Sex lethal
Repression of Sex-lethal by Extramachrochaete involves the inhibition of the formation of Daughterless/Sisterless-b heterodimers. Sexual identity in Drosophila is determined by zygotic X-chromosome dose. Two potent indicators
of X-chromosome dose are Sisterless-a (SIS-a) and Sisterless-b (SIS-b). SIS-a encodes a bZIP protein homolog that
functions in all somatic nuclei to activate Sxl transcription. In contrast with other elements of the
sex-determination signal, the functioning of this transcription factor in somatic cells may be specific
to X-chromosome counting. The pattern of SIS-a RNA
accumulation is very similar to that for SIS-b, with a peak in nuclear cycle 12 at about the time of
onset of Sxl transcription (Erickson, 1993). SIS-a and SIS-b form functional heterodimers (Hoshijima, 1995).
The X-linked gene runt plays a role in the regulation of Sex lethal. Reduced function of runt results in female-specific lethality and sexual transformation of XX animals that are heterozygous for Sxl. The presence of a loss-of-function runt mutation masculinizes triploid intersexes. In contrast, runt duplications cause a reduction in male viability by ectopic activation of Sex-lethal. Runt is needed for the initial step of Sex-lethal activation, but does not have a major role as an X-counting element (Torres, 1992). scute is another X linked activator of Sxl.
In D. melanogaster, a set of 'X:A numerator genes,' which includes sisterlessA (sisA ), determines sex
by controlling the transcription of Sex-lethal (Sxl). sisA was characterized from D. pseudoobscura and
D. virilis and the timing of sisA and Sxl expression was studied with single cell-cycle resolution in D. virilis,
both to guide structure-function studies of sisA and to help understand sex determination evolution. D. virilis sisA is shown to shares 58% amino acid identity with its D. melanogaster ortholog. The identities
confirm sisA as an atypical bZIP transcription factor. Although D. virilis sisA can substitute for
D. melanogaster sisA, the protein is not fully functional in a heterologous context.
A single copy of a D. virilis sisA expressing chimeric transgene fully
rescues females homozygous for the hypomorphic, female-specific
lethal allele sisA1; however, the chimeric transgene
is not fully effective at rescuing hemizygous sisA1 females,
a more stringent test of sisA function (Erickson, 1998).
The protein-coding sequences of sisA display a commonly
observed pattern: highly conserved regions separated by
completely diverged sequences. Conservation of the
bZIP domain confirms the validity of this motif assignment,
despite the occurrence of non-canonical residues in key
positions. The N-terminal region of the protein was also found
to be conserved, notwithstanding the fact that the N-terminal
15 amino acids can be deleted with only modest effects on in
vivo function. An intriguing feature
of the D. melanogaster protein is a run of six glycine-serine
pairs hypothesized to serve as a flexible spacer linking two
functional domains. Although this run is not conserved, it
occurs in a region that is notable for its variable length and lack
of conservation, consistent with such a spacer role.
The level of amino acid sequence conservation for SisA
protein within the genus (55-68% identity) is comparable to
that seen with the Drosophila sex determination proteins SisB (Scute) and
Tra-2: less than that for Runt and Sxl (80-90%), and is
considerably greater than that for Rra (36%). Thus sisA, like
most other sex-signal genes of Drosophila, does not display the
remarkably rapid evolution observed for the mammalian sex-signal
gene, SRY (Erickson, 1998).
Temporal and spatial features of sisA and Sxl early promoter
expression are strikingly conserved. D. virilis
sisA transcripts are apparent in nuclear cycle 8. At this point, the
nuclei are migrating outward to the cortex. Although
differences were noted among nuclei at the time of onset, all
nuclei begin to transcribe sisA within one cell cycle.
Initially, sisA transcripts are found primarily within nuclei.
Unambiguous cytoplasmic signals arise only later during cycle
10. Other Drosophila genes that turn on at a comparably early
stage display similar behavior.
Nuclei near the posterior pole express lower levels of SISA
mRNA than do nuclei elsewhere. All somatic and yolk nuclei
continue to express sisA in the subsequent cleavage cycles. In
contrast, expression is extinguished in the germline
precursors, the pole cells, during cycle 9. Transcripts are
readily apparent in prepole cell nuclei as the pole buds form,
but disappear midway through bud formation.
Transcription of sisA is maintained during the subsequent
cycles of the syncytial blastoderm. For somatic nuclei,
the level of SISA mRNA peaks in late cycle 12 or early cycle
13. By this point, the sisA regulatory target, SxlPe, has become
active. In some embryos, focused dots of sisA
hybridization can be seen in the nuclei. Such dots represent
nascent transcripts. After cycle 13, nuclear dots disappear, signaling the
cessation of sisA transcription. The cytoplasmic hybridization
signals begin to disappear from the periphery of the embryo
first, but by early to mid cycle 14, the last traces of SISA mRNA
have disappeared from all surface nuclei. As had been the case
with D. melanogaster, no subsequent somatic or germ-cell
transcription of sisA is observed in D. virilis embryos.
In D. virilis as in D. melanogaster, sisA transcripts persist
in the yolk nuclei until the demise of those nuclei 10 to 12 hours
after fertilization. The primary yolk nuclei derive from
70-100 nuclei that fail to migrate with the majority of nuclei
toward the periphery of the embryo during cycles 8 and 9. Instead
they fall back into the central yolk mass and eventually become
polyploid. During cycle
14, sisA transcript levels in these nuclei increase abruptly and
remain high thereafter. During normal embryogenesis, some
nuclei drop from the periphery into the surface of the yolk mass
to become 'secondary' yolk nuclei, perhaps as a consequence of
errors in replication or division. Although not highly polyploid, these nuclei also
express high levels of sisA. Probable secondary yolk nuclei can
be seen in cycle 14 near the periphery and at the anterior
pole of the embryo. Their behavior suggests that persistence of
sisA transcription is likely to be a
consequence of environmental
differences between the central yolk
mass and the yolk-free periphery, rather
than polyploidy per se. In D. melanogaster, the early onset of sisA transcription, the
extinction of sisA transcription in pole cells and the abrupt
cessation of sisA transcription during nuclear cycle 14 are all
reflections of this gene's participation in sex determination
through its regulation of Sxl. The persistence of sisA
transcription in D. melanogaster yolk nuclei suggests that the
gene might also have functions that are not sex-specific. All four of these unusual features have been
conserved over the >40 Myr separating D. melanogaster and D. virilis. Expression of sisA and Sxl is as tightly coupled in D. virilis as it is in
