tramtrack
Tramtrack RNA is alternatively spliced, giving rise to two forms. One is a protein of 69 kDa that binds the fushi tarazu promoter. A second is a protein of 88 kDa with an alternative set of zinc
fingers, having a DNA binding specificity distinct from that of the first protein. Tramtrack (Ttk88) expression represses neuronal fate determination in the developing Drosophila eye. Ttk88 was ectopically expressed in R3, R4 and R7 photoreceptor precursors and the four cone cell precursors using a Sevenless enhancer. The resultant transgenic flies have three missing photoreceptor precursors per ommatidium. Ectopic expression of Ttk88 in all cells posterior to the morphogenetic furrow results in flies devoid of photoreceptors (Tang, 1997).
Phyllopod acts to antagonize this repression by a mechanism that requires Seven In Absentia and is associated with decreased Ttk88 protein levels, but not reduced ttk88 gene transcription or mRNA stability. Sina, Phyl, and Ttk88 physically interact. Phyl interacts with Sina and Phyl interacts strongly with Ttk88 (but not with TTK69). Sina and TTK88 show a weak interaction. Sina interacts genetically and physically with UBCD1, a component of the ubiquitin-dependent protein degradation pathway (Treier, 1992). These results suggest a model in which activation of the Sevenless receptor tyrosine kinase induces Phyl expression, which then acts with Sina to target the transcriptional repressor Ttk88 for degradation, thereby promoting R7 cell fate specification (Tang, 1997).
The transcription repressor Tramtrack (Ttk) is found in cone cells but not photoreceptor cells of the Drosophila eye. Down-regulation of Ttk expression occurs in photoreceptor cells and is required for their fate determination. Down-regulation requires the presence of Phyllopod, which is induced by the ras pathway, and Seven In Absentia (Sina). Loss of either gene causes accumulation of Ttk in photoreceptor cells, and Ttk does not accumulate in cone cells if both Phyl and Sina are present. Reduction of Ttk levels by Phyl depends on sina function. Reduction of Ttk expression induced by ectopic phyl does not occur in a sina mutant background. Down-regulation of Ttk expression by phyl does not occur at the transcriptional level. That is, the level of ttk expression as detected using a ttk expression vector is uneffected by alteration in phyl function. Sina and Phyl promote ubiquitination and rapid degradation of Ttk by the proteasome pathway in cell culture. Both Sina and Phyl bind to the N-terminal domain of Ttk. Deletion of amino acids 1-286 of Ttk88 abolishes the interaction with Sina, whereas a polypeptide containing amino acids 1-116 of Ttk88 results in a strong interaction. Interestingly, this region corresponds precisely to the BTB domain shared by both Ttk69 an Ttk88. These results argue that photoreceptor differentiation is regulated by the RAS pathway through targeted proteolysis of the Ttk repressor (S. Li, 1997).
Musashi
(Msi), the Drosophila neural RNA-binding protein, plays a part in eye development. Msi expression is observed in
the nuclei of all photoreceptor cells (R1-R8). Although a msi
loss-of-function mutation results in only weak abnormalities in
photoreceptor differentiation, the msi eye phenotype is
significantly enhanced in a seven in absentia (sina) background.
sina is known to be involved in the degradation of the Tramtrack
(Ttk) protein, leading to the specification of the R7 fate. Msi also functions
to regulate Ttk expression. The sina msi mutants show significantly
high ectopic expression of Ttk69 and failure in the determination of the R1,
R6, and R7 fates. Other photoreceptor cells also fail to differentiate, with
abnormalities occurring late in the differentiation process. These results
suggest that Msi and Sina function redundantly to downregulate Ttk in
developing photoreceptor cells (Hirota, 1999).
To examine the pattern of Msi expression in eye development, a
monoclonal antibody was generated against the Msi protein. The N-terminal
region of Msi (210 amino acids) was used as the antigen for immunization.
The antibody 3A5 recognizes three protein species with relative molecular
masses of 60-70 kDa in immunoblots of protein extracts from wild-type eye
discs. These sizes are compatible with the molecular weight predicted from
the cDNA sequence (63 kDa, 606 amino acids). Msi may receive three
different types of modification, resulting in the three bands observed. In the
immunoblot analysis, the intensities of these bands were reduced in
msi1/+ extracts and undetectable in msi1/msi1extracts.
Immunohistochemistry with this antibody does not detect any signals in
msi1/msi1 discs. Taken together, these results suggest that
the antibody 3A5 recognizes the Msi protein specifically. To determine the
types of cells expressing Msi in developing eye discs, double staining with
antibodies against Msi and a neuronal marker, Elav (Robinow and White,
1991), was performed. In eye imaginal discs from third-instar larvae, a low
level of Msi expression is observed in cells forming a stripe immediately
anterior to the morphogenetic furrow (MF); a more intense signal is also observed in the
developing ommatidia posterior to the MF. Msi expression is restricted to
nuclei in cells posterior to the MF, although it appears not to be restricted to
nuclei anterior to it. Compared with Elav staining, some cells anterior and
just posterior to the MF are immunopositive for Msi, but negative for Elav.
To compare Msi and Elav expression in detail, discs were stained with both
Msi and Elav antibodies using HRP labeled secondary antibodies. The
ommatidial clusters located 4 to 6 rows posterior to the MF contain three
Elav-positive neurons: R8, R2, and R5. These three cells are also positive for
Msi, and Msi expression is also detectable in the presumptive R3 and R4 cells
prior to their Elav expression. At 7 to 10 rows posterior to the MF, the
ommatidial clusters contain five Elav-positive neurons: R8, R2, R5, R3, and
R4. In addition to these five cells, three Msi-positive cells are located basally
adjacent to R2, R8, and R5; these locations correspond to the presumptive R1, R6, and
R7 cells. This result suggests that expression of Msi begins earlier than that
of Elav during the neuronal differentiation of photoreceptor cells. Staining of
pupal retinas 40 h after puparium formation (40 h APF) with the anti-Msi
antibody shows that all photoreceptor cells continue to express Msi protein
during pupal eye development. Msi is also expressed in the photoreceptor
cells of adult flies (Hirota, 1999).
To investigate the functions of Msi during eye development, the eye
phenotype of the msi1/msi1mutants was examined.
msi1/msi1eyes contain abnormal ommatidia, with deformed
rhabdomeres and/or irregular orientation, at a low frequency (3.05%).
