of amnioserosa cells in early and late stage embryos, respectively. These observations suggest that Med is limiting for signaling, in which case failure to recycle SUMO-modified Med would have a significant impact on the Med pool (Miles, 2008).
Med, which constitutively shuttles between the nucleus and cytoplasm in the absence of signal, is also SUMO modified in the nucleus. There is evidence that in the absence of signal, the Sno corepressor is recruited to nuclear Smad4 to prevent signal-independent transcriptional activation. By limiting Meds time in the nucleus, SUMO-mediated nuclear export may be an additional strategy deployed to further protect against inappropriate transcriptional responses. Interestingly, the results suggest that activation of the Dpp pathway inhibits Med constitutive shuttling. This scenario is different from that described for vertebrate Smad4, which can shuttle independently of an R-Smad upon active TGF-β signaling. Recently, basal shuttling of Smad4 has been shown to require Importin7/8, whereas the mechanism of nuclear import of constitutively shuttling Med is independent of Moleskin, the Drosophila ortholog of Importin7/8. These findings provide further support to the conclusion that there are inherent differences between the constitutive shuttling properties of Med and Smad4 (Miles, 2008).
These data identify a central role for SUMO in modulating the nuclear-cytoplasmic partitioning of the Smad transcription factors. Precedents already exist for SUMO in regulating both the import and export of proteins. For example, SUMO has been implicated in promoting the nuclear retention of the Elk-1 transcription factor, adenoviral E1B-55K protein, and CtBP1 corepressor. In terms of SUMO promoting nuclear export, as the data suggest for Med, examples include the TEL repressor protein, MEK1 kinase, ribosome biogenesis factors, and p53 transcription factor (Miles, 2008).
Following genotoxic stress, SUMOylation of the IkappaB kinase regulator NEMO triggers a cascade of additional modifications including phosphorylation and ubiquitination that ultimately promote NEMOs nuclear export. Ectodermin, a nuclear ubiquitin ligase, constrains BMP signaling by promoting nuclear clearance of Smad4. Whether the fly ortholog of Ectodermin has a similar role, and indeed if there is any interplay between Ectoderminmediated ubiquitination and SUMOylation of Med in its nuclear export, remains to be determined. An alternative mechanism by which SUMO promotes Med export is based on that described for p53. p53 is monoubiquitinated by MDM2, which exposes the NES and allows recruitment of the PIASy E3 ligase leading to p53 SUMOylation. As a result, MDM2 dissociates and p53 nuclear export occurs. SUMOylation may re-expose the Med NES that has been inactivated upon signaling (Watanabe, 2000), promoting nuclear export. The location of the Med NES in between SUMO sites A and B may lend itself to this type of regulation. Interestingly, SUMO sites A and B are the two that are conserved in vertebrate Smad4, as is the position of the NES. It is speculated that SUMOylation will also direct nuclear export of vertebrate Smad4 (Miles, 2008).
Although SUMO modification of Smad4 has been postulated to have both positive and negative effects on gene expression, Med SUMOylation leads to a reduction in its transcriptional activity in the context of Dpp signaling in the Drosophila embryo. These differences may reflect promoter-specific effects or particular characteristics of the transcription factor complex that depend on which receptor-activated Smad is associated with Med/Smad4 (Miles, 2008).
Studies of extracellular signals such as Dpp and Hedgehog support the generation of different gene activity thresholds by a 'French flag' model of positional information. Signal concentration provides positional information so that cells located nearest the source activate a peak threshold of gene activity and adopt a specific cell fate, whereas cells located further from the source express different threshold responses and assume distinct fates. Morphogen concentration at the source and sink is therefore crucial, and mechanisms that have been characterized for regulating patterning by morphogens have intuitively focused on the morphogen itself. However, the current results identify a twist on the French flag model whereby the positional information provided by a specific concentration of morphogen can be refined by modulating the activity of an intracellular transducer. In this way the French flag floats in relation to Dpp activity, since the absolute amount of Dpp required for each fate is influenced by the activity of the SUMOylation pathway. Although this study has concentrated on the SUMO post-translational modification, any mechanism that hones the activity or distribution of an intracellular transducer will affect the interpretation of positional information and pattern formation in a similar way. Moreover, it is predicted that SUMO itself will be used to modulate the signaling outputs by other morphogens in different developmental contexts. A good candidate appears to be the Wnt morphogen, as links between SUMO and the Wnt pathway during Xenopus development been suggested (Miles, 2008).
The spatial and temporal range of the Dpp/BMP signal is controlled not only by Med SUMOylation but also by PDP dephosphorylation of pMad and dSmurf-dependent ubiquitination of cytoplasmic Mad. Therefore, multiple mechanisms exist for constraining the activity of the Smad transcription factors, all of which are wasteful in terms of signal. Although wasteful, having a dedicated dampener in the form of SUMO modification may be tolerated so that the Dpp signaling pathway can be controlled somewhat in the event of inappropriate activation. This may be essential given the potency of Dpp signaling in inducing cell fates. Another possibility is that the disadvantage of losing signal through this built-in dampener is far outweighed by its use as a mechanism through which the presence of an extracellular signal can constantly be sensed (Miles, 2008).
In addition to the central role of Med/Smad4 in mediating the appropriate transcriptional outputs in response to signaling by all TGF-β ligands, the function of Smad4 as an essential tumor suppressor protein in humans has been well documented. As well as SUMOylation, ubiquitination of the Med/Smad4 transcription factor has been described. Therefore, it appears that multiple mechanisms are deployed during development to harness the activity of this pivotal signal-responsive transcription factor (Miles, 2008).