D. melanogaster (Erickson, 1998).
One aim of this study was to use DNA sequence conservation
to identify potential regulatory regions that allow genes to
function as X:A numerator elements. The heptomeric sequence
CAGGTAG, which is conserved in all three sisA genes
examined here, is a promising candidate. It appears in pairs less
than 200 bp upstream of the transcription start site, not only for
sisA, but also for sisB (scute) and Sxl. These three genes have
in common an unusually early onset of expression that allows
the embryo to establish X-chromosome dosage compensation
by the time general transcription begins. Perhaps this sequence
provides for such an early start of transcription. Taken together, these data indicate that the same primary sex determination mechanism
exists throughout the genus Drosophila (Erickson, 1998).
Brother and Big brother were isolated as Runt-interacting
proteins and are homologous to CBFb, which interacts with
the mammalian CBFa Runt-domain proteins. In vitro
experiments indicate that Brother family proteins regulate
the DNA binding activity of Runt-domain proteins without
contacting DNA. Functional interactions between Brother
proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt
interaction with Brother proteins but does not affect DNA
binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays.
Interestingly, this mutant protein acts dominantly to
interfere with the Runt-dependent activation of Sex-lethal
transcription. To investigate further the requirements for
Brother proteins in Drosophila development, an
examination was carried out of the effects of expression of a Brother fusion protein
homologous to the dominant negative CBFb::SMMHC
fusion protein that is associated with leukemia in humans.
This Bro::SMMHC fusion protein interferes with the
activity of Runt and a second Runt domain protein,
Lozenge. The effects of lozenge
mutations on eye development are suppressed by
expression of wild-type Brother proteins, suggesting that
Brother/Big brother dosage is limiting in this
developmental context. Results obtained when Runt is
expressed in developing eye discs further support this
hypothesis. These results firmly establish the importance of
the Brother and Big brother proteins for the biological
activities of Runt and Lozenge, and further suggest that
Brother protein function is not restricted to enhancing
DNA-binding (Li, 1999).
Repression of Sex lethalPe expression was observed in female embryos
injected with Runt[G163R] mRNA. This dominant negative
activity indicates that the Runt[G163R] protein interacts with
some other factor(s) in the Drosophila embryos in a manner
that interferes with the activity of the wild-type Runt protein.
In contrast to this, no dominant negative interference is
observed when runt[CK], a Runt derivative that is specifically
impaired for DNA-binding, is used in this assay. If the Runt[G163R] protein is interfering by
competing for interaction with some other limiting protein
factors then Runt[CK] protein would also be expected to
behave as a dominant negative. Taken together, these results
suggest that DNA binding is required for the dominant negative
activity of Runt[G163R]. This is somewhat surprising as the
prevailing view, primarily from in vitro experiments, has been
that the central function of the Bro/Bgb and CBFbeta proteins is
to enhance DNA-binding by the Runt domain proteins. The data in this paper strongly suggest that the Bro proteins have other functions
in addition to enhancement of DNA binding by Runt. What
then might be the other functions of the Bro/Bgb proteins? One
possibility is that Bro induces a conformational change in Runt
that is required for transcriptional activation. Runt/Bro
complexes induce a bend in DNA that is greater than that
observed by the binding of Runt alone.
Perhaps DNA-bending is critical for interactions between Runt
and other transcription factors on the Sex lethalPe promoter. An
alternative possibility is that Bro/Bgb may be a bridge between
Runt and other proteins that are critical for transcription
regulation. In this model Runt[G163R] would compete for
binding to the early promoter region of Sex lethalPe-lacZ but when
bound would fail to activate transcription because other Bro-interacting
proteins are not recruited. In a two-hybrid screen
for Bro-interacting proteins a number of
proteins have been identified that appear to be members of the Trithorax group
of transcriptional regulators
(G. Golling, personal
communication to Li, 1999) have been identified. Trithorax group proteins have been
implicated as having widespread roles in transcription
activation in Drosophila development and it is attractive to
speculate that recruitment of such proteins by Runt and Bro
contributes to the activation of Sxl transcription. It is clear from
the results presented here that interactions between Runt
domain proteins and Bro/Bgb/CBFbeta proteins are important for
the functions of these conserved transcriptional regulators.
Experiments that further address the functions of the Bro/Bgb
and CBFbeta proteins will be essential for understanding the
mechanisms that account for the pivotal regulatory roles of
these proteins in diverse developmental contexts (Li, 1999).
Do Hairy and Runt repress target gene transcription independently of DNA binding, or as promoter bound regulators? Hairy-related transcriptional repressors show similar basic and HLH domains, and all terminate with an identical C-terminal tetrapeptide (WRPW), mutations of which largely or completely abolish repressor activity. It has proved difficult to define the precise molecular mechanism of Hairy action during segmentation. In order to explore the ability of Hairy and Runt to act as promoter-bound transcriptional regulators, heterologous transcriptional activation domains (Act) were substituted for the WRPW repression domain (of Hairy) and the activation domain of Runt and the effects of such substitution were examined on presumed targets of Hairy and Runt. Hairy-Act was used to study sex determination. Ectopic Hairy mimics the activity of Deadpan in repressing early Sex-lethal transcription. Expression of Hairy-Act activates Sxl and causes male lethality, implying that Deadpan recognizes the Sxl promoter directly, and excludes models for Sxl regulation in which DPN functions as a passive repressor (Jiménez, 1996).