Staining developing msi1/msi1-eye discs with antibodies
against several neuronal markers reveals that the number of photoreceptor
cells is not affected, suggesting that msi is involved in late processes
of photoreceptor cells differentiation, including the formation of
rhabdomeres. However, the penetrance of the
msi1/msi1phenotype is so low that it was difficult to
investigate the Msi function. Also examined were the genetic interactions of msi with
ttk, a possible target gene of Msi, and sina, a factor involved
in the degradation of Ttk. Strong genetic interactions occur
between msi and sina mutations in eye development. In
wild-type flies, the compound eye has a regular array of ommatidia, each of
which contains eight photoreceptor cells (R1-R8). At the R7 level, the
rhabdomeres of seven photoreceptor cells (R1-R7) are arranged in a
characteristic asymmetrical trapezoid. Since Sina is essential for the
differentiation of R7, R7 is missing in 90% of the ommatidia of
sina2/sina3 mutants (in which little if any functional gene products
are produced) and the external morphology of the eyes shows a slight
roughness. Notably, the eye phenotype of the double homozygous mutants
of msi and sina (sina2msi1/sina3msi1) show
synergistic, but not additive, enhancement. The external morphology of the
double homozygous mutants shows strikingly disturbed ommatidial arrays.
Most of the rhabdomeres are severely deformed and no ommatidia contain
more than five rhabdomeres, suggesting that msi and sina
have important and distinct roles in the differentiation of photoreceptor
cells. To confirm the function of Msi, rescue experiments of sina msi
double mutants by Msi were performed. A genomic region containing the
wild-type msi gene (referred to as P[msi1+]) was introduced
into the sina msi mutant flies by P element-mediated germline
transformation. The P[msi1+] fragment significantly rescues the
defects in the sina msi eyes. The external morphology of the eye and
the formation of rhabdomeres are nearly normal. This result indicates that
the sina msi eye phenotype is partially due to the loss of msi
function. Since Msi contains two RNA recognition motifs (RRMs), RRM-A and
RRM-B, the RNA-binding activity of Msi is likely to be essential for its
rescuing activity. To test this possibility, a transgenic rescue experiment was
performed in which mutant Msi proteins whose RNA-binding activities were
designed to have been abolished were expressed. The RRM domains contain a
consensus sequence that is composed of two highly conserved short
segments, referred to as RNP1 (ribonucleoprotein octamer consensus) and
RNP2. Since the aromatic side chains of RNP1 are known to be crucial for
RNA binding, mutations that change phenylalanine to alanine in three places
in the RNP1 were induced into both of the two RRMs of Msi (referred to as
P[msiA*B*]). The P[msiA*B*] fragment does not rescue the
sina msi eye phenotype. Additionally, P[msi1+] fully rescues
the weak defects in the msi1eyes described above, while
P[msiA*B*] had no effect. Taken together, it is concluded that the
RNA-binding ability of Msi is involved in normal eye development (Hirota,
1999).
To examine how the sina and msi mutations causes the
severe eye defects described above, the neuronal differentiation of this
double mutant was examined by staining with anti-Elav antibody. All the
ommatidia posterior to the eighth row from the MF contain eight
photoreceptor cells in wild-type eye discs. In the same region, 90% of the
ommatidia of sina2/sina3 lack R7. In the same region, all the
ommatidia of sina2msi1/sina3msi1contain only five
cells, consistent with the appearance of the phenotype in adult eyes. In the
sina2msi1/sina3msi1eye discs, a nearly normal
pattern of Elav staining was observed in developing ommatidia up to the
five-cell precluster stage, which is composed of R2, R3, R4, R5, and R8.
Subsequently, however, R1, R6, and R7 are never added to the ommatidia.
These results suggest that Msi and Sina have redundant functions in the cell
fate determination of R1, R6, and R7. Furthermore, in the posterior region of
the eye discs, the spatial arrangement of the five cells in each ommatidium
changes and overlaps abnormally. In the most posterior region of the eye
discs, many ommatidia with reduced numbers of Elav-positive cells are
observed. These results indicate that R2, R3, R4, R5, and R8, which had once
expressed Elav, gradually have their spatial arrangement in the ommatidia
disrupted, and that some of the photoreceptor cells fail to maintain Elav
expression, resulting in a strikingly deformed eye (Hirota, 1999).
To confirm the requirement of Sina and Msi for the cell fate
determination of R1 and R6, the sina msi eye discs were stained with
antibody against Bar, an R1/R6-specific marker. Bar is expressed in R1 and
R6 in wild-type eye discs. In 40% of the ommatidia of sina2/sina3 eye
discs, the number of Bar-positive cells is reduced to one or zero,
suggesting that Sina has some roles in the cell fate determination of R1 and
R6, where Sina is known to be expressed. Consistent with anti-Elav staining,
Bar-positive cells completely disappear in the
sina2msi1/sina3msi1eye discs. In combination with
sina1, a hypomorphic allele, instead of sina2, one or two
Bar-positive cells per ommatidium are detected in 50% of the
sina2msi1/sina3msi1ommatidia, confirming that Msi
and Sina have redundant functions required for the cell fate determination
of R1 and R6 (Hirota, 1999).
Sina has been suggested to be involved in the degradation of Ttk, a
general inhibitor of neuronal differentiation. Since sina msi double
mutants show defects in neuronal differentiation, the possibility that the
expression pattern of Ttk69 is different in these animals was tested by
examining the Ttk expression pattern in eye discs stained with anti-Ttk69
antibody. In wild-type eye discs, Ttk69 is detected in four cone cells per
ommatidium. The expression pattern of Ttk69 in the
msi1/msi1eye discs is indistinguishable from that of
wild-type. In sina2/sina3 eye discs, five cells were labeled in 5% of
the ommatidia, suggesting that degradation of Ttk69 is reduced. Notably,
50% of the ommatidia in the sina2msi1/sina3msi1eye
discs contain additional Ttk69-expressing cells, indicating that the average
number of cells per ommatidium that express Ttk69 ectopically is larger in
sina2msi1/sina3msi1 than in sina2/sina3 eye discs.
Since both photoreceptor and cone cells are deformed at later stages of
development the cell types ectopically expressing Ttk69 could not be
identified. This result suggests that the Ttk69 expression is negatively
regulated by both Msi and Sina. Consistent with this idea, the morphology of
sina msi double mutants is significantly recovered in a heterozygous
background for ttkosn (sina2msi1ttk osn/sina3msi1), a
mutation that disrupts expression of both the Ttk69 and Ttk88 proteins.