The TGF-β/BMP signaling cascades control a wide range of developmental and physiological functions in vertebrates and invertebrates. In Drosophila melanogaster, members of this pathway can be divided into a Bone Morphogenic Protein (BMP) and an Activin-β (Act-β) branch, where Decapentaplegic (Dpp), a member of the BMP family has been most intensively studied. They differ in ligands, receptors and transmitting proteins, but also share some components, such as the Co-Smad Medea (Med). The essential role of Med is to form a complex with one of the two activating Smads, Mothers against decapentaplegic (Mad) or dSmad, and to translocate together to the nucleus where they can function as transcriptional regulators of downstream target genes. This signaling cascade underlies different mechanisms of negative regulation, which can be exerted by inhibitory Smads, such as Daughters against decapentaplegic (Dad), but also by the Ski-Sno family. This work identified and functionally analyzed a new member of the Ski/Sno-family, fussel (fuss, annotated as CG11093, currently termed CORL), the Drosophila homolog of the human functional suppressing element 15 (fussel-15). fuss codes for two differentially spliced transcripts with a neuronal expression pattern. The proteins are characterized by a Ski-Sno and a SAND homology domain. Overexpression studies and genetic interaction experiments clearly reveal an interaction of fuss with members of the BMP pathway, leading to a strong repression of BMP-signaling. The protein interacts directly with Medea and seems to reprogram the Smad pathway through its influence upon the formation of functional Mad/Medea complexes. This leads among others to a repression of downstream target genes of the Dpp pathway, such as optomotor blind (omb). Taken together shows that fuss exerts a pivotal role as an antagonist of BMP signaling in Drosophila melanogaster (Fischer, 2012). The transforming growth factor beta (TGF-beta) and bone morphogenic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. This study has mapped Drosophila TGF-beta signaling factors Mad, dSmad2, Medea and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF
Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling (Van Bortle, 2015).
TGF-β effector proteins have been shown to co-localize with mammalian CTCF in a CTCF-dependent manner at just 2 individual loci. This observation has been extended to Drosophila using a genome-wide approach, providing evidence that architectural protein CTCF and canonical Smad signaling proteins, both highly conserved from fly to humans, co-localize on a global scale. Context-specific features were uncovered in which Smad localization is dependent or independent of CTCF binding. Interestingly, genome-wide analysis identifies Mad, dSmad2, Medea, and Schnurri binding to previously characterized response elements even in the absence of DPP ligand, in which levels of phosphorylated Mad are undetectable. This signal-independent clustering of signaling proteins suggests that the genomic TGF-β signaling response is not as simple as regulating binary 'off vs. on' states, dependent on phosphorylated Mad. However, attempts to map the genomic landscape of phosphorylated-Mad before and after DPP stimulation were unsuccessful, likely due to issues with currently available p-Mad antibodies. Though it was not possible to determine the role of phosphorylation as a determinant in Mad localization, it is conceivable that phosphorylation of Mad might play a role in regulating the resident time of DNA-binding, the recruitment of additional regulatory partners, or the ability to establish functional long-range interactions (Van Bortle, 2015).
Smad co-binding at dCTCF sites is sensitive to dCTCF depletion at low occupancy dCTCF target sequences for which Smad consensus sequences are depleted, whereas high occupancy dCTCF binding sites co-bound by additional architectural proteins remain unaffected. The dCTCF-independent recruitment of Smads to high occupancy APBSs suggests that additional architectural proteins may redundantly recruit Smads, or simply provide an accessible chromatin landscape to which Mad, Medea, and dSmad2 can associate. Nevertheless, dCTCF-dependent localization of Smad proteins to specific low occupancy elements is consistent with the CTCF-dependent nature of Smad binding at both the APP and H19 promoters in humans. It is speculated that dCTCF-dependent Smad localization to low occupancy APBSs within topological domains may represent regulatory elements involved in enhancer-promoter interactions, whereas dCTCF-independent high occupancy APBSs are involved in establishing higher-order chromosome organization. What role Smads might play in establishing or maintaining such long-range interactions relevant to chromosome architecture, or whether Smads and other transcription factors simply localize to high occupancy APBSs due to chromatin accessibility, remains difficult to address. However, it has been recently shown that high occupancy APBSs are distinct from analogous transcription factor hotspots, suggesting some level of specificity, most likely governed by protein-protein interactions, decides which factors can associate and where. Alternatively, the enrichment of ChIP-seq signal at high occupancy APBSs may, to some degree, reflect indirect association via long-range interactions with regulatory elements directly bound by Smad proteins. This possibility raises a potential explanation for why Smad ChIP signal is independent of dCTCF binding at high occupancy APBSs (Van Bortle, 2015).
Surprisingly, DPP-activated phosphorylation of Mad does not lead to significant changes in dCTCF binding, whereas Mad, Medea, and Schnurri levels increase at regulatory elements away from dCTCF. These results suggest that TGF-β signaling in Kc167 cells redirects Smad binding to genomic loci independent of architectural proteins, and that architectural proteins may facilitate binding of nuclear Smad proteins in the absence of signaling. The complete loss of Smad ChIP signal at numerous dCTCF binding sites enriched for the core dCTCF consensus sequence nevertheless provides compelling evidence that recruitment of Smad proteins is directly governed by Drosophila CTCF at a subset of binding sites. These results establish CTCF as an important determinant of Smad localization and, depending on the cell-type specific binding patterns of CTCF, suggest that CTCF might also influence the tissue-specific localization of Smad proteins analogous to master regulatory transcription factors in multi-potent stem cells (Van Bortle, 2015).
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