The hermaphrodite locus, has both maternal and zygotic functions required for normal female
development in Drosophila. Maternal her function is needed for the viability of female offspring, while
zygotic her function is needed for female sexual differentiation. Maternal her function is needed early in the
hierarchy: genetic interactions of her with the sisterless genes (sis-a and sis-b), with function-specific
Sex-lethal alleles suggest that maternal her function is
needed for Sxl initiation. When mothers are defective for her function, their daughters fail to activate a
reporter gene for the Sxl early promoter and are deficient in SXL protein expression. Dosage
compensation is misregulated in the moribund daughters: some salivary gland cells show binding of
the Maleless (MLE) dosage compensation regulatory protein to the X chromosome, a binding pattern
normally seen only in males. Thus maternal her function is needed early in the hierarchy as a positive
regulator of Sxl, and the maternal effects of her on female viability probably reflect Sxl's role in
regulating dosage compensation (Pultz, 1995).
Germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several
germline-specific markers. These defects have been traced back to the syncytial blastoderm stage. Pole cells in nos minus
embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped
are ectopically activated in nos minus germ cells. nos minus germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal
of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos minus germ cells. Supporting the conclusion that Sxl is an important target for nos
repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity (Deshpande, 1999b).
In wild-type embryos, transcription factors, such as Runt, Sisterless-a, and Scute, are responsible for activating the Sxl establishment promoter, Sxl-Pe. The genes encoding these positive regulators are on the X chromosome, and they are expressed in the early precellular zygote in direct proportion to the number of gene copies. Sufficient quantities of the X-linked activators are produced by 2X/2A nuclei to activate Sxl-Pe, while quantities produced by 1X/2A nuclei are insufficient to activate Sxl-Pe. Pole cells differ from the surrounding soma in that these activators are not expressed at detectable levels in the germline precursors, and Sxl-Pe remains off in both sexes. The failure to express these activators most likely reflects the global downregulation of RNA polymerase II transcription in wild-type pole cells. Thus, one mechanism that might account for the inappropriate activation of Sxl-Pe in nos- pole cells would be a general derepression of somatically active genes. As a consequence, the genes encoding the X-linked activators would be expressed, and these in turn would activate Sxl-Pe. Although it seems reasonable to believe that the ectopic expression of the X-linked activators could contribute to the activation of Sxl-Pe in nos- pole cells, it does not readily explain why Sxl-Pe is turned on not only in 2X but also in 1X pole cells. Moreover, when Scute protein expression was examined in nos- embryos, the level of Scute protein in pole cells was less than that seen in 1X/2A somatic nuclei. For this reason, it is suspected that Sxl-Pe may be activated in nos- pole cells by a mechanism that, at least in part, bypasses the normal regulation of this promoter by the X/A counting system (Deshpande, 1999b).
Although Nos protein is likely to control Sxl-Pe activity by an indirect mechanism, a number of lines of evidence indicate that Sxl is an important nos regulatory target. In wild-type, Sxl proteins are normally not expressed in the germline until after the formation of the primitive gonad, and at this stage expression is restricted to the female germline. As a consequence of the ectopic activation of Sxl-Pe, Sxl proteins are present in nos- pole cells at the blastoderm stage. It would appear that the premature appearance of Sxl proteins in the pole cells is an important contributing factor to the nos- phenotype. The migration and cell cycle defects of nos- germ cells can be alleviated by the elimination of the Sxl gene. Conversely, it is possible to induce both of these defects in wild-type germ cells by ectopically expressing Sxl protein. While the removal of Sxl mitigates some of the defects of nos- germ cells, it should be noted that these cells are still abnormal. They fail to establish/maintain transcriptional quiescence, and they cannot form a functional adult germline. This finding indicates that Sxl is not the only target for nos regulation (Deshpande, 1999b).
Why does ectopic expression of Sxl protein (either in the absence of nos or in the presence of the Sxl transgene) disrupt germ cell migration and induce cell cycle defects? Sxl encodes an RNA-binding protein that functions in the soma as both a splicing and translational regulator. Since the Sxl protein is predominantly localized in the cytoplasm of nos- pole cells, it is imagined that Sxl also functions to regulate the translation of mRNAs encoding proteins critical to migration or cell cycle control. An important goal for future study will be the identification of these Sxl targets (Deshpande, 1999b).
Drosophila sex is determined by the action of the X:A chromosome balance on transcription of Sex-lethal (Sxl), a feminizing switch gene. Loss-of-function mutations in denominator elements of the X:A signal were obtained by selecting for dominant suppressors of a female-specific lethal mutation in the numerator
element, sisterlessA (sisA). Ten suppressors were recovered in this extensive genome-wide selection. All were mutations in deadpan, a pleiotropic locus
previously discovered to be a denominator element. Detailed genetic and molecular characterization is presented of this diverse set of new dpn alleles including their
effects on Sxl. Although selected only for impairment of sex-specific functions, all are also impaired in nonsex-specific functions. Male-lethal effects were
anticipated for mutations in a major denominator element, but viability of males lacking dpn function is reduced no more than 50% relative to their
dpn- sisters. Moreover, loss of dpn activity in males causes only a modest derepression of the Sxl 'establishment' promoter (Sxlpe), the X:A target. By itself, dpn
cannot account for the masculinizing effect of increased autosomal ploidy, the effect that gives rise to the concept of the X:A ratio; nevertheless, if there are other
denominator elements, these results suggest that their individual contributions to the sex-determination signal are even less than those of dpn. The time course of
expression of dpn and of Sxl in dpn mutant backgrounds suggests that dpn is required for sex determination only during the later stages of X:A signaling in males to
prevent inappropriate expression of Sxlpe in the face of increasing sis gene product levels (Barbash, 1996).
nanos (nos) specifies posterior development in the
Drosophila embryo by repressing the translation of maternal hb
mRNA. In addition to this somatic function, nos is required in the
germline progenitors, the pole cells, to establish transcriptional quiescence.
nos has been shown to be required to keep turned off in the germline of
both sexes the Sex-lethal establishment promoter, Sxl-Pe. nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the
soma since the effects of nos on promoter activity are correlated with
changes in the phosphorylation status of the carboxy terminal domain (CTD)
repeats of the large RNA polymerase II subunit. Finally, evidence is presented
indicating that the suppression of transcription in the soma by Nos protein is
important for normal embryonic development (Deshpande, 2005).