The external morphology of the eye and the formation of the rhabdomeres
are nearly normal. Thirty percent of the
sina2msi1ttkosn/sina3msi1ommatidia show the normal
number and arrangement of photoreceptor cells and are indistinguishable
from wild-type ommatidia. These results suggest that the severe eye
phenotype of the sina2msi1/sina3msi1 double mutant may result
from an elevation in Ttk expression levels (Hirota, 1999).
These results demonstrate that Msi and Sina redundantly function as
factors required for the downregulation of Ttk69; however, the mechanism
remains unknown. A recent study, exploring the target RNA for Msi,
indicates that TTK69 mRNA contains multiple sites that could
potentially be recognized by Msi. Therefore, it is likely that Msi binds to the
TTK69 mRNA to inhibit its translation or reduce its stability. In
contrast, Sina has been shown to function with Phyl to target Ttk protein for
degradation. Thus, Msi and Sina are likely to function to down-regulate Ttk
at different levels in independent manners. If one functions via the other, the
phenotype of the sina msi double null mutants would be identical to
the phenotype of single null mutants for the gene that functions downstream
of the other. Instead, the sina msi mutants show a synergistically
enhanced eye phenotype that is much more severe than that of the single
mutants. Furthermore, the finding that a half-reduction in the gene dosage
of ttk suppresses the sina msi double null mutants indicates
that ttk is downstream of msi and sina. Taken
together, the expression of Ttk is likely to be regulated posttranscriptionally
by factors including Msi, and posttranslationally by Sina, Phyl, and Ebi (a WD
repeats protein). The negative regulation of Ttk by Msi and Sina is required
for both the early processes of R1, R6, and R7 differentiation and the late
processes of the differentiation of other photoreceptor cells in eye
development. Further studies will extend knowledge of how the
posttranscriptional regulation of gene expression by Msi controls ommatidial
development (Hirota, 1999).
The BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain is an evolutionarily conserved
protein-protein interaction motif. Many BTB-containing proteins are transcriptional regulators involved in a wide range of
developmental processes. However, the significance of the BTB domain in development has not been evaluated. Evidence is presented that overexpression of the Tramtrack69 (Ttk69) protein not only blocks neuronal photoreceptor
differentiation but also promotes nonneuronal cone cell specification in early Drosophila eye development (Wen, 2000).
To investigate the role of Ttk69 in early eye development, the full-length Ttk69 was overexpressed in cells of the R7 equivalent group that
include the R3/R4, R7, and cone cell precursors by using the Gal4/UAS expression system. In sev-Gal4/UAS-ttk69
eye discs, all R3/R4 and many R7 precursor cells fail to express the Elav protein, which has been commonly used as a neural-specific marker. Consequently, these cells do not become R cells. With one copy of the ttk69 transgene, all
ommatidia contain four or fewer outer R cells (on average 3.3 outer R cells per ommatidium), and 43% of the ommatidia have no R7 cell. When
driven by ey-Gal4 for expression in the anterior region of the developing eye, Ttk69 effectively blocks eye formation. Thus, Ttk69 is sufficient to
block neural differentiation in eye discs, which is consistent with the idea that Ttk69 is a negative regulator of R cell fate during the larval stages of eye
development. At late pupal stages, Ttk69 is positively and autonomously required for facilitating R cell differentiation (Wen, 2000).
To determine if Ttk69 also plays a role in cone cell specification, sev-Gal4/UAS-ttk69 eye discs were stained with an antibody made against the Cut protein, which is normally expressed in all cone cells and has been commonly used as a cone cell marker. All R3/R4 and many R7 precursor cells are positive for Cut expression. The anterior and posterior cone cell precursors are not recruited into the ommatidial clusters in the mutant eye disc. However, it appears that the polar and equatorial cone cells can be occasionally recruited. Consequently, each
ommatidium contains two to five cone cells. Mystery cells often become Cut positive as well, but they appear only for several hours before being
excluded from the clusters. Thus, ttk69 overexpression appears to transform photoreceptor neurons to nonneuronal cone cells. Consistent with this
observation, ttk69 has been shown to be sufficient to transform neurons into support cells in sensory organs. Loss-of-function analysis indicates that Ttk69 is necessary for the development of cone cells (Wen, 2000).
The BTB domain is essential for Ttk69 function; single amino acid changes in highly conserved residues in this domain abolish Ttk69 activity. Interestingly,
the Ttk69 BTB can be substituted by the BTB of the human Bcl-6 protein, suggesting that BTB function has been conserved between Drosophila and humans. The Ttk69 BTB domain is critical for mediating interaction with the Drosophila homolog of C-terminal-binding protein (dCtBP) in vitro, and
dCtBP minus mutations genetically interact with ttk69. Furthermore, the C-terminal region downstream of the DNA-binding zinc fingers has been shown to be essential for
Ttk69 function. A dCtBP consensus binding motif in the C terminus appears to contribute to Ttk69 activity, but it cannot be fully responsible for the function
of the C terminus (Wen, 2000).
ebi (the Japanese term for 'shrimp') regulates the Epidermal growth factor receptor signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar
phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical
Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly
arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved
biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes Egfr-dependent down-regulation
of Tramtrack88, an antagonist of neuronal development (Dong, 1999).
The similarity of Ebi to F-box/WD40 repeat-containing
proteins and its nuclear localization suggests that Ebi may regulate Egfr signaling through degradation of nuclear proteins. Recent studies have
revealed an important role for both Egfr and
degradation of a specific transcription factor Tramtrack88, for R7 development. The structural similarity between Ebi and
F-box/WD40-repeat proteins involved in protein
degradation prompted an exploration of the relationship between
ebi and Ttk88 protein levels in the developing eye. Ttk88 is expressed at very low levels in
undifferentiated cells in the developing eye disc and at high levels in
developing cone cell nuclei; it is not expressed in developing
photoreceptor cells. The protein Phyllopod, promotes the degradation of Tramtrack. Transformation of cone cells into R7 by misexpression of phyllopod under the sevenless promoter leads to Ttk88 degradation.
Ectopic R7 induction by constitutively active Egfr (TorDEgfr) driven by the
sev promoter also leads to marked degradation of Ttk88.
sev-TorDEgfr-induced Ttk88 degradation is
dominantly suppressed by ebi. Similarly, ebi
dominantly suppresses the pGMR-phyl-induced decrease in
Ttk88, as well as the pGMR-phyl-induced eye phenotype (Dong, 1999).
The role of ebi in regulating Ttk88
levels in an otherwise wild-type eye disc was examined. Analysis of Ttk88 levels in
the small mutant clones generated with ey-Flp reveals no obvious differences. To explore this issue further, reduction in ebi
was achieved by expressing the dominant-negative form of ebi
in all cells posterior to the morphogenetic furrow in an ebi
heterozygous background. Dominant-negative
ebi contains the amino-terminal half of the protein from
amino acids 1-334 expressed under the control of
the pGMR promoter. In wild-type eye discs, Ttk88 staining is not
observed in a focal plane in which photoreceptor cell nuclei are
located. In contrast, in mutant discs, an average of 36 ± 6 Ttk88-positive nuclei are observed in this
region. Most Ttk88-positive nuclei are found 8-10 rows posterior to
the morphogenetic furrow. This increase in Ttk88-positive cells also parallels a concomitant
decrease in the number of cells stained with the pan-neuronal stain,
anti-Elav. In wild-type eye discs, all ommatidia 8-10 rows posterior to the morphogenetic furrow have at least seven
Elav-positive cells (R1-R6 and R8). However, in mutant discs,
many ommatidia in this region contain less than seven stained cells. Interestingly, a
considerably smaller fraction of ommatidia in
rows 11-13 contain less than eight Elav-positive R cells;
in wild-type discs, all clusters contain eight Elav-positive cells in
this region. Hence, a reduction in ebi activity delays neuronal development and this is correlated with persistent nuclear expression of the Ttk88 protein. In summary, both ebi and
Egfr promote Ttk88 down-regulation, thereby promoting neuronal
development. Further work in other
developmental contexts is required to assess the relationship
between ebi and ttk in Egfr signaling (Dong, 1999).
Three lines of evidence were found that link Ebi to Sina and Pyllopod-dependent degradation of Ttk88. The first line of evidence comes from transient transfection experiments. Sina and Phyl are able to target Ttk88 for ubiquitin-dependent degradation when expressed in transient transfection experiments in S2 cells. S2 cells contain high levels of endogenous Ebi. To interfere with the activity of this protein, an N-terminal fragment (EbiN) was expressed that has been used (Dong, 1999) as a dominant-negative mutant to interfere with Ttk88 degradation in the eye disk. Cells were co-transfected with pIZT vectors constitutively expressing either Cat or EbiN from the OpIE2 promotor, in the presence of metallothionine-inducible vectors containing Phyl, Sina and Ttk88-myc. Following transfection and copper induction, cells were metabolically labeled with [35S]methionine and then chased with cold methionine for various time points to monitor Ttk88 degradation. In the presence of Sina and Phyl, Ttk88 is degraded with a half life of ~25 min, a process that can be blocked by the proteosome inhibitor MG132. Ttk88 degradation is blocked in cells expressing the EbiN dominant-negative form of pIZT, whereas a control pIZT vector that constitutively expressed Cat gave no effect (Boulton, 2000).
The second line of evidence stems from an in vitro degradation assay. To investigate whether Ebi is linked to Ttk88 degradation, attempts were made to reconstitute Ttk88 degradation in vitro. These studies show that Sina and Phyl are needed for Ttk degradation, mirroring their requirement in vivo. The results of the in vitro degradation assay suggested that Ebi might physically associate with Ttk88, Sina and Phyl. Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. When Sina, Phyl and Ttk88 are co-expressed in the same lysate, Ebi is able to pull down Ttk88 with a much higher affinity, compared with Ttk88 when expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is likely that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association (Boulton, 2000).
If stabilization of Ttk88 is responsible for the cell cycle phenotypes associated with Ebi, a consistent pattern of genetic interactions between GMR-p21 or GMR-E2F-DP-p35 and mutations in the Egfr pathway would be expected that function to regulate Ttk88 levels. However, unlike mutations in Ebi, loss-of-function alleles of egfr, gap1, raf1, ras1, mapk, yan, sina, phyl and ttk have no strong effect on either the GMR-p21 or the GMR-E2F-DP-p35 phenotype. Mutations in the Ets-domain transcription factor Pointed (pnt) enhance the GMR-E2F-DP-p35 phenotype but fail to modify the GMR-p21 phenotype. While it is possible that Ebi is the only dosage-sensitive component of the Egfr pathway whose levels affect cell proliferation, these results suggest that Ebi might have an activity that is independent of Ttk88 degradation (Boulton, 2000).
To test directly whether stabilization of Ttk88 is likely to be responsible for the changes in cell cycle control caused by ebi mutant alleles, the effects of elevating the levels of Ttk88 were tested. Ectopic expression of Ttk88 was induced by heat shock for 30 min at 39°C in embryos carrying hs-Ttk88. Following recovery, embryos were either pulsed with BrdU or aged for immunohistochemistry. The ectopic expression of Ttk88 from a heat shock-regulated transgene is sufficient to disrupt neuronal differentiation in stage 13-14 embryos. Unlike the ebi mutant embryos, BrdU incorporation demonstrates that the block to differentiation in hs-Ttk88 embryos does not result in a failure to exit the cell cycle in the PNS and CNS. Furthermore, expression of Ttk88 from a GMR transgene inhibits differentiation in the eye disc. However, the GMR-Ttk88 transgene fails to suppress the GMR-p21 eye phenotype and is unable to restore S phases in the GMR-p21 eye disc. In contrast, halving the dosage of Ebi or expressing cyclin E from the GMR promoter is sufficient to restore the second mitotic wave of S phases. It is concluded that increasing Ttk88 protein to a level where differentiation is perturbed in either the embryo or the eye disc is insufficient to promote S phase entry in either of the situations where ebi mutations give this effect. It is inferred that Ebi must have a second function, independent of Ttk88 degradation, that is important for regulating cell cycle exit (Boulton, 2000).
To investigate whether Ebi is linked to Ttk88, attempts were made to reconstitute Ttk88 degradation in vitro. Initial experiments showed that Ttk88 is not degraded when co-expressed with Sina and Phyl in rabbit reticulocyte lysate. In an attempt to stimulate the degradation activity, GST (glutathione S-transferase) or GST-Ebi beads were incubated together with ubiquitin, an ATP regeneration system and Sina-Phyl-Ttk co-expressed in a reticulocyte lysate. Neither GST nor GST-Ebi is able to promote Ttk88 degradation in this setting. It was speculated that Ebi may function as a bridge between the Sina-Phyl-Ttk88 complex and an activity required for ubiquitylation and subsequent degradation of Ttk88. In order to provide this missing activity, GST or GST-Ebi beads were preincubated with S2 extracts (now referred to as loaded beads) prior to performing the degradation reaction. Loaded GST-Ebi beads are unable to degrade Ttk88 when expressed alone. However, when Sina and Phyl are co-translated with Ttk88, loaded GST-Ebi beads are able to promote degradation of TtK88 by a mechanism that is blocked by the proteosome inhibitor, LLnL, whereas loaded GST beads cannot. The need for Sina and Phyl for Ttk degradation mirrors their requirement in vivo. In vitro-translated E2F, dDP or cyclin E is not degraded in any of the experiments described. In order to map the region of Ebi that associates with the activity required for Ttk88 degradation, loaded GST-EbiN and GST-EbiC beads were pre-incubated and then the degradation assay was performed. Interestingly, neither half of Ebi alone is capable of targeting Ttk88 for degradation. These data suggest that the full-length Ebi protein may act to bring the Sina-Phyl-Ttk88 complex and a ubiquitylation activity into close proximity (Boulton, 2000).