During the rapid nuclear division cycles in cleavage stage Drosophila
embryos, RNA polymerase II transcription is largely shut down and only a few
genes are actively transcribed. RNA polymerase II transcription in somatic
nuclei is upregulated soon after they migrate to the periphery of the embryo at
stage 9 and by nuclear cycle 10 and 11 many of the key segmentation genes are
already actively transcribed.
While RNA polymerase II activity is substantially augmented when the nuclei
reach the periphery of the embryo in the soma, the opposite occurs in the
germline pole cell nuclei. When these nuclei migrate into the posterior pole
plasm and pole cells are formed, transcription is shut down rather than
activated. Previous studies have implicated the posterior determinants
nos and pum in establishing/maintaining transcriptional
quiescence in pole cells. In embryos derived
from mothers mutant for either nos or pum, RNA polymerase II
transcription is not properly downregulated in the pole cells and several genes
that are normally active only in somatic nuclei are ectopically expressed (Deshpande, 2005).
Since only nos mRNA localized at the posterior pole is translated, pole cells have
the highest levels of Nos. However, translation of the localized message
generates a Nos gradient that extends to the center of the embryo. An obvious
question is whether this Nos gradient also affects RNA polymerase II activity
in somatic nuclei. Indeed, transcription of Sxl-Pe is upregulated in
somatic nuclei when Nos protein is removed, and is repressed when Nos protein
is ectopically expressed. The role of Nos protein in repressing transcription
is not restricted to the sex determination pathway since the activity of other
promoters also appears to be increased in the absence of nos function (Deshpande, 2005).
Several mechanisms
could potentially explain the ectopic activation of Sxl-Pe in the soma
and germline of nos mutant embryos. The most obvious is that this
promoter is turned-on by maternal Hb expressed in the absence of nos.
However, Sxl-Pe is upregulated in nos− embryos
even when maternal Hb is eliminated. In addition, ectopic expression of Hb from
a transgene lacking NREs seemed to repress rather than activate Sxl-Pe.
Another possibility is that the zygotic expression of one or more
of the X-linked numerators is elevated in nos embryos, upsetting X
chromosome to autosome counting. However, since none of the known numerators has a
recognizable NRE in the 3′UTR of its message, it seems unlikely that
these genes are direct targets for translational repression by Nos protein. In
addition, it is not at all clear why numerator genes (which are transcribed in
the zygote) would be subject to translational repression by Nos, while
autosomal denominator genes such as deadpan (which turns off
Sxl-Pe) would not (Deshpande, 2005).
For this reason, the idea is favored that
Sxl-Pe is activated in nosm− embryos at
least in part because RNA polymerase II activity is upregulated. Support for
this idea comes from analysis of CTD phosphorylation. When RNA polymerase is
transcriptionally engaged the CTD domain is phosphorylated on serine 2 and 5.
In wild-type pole cells, phospho-ser2 cannot be detected, while there is only
little phospho-ser5. In contrast, phospho-ser2 is found in
nosm− pole cells, while the level of
phospho-ser5 is increased. nos-dependent alterations in CTD
phosphorylation are also evident in the soma. When Nos is absent, the level of
ser2 and ser5 CTD phosphorylation is elevated, while both types of CTD
phosphorylation are reduced by ectopic Nos protein (Deshpande, 2005).
Additional evidence that nos has a global effect on transcription comes from the finding that nos regulates the methylation of histone H3 in the germline of worms and flies. In both
organisms, the methylation of histone H3 on lysine 4 (H3meK4) is upregulated in
the soma when zygotic transcription commences in early embryogenesis. In
contrast, little or no methylation H3 K4 is observed in the transcriptionally
quiescent germline. Inhibition of H3 K4 methylation in germ cells requires
nos and H3meK4 is markedly upregulated in nos−
germ cells. In light of these findings, K4 methylation was examined in the soma of
nosm− embryos. As might be expected from the
effects of nos on CTD phosphorylation, somatic H3meK4 is elevated
compared to wild type (Deshpande, 2005).
Since phosphorylation of
serines 2 and 5 are correlated with transcription, the nos-dependent
alterations in CTD phosphorylation are consistent with the idea that nos
has a global impact on RNA polymerase II activity. If this is the case, an
important question is whether CTD phosphorylation is the cause or the
consequence of nos induced changes in the activity of the
transcriptional apparatus. Because actively transcribing RNA polymerase has a
hyperphosphorylated CTD domain, any mechanism, which leads to a general
increase (or decrease) in transcription, would likely alter the level of CTD
phosphorylation. This makes it difficult to distinguish between cause and
effect. In contrast, besides being a characteristic feature of elongating
polymerase, CTD phosphorylation has been linked to the last steps in the
initiation process, promoter clearance and the formation of an elongation
competent RNA polymerase complex. Moreover, there is growing evidence that
these steps in the transcription cycle are subject to regulation (Deshpande, 2005).
The fact that CTD
phosphorylation may be a key control point in the transcriptional cycle raises
the possibility that nos exerts its effects on polymerase activity by
inhibiting the translation of some factor which promotes CTD phosphorylation.
In nos mutants, the level of this factor would increase, leading to a
general derepression of transcription. Conversely, the level of this factor
would decrease by ectopic Nos, reducing overall transcription (Deshpande, 2005).
While the results clearly
show that Sxl-Pe is inappropriately turned on in the soma of male
embryos and upregulated in the soma of female embryos in the absence of
nos activity, it was initially surprising to find that there is usually
not a very pronounced posterior-anterior activation gradient. In fact,
the smaller Sxl-Pe0.4 kb promoter is clearly
activated not only in the posterior but also in the anterior of nos
embryos, while the larger Sxl-Pe3.0, usually shows at
most only a very shallow posterior–anterior gradient of
β-galactosidase expression. Since the Nos gradient does not extend beyond
the midpoint of the embryo, and the repressive effects of Nos on hb mRNA
translation are restricted to this posterior domain, one might have expected
that the activation of Sxl-Pe in would be tightly restricted to the
posterior half of nos mutant embryos. However, proteins of average size
would be expected to diffuse (in water or even in cytoplasm) through the volume
of a fly embryo over a time scale of minutes, and the establishment of
gradients like those seen for Nos or Bcd are likely to require special
mechanisms including a localize source of product, as well as the sequestration
(e.g. nuclear localization) and degradation of the product. If the Nos target
for inhibiting general RNA Pol II activity is translated from a uniformly
distributed maternal mRNA and is able to equilibrate through the embryo during
the time between the onset of the very rapid nuclear divisions and the
formation of the cellular blastoderm, only a shallow gradient of this factor in
the soma might be expected at any one time in the presence or absence of Nos.