The results of the in vitro degradation assay suggest that Ebi might physically associate with Ttk88, Sina and Phyl. This was tested using the in vitro translated Sina, Phyl and Ttk88, and the GST-Ebi fusion proteins described above. GST-Ebi was found to interact strongly with Sina and Phyl, and weakly with Ttk88, when these proteins were expressed and assayed individually. The GST control shows no association. Interestingly, when Sina, Phyl and Ttk88 are co-expressed in the same lysate, GST-Ebi is able to pull down Ttk88 with a much higher affinity compared with when Ttk88 is expressed and bound alone. This suggests that the Ebi-Ttk interaction is indirect and may require Sina and Phyl to facilitate association. To define the region of Ebi required for Sina, Phyl and Ttk association, GST-EbiN (N-terminal domain) and GST-EbiC (C-terminal WD-repeat domain) fusion proteins were tested for binding. GST-EbiN does not associate with any of the proteins, whereas GST-EbiC is able to bind to all three proteins, although with a slightly reduced affinity when compared with the full-length protein. Since the ß-propeller structure formed by WD-repeat domains provides a surface for many protein-protein interactions, it is possible that this domain in Ebi provides a scaffold for Sina, Phyl and Ttk88 association. To provide further evidence for these interactions, pIZT-Ebi with pIZT-V5His-Sina or pIZT-V5His-Phyl, or all three constructs were transiently co-transfected into S2 cells. Sina and Phyl (His6- and V5-tagged) were purified from lysates from the various transfected populations by Ni-NTA agarose chromatography. The beads were then subjected to Western blotting using a monoclonal antibody to Ebi. Ebi was found to co-precipitate with Sina and Phyl but was not observed in the untransfected control (Boulton, 2000).
Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signaling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (Ttk69) acts downstream of Notch as a determinant of non-neuronal identity. Repression of Ttk69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (Msi). Although msi can act downstream of Notch, Notch signaling does not affect Msi expression. Thus, Notch signaling is likely to regulate Msi activity rather than its expression. These results define cell-type-specific translational control of ttk69 by Msi as a downstream event of Notch signaling in asymmetric cell division (Okabe, 2001).
Mechanosensory bristle development in Drosophila is an excellent model system in which to address the molecular mechanisms of asymmetric cell division. Four successive asymmetric cell divisions from a common precursor cell, called the sensory organ precursor (SOP) generate a sensory bristle comprising four different non-neuronal support cells and one neuron. The first asymmetric cell division of SOP into IIa non-neuronal and IIb neuronal precursors is regulated by Notch signaling; specific activation of Notch occurs in IIa owing to inhibition in IIb by Numb, an intracellular negative regulator of Notch. Activation of Notch signaling results in the appearance of Ttk69 protein in the IIa precursor, but not in IIb. The expression pattern of Ttk69, phenotypes of ttk69 loss-of-function mutants, and the overexpression of Ttk69 suggest that Ttk69 is necessary and sufficient to specify the IIa non-neuronal lineage; however, the mechanism through which Notch signaling regulates Ttk69 expression has remained elusive (Okabe, 2001).
Although Ttk69 protein is not detected in the IIb neuronal precursor in which Notch signaling is inactive, similar levels of ttk69 mRNA are present in the IIb precursor and its non-neuronal sibling. Using a lacZ reporter in a ttk enhancer trap line, it has been confirmed that there are indistinguishable levels of expression between IIa and IIb. Selective repression of Ttk69 protein expression in the IIb neuronal precursor thus must occur post-transcriptionally, either by post-translational degradation or translational repression (Okabe, 2001).
Ttk69 protein also regulates neural development in developing adult photoreceptor cells, where Ttk69 expression is controlled by selective degradation in neuronal cells dependent on seven in absentia (sina) and phyllopod functions. These two genes are also involved in the cell-fate decision of mechanosensory bristle lineage; loss of either gene function causes a 'double-bristle phenotype', suggesting that the IIb precursor is transformed into IIa. However, as the penetrance of the sina-null mutant phenotype (12.1% transformation of IIb precursor into IIa) is much less than that of the phenotype of overexpression of ttk69, other mechanisms must exist for post-transcriptionally regulating ttk69 (Okabe, 2001).
A good candidate for a repressor of ttk69 is the msi gene product. Loss of msi function causes a double-bristle phenotype similar to that of overexpression of Ttk69 (36.3%). Furthermore, it was found that in msi mutants Ttk69 protein is ectopically expressed in IIb precursors, and that a reduction of one copy of the ttk69 gene dominantly suppresses the msi double-bristle phenotype (14.3%). Loss of ttk transforms IIa precursor into IIb precursor even in the absence of msi, confirming that ttk69 acts downstream of msi in the first asymmetric cell division (Okabe, 2001).
These results indicate that the msi phenotype is caused by a derepression of Ttk69 protein expression in the IIb neuronal precursor. This msi-dependent pathway acts parallel to the sina-dependent pathway, because a sina;msi double-null mutation shows a much more severe phenotype than either one alone (the production of extra outer support cells in 95.3% of bristles. Since msi encodes an RNA-binding protein containing two RNA recognition motifs, Msi may act directly on cis-acting sequences of the ttk69 mRNA and regulate expression of Ttk69 protein at the translational level (Okabe, 2001).
To identify cis-element(s) crucial for the post-transcriptional regulation of ttk69 mRNA, advantage was taken of ttk1, an allele of ttk with a transposable element (P-element) insertion in the 3' untranslated region (UTR) of ttk69 mRNA. Although this insertion causes the loss of another isoform of TTK (Ttk88), it also behaves as a gain-of-function allele of ttk69; Ttk69 protein is ectopically expressed in the IIb precursor in ttk1 pupal nota. In fact, it was found that ttk1 mutants display a semi-dominant double-bristle phenotype (5.3% in heterozygous mutants; 26.2% in homozygous mutants), a phenotype identical to overexpression of either Ttk69 or Ttk88. Thus, the bristle phenotype in ttk is caused by ectopic expression of Ttk69 rather than the loss of Ttk88 (Okabe, 2001).