In contrast, in pole cells, where repression of this factor by Nos would
presumably be required to impose transcriptional quiescence, the formation of
the cell membrane would prevent factor synthesized in the soma from influencing
polymerase activity. This would enable Nos in the pole cells to reduce the
level of this factor below the threshold required for transcriptional
activation (Deshpande, 2005).
As observed in many species, establishing transcriptional
quiescence in the newly formed pole cells during early embryogenesis is a
critical step in the development of the Drosophila germline. However, it
is not immediately obvious what role nos mediated down regulation of
polymerase activity would have in the development of the soma. Obviously,
hyperactivation of Sxl-Pe in nosm−
embryos could inappropriately switch on the Sxl autoregulatory feedback
loop in males. However, analysis of Sxl accumulation in post-blastoderm
stages suggests that only very few nosm− 1X/2A
embryos actually make the wrong choice in sexual identity. There is also little
evidence of a sex-bias in adult progeny of nos−
hb− germline clone mothers. The fact that the
Sxl autoregulatory loop is usually not activated in male
nosm− embryos, which are hemizygous for
Sxl, is not altogether surprising. In females that have only a single
wild type Sxl gene, activation of the autoregulatory loop is severely
compromised by conditions which diminish Sxl-Pe activity. Since the amount of Sxl
produced by Sxl-Pe in nosm− male embryos
is much less than that in wild-type females, the autoregulatory loop should be
activated infrequently (Deshpande, 2005).
While nosm− males
largely escape the effects of activating Sxl-Pe, the increased
polymerase activity appears to have other consequences. Previous studies have
shown that removal of maternal hb suppresses the posterior defects of
nosm− embryos. However, it has been shown that only about 40% of
the hb+/hb− embryos from
nos− hb− mothers survive to
adults. Likewise, it was found that only 60% of the embryos produced by
hb− nos− mothers hatch as
first instar larva, and that an even lower number survive to the adult stage.
Taken together, these experiments argue that nos has important functions
in the soma besides blocking translation of maternal hb mRNA. It seems
possible that the segmentation/developmental defects evident in progeny of
hb− nos− clone mothers could
arise from the upregulation of various patterning genes in the absence of
nos activity. It is presumed that in wild-type embryos the activity of the
transcriptional apparatus and of target zygote promoters is appropriately
adjusted to compensate for the repressive effects exerted by Nos. Because the
transcriptional apparatus is hyperactivated in the absence of Nos function,
this balance is perturbed and many genes are overexpressed (Deshpande, 2005).
X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway. Although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal (Avila, 2007).
The question of how embryos differentiate between precise 2-fold differences in X-linked signal element (XSE) doses is central to understanding how genetic constitution defines sexual fate. Current X-chromosome-counting models posit that the female fate is set when XSE proteins exceed a threshold concentration and activate SxlPe. The XSE threshold is set by interactions between the XSEs and other proteins in the embryo. Some XSEs interact with maternally supplied proteins to form dose-sensitive transcription factors, such as Scute/Daughterless, that bind SxlPe, but XSE doses are also assessed with reference to maternally and zygotically expressed repressors. Three XSE proteins, SisA, Scute, and Runt, are viewed as acting similarly by binding directly to and activating SxlPe. The fourth XSE, unpaired (upd, also called outstretched or sisC), encodes a secreted ligand that signals through the JAK kinase (hopscotch) to activate the Stat92E transcription factor. Although upd meets the criteria of an XSE, its effects on sex determination are weaker than those of sisA, scute, and runt, and changes in its gene dose have only moderate effects on Sxl. To understand how this comparatively dose-insensitive XSE regulates sex, when and where upd, JAK, and STAT act on the Sxl switch was examined (Avila, 2007).
Using in situ hybridization, the early embryonic expression pattern of upd was defined. No evidence was found for maternally supplied transcripts and it was observed that upd mRNA was first detectable in nuclear cycle 13. The fact that the first upd transcripts are present throughout the embryo, including at the poles, is consistent with the distribution of phosphorylated Stat92E. As cellularization progresses past early cycle 14, the upd pattern resolves into indistinct stripes that developed into a 14 stripe pattern during gastrulation. These results show that upd expression begins later than that of the other XSEs (sisA in cycle 8; scute in cycle 9) and also, paradoxically, that it begins after the onset of transcription of its target, Sxl, in cycle 12 (Avila, 2007).
To understand how upd functions in Sxl activation and how it differs from other XSEs, upd mutations were examined for their effects on SxlPe by using in situ hybridization and on Sxl protein levels by using immunostaining with SXL antibody. Significantly, the RNA probes detected nascent Sxl transcripts, allowing monitoring of both the spatial and temporal responses of SxlPe on a cell-cycle by cell-cycle basis (Avila, 2007).
updsisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous updsisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5-15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because updsisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, updYC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were updsisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of updYC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3-,upd-) appeared identical to those of updYC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).
Except for the ligands, each component of the JAK/STAT pathway is maternally deposited into the embryo. To eliminate JAK/STAT activity completely, the dominant female-sterile technique was used to generate females lacking maternal hopscotch (hop) or Stat92E, which encode the only JAK kinase and STAT in Drosophila. It was expected that by removing maternal hop, STAT would remain unphosphoryated, allowing a determination of the effects of the loss of the entire pathway on SxlPe (Avila, 2007).
When Sxl expression was examined in cycle 14 embryos derived from hopC111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hopC111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).
The findings with hopC111 were confirmed by using the Stat92E06346 mutation. Cycle 14 embryos derived from Stat92E06346 germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hopC111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).
Although the hopC111 and Stat92E06346 mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hopC111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E06346 germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).
When the progeny of hopC111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).