Analysis of the ttk69 cDNA from ttk1 reveals that its ttk69 mRNA lacks the distal 80% of the 3' UTR sequence, owing to a read-through into P-element sequences. These results indicate the importance of the 3' UTR of ttk69 mRNA in vivo for preventing Ttk69 protein expression in the IIb precursor. Thus, the absence of msi or the ttk69 mRNA 3' UTR similarly results in ectopic expression of Ttk69 protein in the IIb precursor and the double-bristle phenotype (Okabe, 2001).
To test whether Msi binds ttk69 mRNA directly, Msi target sequences in vitro were analyzed in vitro, and these sequences were sought in the ttk69 mRNA. Random 30- or 50-base-pair RNA polymers were selected by using recombinant gluthathione S-transferase (GST)-Msi fusion protein immobilized to glutathione beads, and candidate RNAs were concentrated by five cycles of polymerase chain reaction with reverse transcription (RT-PCR) and in vitro transcription. 48 independent RNA polymers were isolated and sequenced, and it was found that all contained a common motif of GU3-6(G/AG) (Okabe, 2001).
Fifteen such sequences are present in the 3' UTR of the ttk69 mRNA, and ten of these are located in the region that is absent in the ttk69 mRNA produced in the ttk1 mutant. By gel mobility shift assays, it has been shown that recombinant Msi protein binds specifically to these sequences in ttk69 mRNA. The RNA ß-region, containing three Msi target sequences (corresponding to position 3,871-3,880 of ttk69 cDNA), was bound by Msi but not by a mutant Msi protein with substitutions in the RNA recognition motifs (MsiA*B*). Binding of the RNA ß-region can be competed by a homologous sequence, but not by a region of ttk69 mRNA lacking Msi-binding motifs. These results show that Msi is a sequence-specific RNA-binding protein that binds the 3' UTR of ttk69 mRNA in vitro (Okabe, 2001).
To address the functional significance of the binding of Msi to ttk69 mRNA 3' UTR, a translational reporter assay was performed in Drosophila S2 cells. Luciferase reporter genes that contained the entire 3' UTR of ttk69 (luc-ttkUTR), the proximal 20% of the 3' UTR (luc-ttkUTRdelta) or that of alcohol dehydrogenease gene (luc-AdhUTR) were introduced in S2 cells that express Msi, and translational and transcriptional outputs were quantified by measuring the enzymatic activities and mRNA levels (Okabe, 2001).
In cells expressing Msi, these three reporter genes produce roughly the same amount of mRNA. When the translational output was measured by luciferase activity, however, the luc-ttkUTR reporter produced only 24.7% activity as compared with the Adh 3'UTR. The luc-ttkUTRdelta, which has 5 of the 15 Msi-binding sites present in the ttk69 3' UTR and mimics ttk69 mRNA produced in ttk1, exhibits only a modest decrease in the translational output (69.8% activity as compared with luc-AdhUTR). This is consistent with the sharp reduction in Msi-dependent translational repression in the ttk1 mutant, because the penetrance of ttk1 homozygous is less than that of msi null homozygous. The effect of ttk69 3' UTR is dependent on the RNA-binding activity of Msi; in cells expressing Msi lacking RNA-binding activity (MsiA*B*), translational repression of the ttk69 3' UTR is not observed. The 3' UTR of ttk69 mRNA therefore confers Msi-dependent translational repression in this assay system. Together, these results indicate that Msi inhibits translation of ttk69 mRNA in IIb precursors by binding to its 3' UTR (Okabe, 2001).
Although the translational inhibitory effect of Msi on ttk69 mRNA is specific to the IIb precursors, Msi protein is present in both IIa and IIb precursors. Thus, IIa precursors must somehow be able to escape the action of Msi as a translational repressor of Ttk69, probably through the effect of Notch signaling. Loss of Notch function in the SOP lineage causes the transformation of IIa precursor into IIb, with mutants showing a balding phenotype owing to loss of socket and shaft cells. This phenotype in IIa precursor is dependent on Msi activity; loss of both Notch and msi function results in a dense double-bristle phenotype with no neurons in the subepidermal layer, indicating that the IIb precursor took the non-neuronal fate. msi is thus epistatic to Notch during the asymmetric cell division giving rise to IIa and IIb precursors. Taken together, it is proposed that Notch inhibits Msi activity in the IIa precursor (non-neuronal cell), thus allowing translation of ttk69 mRNA, whereas in the IIb precursor (neuronal cell), where Notch is inactivated by Numb, Msi prevents ttk69 translation (Okabe, 2001).
dMi-2, the ATPase subunit of the Drosophila nucleosome remodelling and histone deacetylation (dNuRD) complex, has been identified in
a two-hybrid screen as an interacting partner of the transcriptional repressor, Tramtrack69 (Ttk69). A short region of Ttk69 is
sufficient to mediate this interaction. Ttk69, but not the Ttk88 isoform, co-purifies with the dNuRD complex isolated from embryo
extracts. dMi-2 and Ttk69 co-immunoprecipitate from embryonic extracts, indicating that they can associate in vivo. Both dMi-2 and
Ttk69 co-localize at a number of discrete sites on polytene chromosomes, showing that they bind common target loci. dMi-2 and Ttk interact genetically, indicating a functional interaction in vivo. It is proposed that Ttk69 represses some
target genes by remodelling chromatin structure through the recruitment of the dNuRD complex (Murawsky, 2001).
Fractionation of 0-24 h embryonic extracts by gel-filtration chromatography and subsequent Western blotting shows that the majority of Ttk69 elutes in a broad peak with a molecular weight of ~1 MDa, much larger than the predicted monomer size of 69 kDa. Antibodies directed against the C-terminal region of dMi-2 show that dMi-2 and Ttk69 are present in an overlapping set of high-molecular-weight fractions. In a separate purification procedure developed to isolate the dMi-2-containing dNuRD complex, Ttk69 consistently co-purified with dMi-2 through multiple fractionation steps. Western analysis has confirmed that Ttk69 is present in the purest dMi-2 fractions. Ttk69 also physically associates with dMi-2 in partially purified nuclear extracts, as shown by co-immunoprecipitation of dMi-2 from these fractions using alpha-Ttk69 antibodies. alpha-Ttk69 antibodies co-precipitate dMi-2. dMi-2 is also precipitated by alpha-dMi-2 and alpha-dRpd3 (HDAC subunit of dNuRD) antibodies (Murawsky, 2001).
To establish if dMi-2 is required for Ttk69-mediated repression in vivo, an examination was made to see whether simultaneous loss of dMi-2 would increase neuron over-production seen in the peripheral nervous system (PNS) of embryos mutant for the hypomorphic ttkrM730 allele. Normally, Ttk is expressed in all non-neuronal cells of PNS sensory organs and prevents these cells from becoming neurons. In ttk mutants, a variable number of these cells are transformed into neurons, causing excess neurons in the PNS. An antibody against the neuronal antigen 22C10 was used to detect neurons of the lateral pentascolopidial sensory organ of the embryonic PNS. In wild-type embryos, five neurons are present. This number is approximately doubled in ttkrM730 mutants. Although homozygous mutant dMi-24 embryos show no defects in PNS development, loss of dMi-2 in a ttkrM730 mutant background significantly increases neuron number. Moreover, a number of tissues that are not stained by mAb 22C10 in wild-type and ttkrM730 mutants now express the antigen. Epidermal staining is increased and body-wall muscles stain strongly. mAb 22C10 staining of the somatic musculature has been reported for strong ttk loss-of-function alleles such as ttkD2-50, providing further evidence that loss of dMi-2 increases the strength of ttk mutant phenotypes (Murawsky, 2001).
ttk also shows dominant interactions with mutations affecting the components of the dNuRD complex. Titration of Ttk function can increase the number of precursor cells that are recruited to initiate sensory-organ development. One manifestation is the production of ectopic bristles along veins of the adult wing. ttkrM730 interacts dominantly with mutants affecting rpd3 to cause bristle de-repression on the adult wing. Further reduction of dMi-2 levels in the presence of one copy of the null allele dMi-24 increases bristle number synergistically, reflecting functional in vivo interactions between Ttk and both Rpd3 and dMi-2 (Murawsky, 2001).
Interestingly, the interaction of Ttk69 with the dNuRD complex parallels that of another BTB/POZ-containing protein, GAGA, with the ISWI-containing remodelling complex NURF. A second Drosophila protein, Hb, has also been shown to interact with dMi-2, both genetically and by two-hybrid analysis. Hb binds to the same C-terminal region of dMi-2 as does Ttk69, suggesting that this region may be a docking platform for proteins that recruit the dNuRD complex. However, thus far Hb has not been detected in the dNuRD complex. This failure may simply reflect the relative abundance of Ttk69 and Hb in Drosophila embryos. Nevertheless, the association of dMi-2 with more than one Drosophila transcriptional repressor implies that it can be recruited by a number of different proteins. Such recruitment may be a general mechanism by which specific repressors silence their targets. Indeed, MBD2, Ikaros and Aiolos, also known repressors, have been shown to purify with the NuRD complex from mammalian cells (Murawsky, 2001 and references therein).
The association of Ttk69 with the dNuRD complex implies that one mechanism by which Ttk69 may repress its targets is to direct the histone deacetylation and chromatin remodelling activities of the dNuRD complex. Such a function would be consistent with the developmental expression pattern of Ttk69. High levels of Ttk69 expression are detected toward the end of embryogenesis, when most cell types have already been specified and the chosen fate needs to be stabilized. These results imply in particular that dMi-2 is involved in ttk-mediated neuronal repression. It is noteworthy that all known transcription factors that interact with Mi-2-containing complexes are deployed when particular cell fates or expression patterns need to be maintained. Thus, Hb mediates stable homeotic repression, Ikaros/Aiolos maintains B and T cell fates and MBD2 is required for DNA-methylation-dependent silencing. It is speculated that Ttk69 also uses this mechanism stably to repress targets incompatible with determined cell fates (Murawsky, 2001).
These results suggest a model by which Ttk69 interacts with the dMi-2 component of the dNuRD complex and subsequently recruits its repressive activities to target genes. It is speculated that in vivo the association of Ttk69 with dMi-2 is probably not the only route by which Ttk69 may repress transcription. It is known that Ttk69 interacts genetically with dCtBP, another transcriptional co-repressor. Moreover, Ttk69 and dMi-2 do not completely co-localize on polytene chromosomes, suggesting that Ttk69 binds a subset of its targets in the absence of dMi-2. Finally, it is noted that only a small fraction of the total amount of Ttk69 present in embryonic extracts co-purifies with the dNuRD complex. Taken together, it is surmised that Ttk69 is a component of more than one repressive complex and that different target genes may be regulated by different mechanisms (Murawsky, 2001).
The ubiquitin-related SUMO-1 modifier can be covalently attached to a variety of proteins. To date, four substrates have been characterized in mammalian cells: RanGAP1, IkappaBalpha, and the two nuclear body-associated PML and Sp100 proteins. SUMO-1 modification has been shown to be involved in protein localization and/or stabilization and to require the activity of specialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologs have been identified in various species and belong to the so-called Smt3 family of proteins. The Drosophila homologs of mammalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9/lesswright, respectively) have been characterized. dUbc9 is the conjugating enzyme for dSmt3 and dSmt3 can covalently modify a number of proteins in Drosophila cells in addition to the human PML substrate. The dSmt3 transcript and protein are maternally deposited in embryos, where the protein accumulates predominantly in nuclei. Similar to its human counterpart, dSmt3 protein is observed in a punctate nuclear pattern. Tramtrack 69 (Ttk69), a repressor of neuronal differentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocalize on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 species can bind at sites of Ttk69 action in vivo. Altogether, these data indicate a high conservation of the
Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of cell differentiation in Drosophila flies (Lehembre, 2000).
The identification of the transcriptional repressor Ttk69 as a substrate of the dSmt3 conjugation pathway suggests that this mode of posttranslational modification may play a direct role in the modulation of transcriptional regulation. Supporting this possibility, the localization of dSmt3 at particular chromosomal sites shows that the dSmt3 modification can be chromosome associated. Its partial colocalization with
Ttk69 and the ability of the dSmt3-modified Ttk69 protein to bind Ttk69 sites are also consistent with the binding of modified Ttk69 to a subset of Ttk69 recognition
elements. Although Ttk69 is the first transcription factor shown to be modified by the SUMO-1/Smt3 homologs, it seems likely that SUMO-1 also modifies several transcription factors in mammalian cells, as suggested by the observed interaction in a two-hybrid assay of Ubc9 with E1A, IB, WT1, Jun, p53, ATF2, ETS-1, the glucocorticoid receptor, and other nuclear proteins and thus may perform a more general role in transcriptional regulation. These data also indicate that the pattern of covalent modification of Ttk69 may be more complex. In particular, Ttk69 can be phosphorylated as well as conjugated with dSmt3. Notably, general inhibition of serine/threonine phosphorylation prevents dSmt3 conjugation, although it is uncertain whether this is a consequence of a reduction in substrate availability or conjugating activity (Lehembre, 2000 and references therein).