SxlPe is thought to have two main functional elements: a proximal 390 bp X-counting region responsible for sex-specific activation, and a more distal (to -1.4 kb) element that elevates Sxl transcription. Three predicted STAT-binding sites are located in these elements at positions -253, -393, and -428 bp. To test their roles, consensus TTC sequences were changed to TTT because such changes block binding by STAT92E and the mammalian homologs STATs 5 and 5a. In situ hybridizations revealed that the mutation in the proximal STAT site, S1, greatly reduced the number of nuclei expressing SxlPe-lacZ, creating a patchy staining pattern and lower overall mRNA levels. Mutations in S1 and S2, or in all three sites together, caused a strong but variable loss of SxlPe-lacZ expression in most nuclei, resulting in dramatically reduced accumulation of lacZ mRNA. Although the S1, S2, S3 mutant appeared to have a slightly stronger effect than the double mutant, both transgenes exhibited phenotypes reminiscent of those seen in embryos derived from Stat92E06346 germline clones. These results show that STAT92E acts through the consensus binding sites at SxlPe (Avila, 2007).
SxlPe is remarkable for both its rapid response and exquisite sensitivity to X-chromosome dose. In male embryos, it is always off. In female embryos, SxlPe is strongly expressed, but only during a 35-40 min period from mid cycle 12 until about 10-15 min into cycle 14. Given these time constraints, many have assumed that all XSEs would function to establish the initial on or off state of SxlPe. However, it was found that upd behaved very differently than sisA and scute, both of which are required for SxlPe activation and expression. Loss of upd or the JAK/STAT pathway had little or no effect on SxlPe during cycles 12 or 13. Instead, JAK/STAT mutations blocked SxlPe expression late in the process, during cycle 14. This observation is interpreted as revealing that SxlPe is regulated in two mechanistically distinct phases: the first controlling the initial response to X-chromosome dose, and the second acting to maintain or reinforce the initial decision (Avila, 2007).
The relatively late actions of upd and hop offer explanations for several puzzling aspects of upd's function in sex determination. First, upd is considered a weak XSE. This is both because Sxl is comparatively insensitive to upd dose and because loss of upd or JAK/STAT function doesn't eliminate Sxl expression. Both effects are consistent with expectations of a two-step, initiation and maintenance, model for SxlPe function. JAK/STAT mutations would not be expected to eliminate all Sxl function in a two-step model because the STAT-independent initiation step would produce Sxl mRNA and protein. The exact gene dose of upd would not be particularly important for sex because excess active STAT could not induce SxlPe without the prior actions of the initiating XSEs and because even a single dose of upd+ could provide enough active STAT to augment an earlier decision to become female. Thus, the proposed STAT maintenance function explains both the failure of the constitutively active hoptum-l allele to induce ectopic SxlPe expression in males and the ability of hoptum-l to further stimulate SxlPe activity in females. Likewise, the requirement for STAT site S2, located just distal to the 390 bp X-counting region of SxlPe, and the finding that upd is first expressed after Sxl can be explained if STAT's role is to bolster transcription from SxlPe in embryos that already have counted two Xs. Although neither essential for SxlPe expression nor highly dose sensitive, upd, hop, and Stat92E nonetheless play important roles at SxlPe. In their absence, the period of SxlPe activity is cut short, reducing the concentration of Sxl and preventing a large fraction of embryos from engaging the maintenance mode of Sxl expression (Avila, 2007).
How might STAT92E function in a two-step model? One possibility is that STAT might antagonize the late-acting repressor Dpn. Alternatively, the STAT transcription factor might augment, stabilize, or replace earlier-acting XSE activator complexes as their concentrations diminish in cycle 14. BAP60, a core component of the Brahma chromatin-remodeling complex, has been shown to interact with two components of the sex-determination signal. If STAT92E also interacts with the Brahma complex, it might maintain SxlPe chromatin in an active state, facilitating the restoration of transcription after the 13th mitosis (Avila, 2007).
Understanding the commonalities and unique mechanisms STATs employ in their multitude of roles is a fundamental goal of research on this ubiquitous signaling pathway. It is also essential for understanding why the pathway has so often been co-opted into new roles during evolution. STATS seem primarily permissive rather than instructive. They are rarely the primary signals defining cell fate. In these respects, comparison of the even-skipped (eve) stripe 3 enhancer and SxlPe reveals interesting parallels. Both SxlPe and eve stripe 3 are regulated by the balance between several activators and repressors. The responses of both elements to JAK/STAT signaling are extremely rapid, occurring within the dynamic environment of the precellular embryo. Stat92E is important for each, but its roles augment the actions of other factors, rather than being responsible for defining the initiating signals (Avila, 2007).
With respect to the evolution of the sex signal, it has been proposed that a diffusible JAK/STAT signal might have been recruited to allow non linear signal amplification or, alternatively, that a diffusible ligand might render SxlPe less sensitive to random fluctuations in cell-autonomous XSE protein concentrations. Although the weak dose dependence of upd argues against signal amplification, a buffering function is consistent with existing data. These findings suggest another possibility. STAT proteins respond rapidly to a range of regulatory signals; it may be this ability to act within a matter of minutes that brought JAK/STAT into the temporally dynamic X-chromosome-counting process (Avila, 2007).
lilliputian, the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. This study identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, it was found that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi–tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. A role is described for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results it is suggested that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context (Vanderzwan-Butler, 2006).