The biological role and consequences of the conjugation of dSmt3 to Ttk69 are unclear. Among several possibilities would be effects on the targeting of the
repressor to specific chromosomal sites or on its interaction with specific protein partners. Another attractive hypothesis is that dSmt3 modification might antagonize
the degradation of Ttk69 by a proteasome-dependent pathway. Indeed, it has recently been suggested that in human cells, SUMO-1 modification of IB might
serve to block signal-induced ubiquitination and thus degradation of IB. In this context it is intriguing that Sina interacts directly with and destabilizes the other
isoform of Ttk, Ttk88, but that no comparable interaction of Sina and Ttk69 was observed in a two-hybrid assay. Nevertheless, Ttk69 levels are
stabilized in SL2 cells by MG132, an inhibitor of proteasome-mediated proteolysis. It is therefore suggested
that dSmt3 modification might provide a mechanism for the differential stabilization of splicing isoforms, such as Ttk69 and Ttk88, that are transcribed from the same
promoter. Genetic analysis of dSmt3 mutants in Drosophila should hopefully lead to a better understanding of the role of dSmt3 modification in the transcriptional
regulation of sense organ development (Lehembre, 2000).
This study reports the interaction of the Drosophila transcription factors Trithorax-like (GAGA) and Tramtrack (TTK). This interaction is documented both in vitro, through GST pull-down assays, as well as in vivo, in yeast and Schneider S2 cells. GAGA and TTK share in common the presence of an N-terminal POZ/BTB domain, found to be necessary and sufficient for GAGA-TTK interaction. Structural models that could account for this interaction are discussed. GAGA is known to activate the expression of many genes in Drosophila. In contrast, TTK has been proposed to act as a maternally provided repressor of several pair-rule genes, such as even-skipped (eve). As with many Drosophila genes, eve contains at its promoter region binding sites for GAGA and TTK. Transient expression experiments show that GAGA activates transcription from the eve stripe 2 promoter element, and TTK inhibits this GAGA-dependent activation. Repression by TTK of the eve promoter requires its activation by GAGA and depends on the presence of the POZ/BTB domains of TTK and GAGA. These results indicate that GAGA-TTK interaction contributes to the regulation of gene expression in Drosophila (Pagans, 2002; full text of article).
What sort of molecular interactions could account for the formation of GAGA–TTK hetero-oligomers? The POZ/BTB domain, that mediates GAGA–TTK interaction, is also responsible for the formation of GAGA–GAGA and TTK–TTK homo-oligomers. Since POZ/BTB is a highly conserved domain, structural comparative models of those present in GAGA and TTK could be built from the crystal structure of the POZ/BTB dimer of PLZF. Actually, the modelled structures of putative GAGA–GAGA and TTK–TTK homo-dimers bear a similar hydrophobic dimerisation interface as in the PLZF dimer, with a slightly lower complementarity. All the contacts involved in dimerisation are conserved and the central cavity is even more hydrophobic than in PLZF. These results strongly suggest that homomeric GAGA–GAGA and TTK–TTK interactions are likely to involve similar molecular interactions as those observed in the PLZF dimer. Moreover, similar models could also be built for heteromeric GAGA–TTK interaction, indicating that a putative GAGA–TTK hetero-dimer would be stabilised by the same interactions involved in homomeric GAGA–GAGA and TTK–TTK interactions. The extensive dimerisation interface suggests, however, that POZ/BTB-containing proteins are obligated homo-dimers. Actually, there is no experimental evidence indicating that either GAGA or TTK could ever exist as monomers in solution. A putative GAGA–TTK hetero-dimer could, therefore, arise from the respective homo-dimers by swapping of the POZ/BTB domains. However, the extension and high hydrophobic character of the contacts involved in these interactions raise the question of whether such a domain-swapping model could actually account for heteromeric GAGA–TTK interaction. In this respect, it must be noted that the crystal structure of the POZ/BTB domain of PLZF was solved independently by two different groups and, in spite of belonging to different crystallographic space groups, both structures showed conserved dimer–dimer interactions, involving the extension of the ß1/ß5'-sheet of one dimer towards the same region of the next one, keeping an antiparallel orientation. Eight hydrogen bonds connect main-chain atoms of the ß1 chains of each dimer. Each dimer buries 700 Å2 in this interface, with a high complementarity score. To address the question of whether these dimer–dimer interactions are also conserved in the POZ/BTB domains of GAGA and TTK, GAGA and TTK homo-tetramers were modelled, as well as a GAGA–TTK hetero-tetramer. The modelled tetramers have an interacting surface and complementarity similar to those of the PLZF homo-tetramer. The GAGA homo-tetramer and the GAGA–TTK hetero-tetramer keep all the eight hydrogen bonds between the ß1/ß5'-sheets, but the TTK homo-tetramer may lack those at the ends. These considerations raise the possibility that, rather than the formation of GAGA–TTK hetero-dimers, GAGA– TTK interaction would actually involve dimer–dimer interactions through the ß1/ß5'-sheets. GAGA–TTK interaction appears to be specific since, as it was found earlier, TTK does not interact in vitro with the POZ/BTB domains of either ZID or ZF5. Interestingly, the ß5-strand of the ß1/ß5' motif is fully identical in GAGA and TTK, a feature not shared by any of the other POZ/BTB-containing proteins analysed. In the crystal of the POZ/BTB domain of PLZF, this dimer–dimer interaction propagates through the lattice, suggesting that it could give rise to the formation of multimers of higher stoichiometry. Actually, bacterially expressed TTK and GAGA, but not ΔPOZGAGA, form in vitro oligomers of high apparent M. In contrast, dimer stability itself strongly relies on the ß1/ß5'-sheets which, in PLZF, involve the formation of five main chain H-bonds between the ß1 chain of one monomer and the ß5' chain of the second. The only other contact that contributes to dimer formation involves the symmetry related α1' helices of each monomer. Therefore, the ß1 residues that could mediate dimer–dimer interactions are themselves essential for dimerisation as it was found by mutational analysis of the POZ/BTB domain of PLZF (Pagans, 2002).
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