This study uncovered Lilli's role in Runt-dependent transcriptional regulation based on the identification of lilli mutations as dose-dependent suppressors of the lethality produced by threshold levels of NGT-driven Runt expression. In contrast to all of the other Runt-interacting genes and deficiency intervals identified as suppressors in this genetic screen, a reduction in maternal lilli dosage has no effect on either the establishment or maintenance of Runt-dependent en repression. The target of Runt that provides the most dramatic and clearest evidence for a functional interaction between runt and lilli is the pair-rule gene ftz. It is notable that the ftz expression is not discernibly altered by the relatively low levels of NGT-driven Runt used in the genetic screen. This raises a question regarding the basis for lilli acting as a dose-dependent suppressor of the lethality associated with ectopic Runt expression. One explanation is that there are subtle changes in ftz expression at the threshold levels of NGT-driven Runt used in the viability assays that contribute to lethality. A second possibility is that there are other targets of Runt and Lilli that contribute to the lethality associated with ectopic Runt expression. Although Sxl would seem to be one obvious candidate for such a target, the developmental window for Sxl activation occurs prior to the stage during which the NGT-drivers are useful for manipulating gene expression. Indeed, it has not been possible to detect activation of Sxl by NGT-driven Runt, even at levels that are tenfold higher than the levels used in the genetic screen. Finally, it is possible that the effect on viability is in part due to a non-specific effect of Lilli on GAL4-dependent activation of Runt. There is some evidence for non-specificity, especially with Df(2L)C144; however, there is also a clear suppression of lethality with other lilli alleles that do not show a comparable reduction in NGT-driven UAS-lacZ expression. Thus it seems likely that the identification of lilli is due to a combination of specific and non-specific effects on the lethality produced by NGT-driven Runt expression. If this interpretation is correct, then it also seems likely that other deficiency intervals that were eliminated from further consideration due to apparent non-specific effects may in fact have specific and interesting effects that would be revealed by more directly assaying the effects of these mutations on the responses of different targets to NGT-driven Runt expression (Vanderzwan-Butler, 2006).
These observations confirm and extend findings regarding a role for Lilli in the transcriptional activation of the pair-rule gene ftz (Tang, 2001). Lilli does not appear to have any role in regulating other pair-rule genes, and the effects of eliminating maternal Lilli on segment-polarity gene expression have been interpreted as an indirect effect due to the loss of Ftz (Tang, 2001). Thus ftz appears to stand out as the sole gene in the segmentation pathway that shares a requirement for both Lilli and Runt. The previous work from Tang did identify two other targets for Lilli in the early Drosophila embryo, serendipity-α (sry-α) and huckebein (hkb). There is no evidence that either of these genes is regulated by Runt. Thus, just as there are targets of Runt in the segmentation pathway whose regulation is Lilli-independent, there are also targets of Lilli that do not involve interactions with Runt (Vanderzwan-Butler, 2006).
This work adds Sxl as an additional candidate target for Lilli. The elimination of maternal Lilli interferes with the activation of the SxlPE:lacZ reporter gene in all somatic cells of the female embryo. This global effect stands in contrast to the more localized role of Runt which is only required for Sxl activation in cells within the pre-segmental region of female embryos. The failure in Sxl activation observed in the absence of maternal Lilli is similar to the phenotype of embryos that are mutant for either sisA or sisB. Indeed the possibility is considered that the primary defect in lilli germline clone embryos was the failure to activate either of these two X-chromosome linked numerators. Expression of sisA was found to be normal in lilli germline clone embryos (VanderZwan, 2003). The low level of sisB expression in wild-type embryos made it difficult to unambiguously determine whether lilli was important for sisB activation. To further investigate the role of Lilli in Sxl activation the expression of the SxlGOF:lacZ reporter gene was examined. Both Runt and SisB contribute to the ectopic expression of this reporter in male embryos. The elimination of maternal Lilli has a more severe effect on the expression of the SxlGOF:lacZ reporter than is observed in embryos that are mutant for either runt or sisB (VanderZwan, 2003). The most straightforward interpretation of these results is that Lilli is directly involved in the transcriptional activation of the early embryonic Sxl promoter, in this case in cooperation with the four different X-linked factors that are responsible for the sex-specific expression of Sxl in female embryos (Vanderzwan-Butler, 2006).
The inclusion of Sxl gives four putative direct targets of Lilli in the Drosophila embryo. These four genes, Sxl, ftz, sry-α and hkb are normally activated at very early stages, and in all four cases this activation is reduced, if not abrogated, in the absence of maternally provided lilli. The notion that Lilli functions primarily in activation is consistent with observations on the properties of the mammalian homologs FMR2 and LAF4 (Hillman, 2001). However, early activation is clearly not the sole identifying characteristic of Lilli's targets. Indeed, for three of these targets, there are other genes in the same developmental pathway that are activated at the same time that do not require Lilli. In the cellularization pathway, Lilli is required for expression of sry-α but has no role in the activation of bottleneck (bnk) or nullo (Tang, 2001). In the segmentation pathway, the gap gene hkb is expressed in the anterior and posterior poles in response to signaling by the terminal pathway. Elimination of maternal Lilli greatly reduces hkb expression, but has no obvious effect on tailless (tll), another gap gene that is activated at the same stage in response to the terminal signaling pathway (Tang, 2001). Finally, the requirement for maternally provided Lilli that is observed for ftz is not shared with the pair-rule segmentation genes eve, hairy and runt (Tang, 2001). This last observation is perhaps most important as the wealth of information that exists on pair-rule gene regulation provides a useful framework for further considering the potential attributes of Lilli-dependent targets (Vanderzwan-Butler, 2006).
Elimination of maternal Lilli reduces, but does not eliminate ftz expression. The reduced expression that remains is similar to what is obtained in embryos that lack Runt, and is presumed to be in response to other activating factors. The complications presented by these other factors are bypassed in experiments in which Runt is over-expressed, either by heat-shock or by NGT-driven expression. Indeed, the inability of ftz to respond to ectopic Runt in the absence of maternal Lilli provides very compelling evidence for the importance of Lilli in ftz activation (Vanderzwan-Butler, 2006).
Lilli is acutely required for mediating Runt-dependent activation of ftz during the blastoderm stage, and this requirement precedes the temporal requirements for Ftz-F1. Ftz-F1 was initially identified as a factor that interacts with sequences within the ftz 'zebra element', a 669-bp, promoter proximal element that drives early expression in response to gap and pair-rule gene transcription factors. However, subsequent studies revealed that Ftz-F1 plays a more significant role in mediating Ftz-dependent auto-regulation by the so-called 'upstream element' during the early stages of germ-band extension. The earlier requirement for Lilli strongly suggests it contributes to the early 'zebra element'-dependent activation of ftz in response to activating inputs from Runt (Vanderzwan-Butler, 2006).
What is the role of Lilli in mediating Runt-dependent activation? Directed yeast two-hybrid assays fail to detect direct interactions between the full length Runt and Lilli proteins. This observation suggests that other factors contribute to the functional interactions described above. A notable conserved feature that Lilli shares with its mammalian homologs is an HMG-box. This structural DNA-binding motif interacts with the minor groove of DNA and modulates DNA structure by bending. These properties have been interpreted to reflect an architectural role for HMG-box proteins in facilitating the formation of higher order chromatin structures that contribute to the regulation of gene expression. It seems likely that chromatin architecture is important for ftz zebra element function. Although the 'zebra element' was one of the very first cis-regulatory elements in the segmentation pathway to be described, there is not yet a clear understanding of the rules that govern its activity. This is in contrast to the relatively simple combinatorial rules that have been elucidated for several of the stripe-specific elements of the pair-rule genes eve, hairy, and runt. It is proposed that the difficulty in identifying discrete regulatory modules within the zebra element reflects the importance of chromatin architecture in conferring high-fidelity regulation of the zebra element in response to inputs from Runt and other gap and pair-rule transcription factors (Vanderzwan-Butler, 2006).
It is interesting to note that the cis-regulatory element responsible for the initial activation of Sxl shares several properties with the ftz zebra element. The minimal DNA element necessary to faithfully recapitulate the strong, early sex-specific activation of the Sxl promoter is 1.7 kb in size. As found for the ftz zebra element, smaller reporter gene constructs do not function properly, although sub-elements that confer sex-specific regulation, and augment this activation have been identified. The on/off regulation of Sxl is in response to a twofold difference in the activity of four different DNA-binding transcription factors. It is easy to imagine that chromatin architecture may be critical in sensing this twofold difference in a robust and reproducible manner. There is one further similarity shared by Sxl and ftz that is intriguing. The initial Lilli-dependent activation of both genes is followed by a second phase of gene expression that involves distinct cis-regulatory components. In the case of ftz, the switch is from regulation by the zebra element to regulation by the upstream element, whereas for Sxl the switch is from expression at the SxlPe promoter to expression at SxlM a different promoter that is activated in all somatic cells of both male and female embryos . Perhaps the unique requirements for Lilli reflect architecture-dependent regulatory elements that retain the ability to be rapidly re-organized in a developmentally dynamic manner. Further studies on the mechanisms by which Lilli participates in the activation of ftz and Sxl in the early Drosophila embryo should provide new insights on the role of chromatin architecture in developmentally regulated gene expression (Vanderzwan-Butler, 2006).
Sex-lethal (Sxl), the Drosophila sex-determination master switch, is on in females and controls sexual
development as a splicing and translational regulator. Hedgehog (Hh) is a
secreted protein that specifies cell fate during development. Sxl protein has been shown to be part of the Hh cytoplasmic signaling
complex and Hh promotes Sxl nuclear entry (Vied, 2001; Horabin, 2003). In the wing disc anterior compartment, Patched (Ptc), the Hh receptor, acts positively in this process. This study shows that the levels and rate of nuclear entry of full-length
Cubitus interruptus (Ci), the Hh signaling target, are enhanced by Sxl. This
effect requires the cholesterol but not palmitoyl modification on Hh, and
expands the zone of full-length Ci expression. Expansion of Ci activation and
its downstream targets, particularly decapentaplegic the
Drosophila TGFß homolog, suggests a mechanism for generating
different body sizes in the sexes; in Drosophila, females are larger
and this difference is controlled by Sxl. Consistent with this
proposal, discs expressing ectopic Sxl show an increase in growth. In keeping
with the idea of the involvement of a signaling system, this growth effect by
Sxl is not cell autonomous. These results have implications for all organisms
that are sexually dimorphic and use Hh for patterning (Horabin, 2005) (Horabin, 2005).
Drosophila Hh is synthesized as a 45 kDa precursor that is
shortened to a mature form with two lipid modifications; palmitic acid at the
N terminus and cholesterol at the C terminus. Maturation involves
autoproteolytic processing under the control of the C-terminal domain of Hh. To test whether either of the lipid modifications plays a role in Hh
promoted Sxl nuclear entry, female wing discs expressing Hh with only a single
modification were examined. HhN encodes the N-terminal region of Hh that is
palmitoylated but, because it does not undergo autoproteolytic processing,
does not contain the cholesterol moiety. This form of Hh is functional for Ci activation and full-length Ci is detected distantly anterior of the AP boundary. Where HhN levels are maximal, there is a reduction of full-length Ci, most likely from the activation of en, which inhibits Ci transcription. HhN
does not increase Sxl nuclear levels, however. The normal high
nuclear levels in the posterior compartment and graded nuclear localization in
the anterior compartment (Horabin, 2003) are detected, with no change in the cells expressing HhN (Horabin, 2005).
The alternative single modification [cholesterol without palmitoyl
(C84S-Hh)], by contrast, is active with respect to Sxl. The dppGAL4 driver was used to drive expression of C84S-Hh. Relative to endogenous Hh, about threefold less of the nuclear export inhibitor Leptomycin B (LMB) was required to detect nuclear Sxl in these discs, suggesting that the nuclear Sxl is effected primarily by the ectopic Hh (Horabin, 2005).
C84S-Hh has been shown to dominantly destabilize Ci, decreasing the
expression of Hh target genes. Patterning of the wing is compromised and the
size of the region between veins L3 and L4 is reduced. C84S-Hh is also unable
to rescue the embryonic segmentation phenotype caused by loss of Hh. C84S-Hh destabilizes Ci, but only in males. Females show
the opposite effect, increasing the levels of full-length Ci (Horabin, 2005).
This sex specificity, coupled with the observation that Sxl is
present in the Hh cytoplasmic complex, suggests that Sxl may be acting to
stabilize Ci on Hh signaling. If this is the case, expressing Sxl in males
should increase the levels of full-length Ci. Indeed, male discs expressing
Sxl (MS3 isoform), as well as C84S-Hh under the control of dppGAL4,
now show higher levels of full-length Ci and the protein is more nuclear, as
seen in females. Taken together, these results suggest that when the cholesterol moiety is present on Hh, Sxl enhances the production of full-length Ci (Horabin, 2005).
Curiously, the presence of Sxl does not temper the wing patterning defect
caused by the ectopic expression of C84S-Hh; the reported narrowing between
wing veins L3 and L4 is the same in the two sexes. The form of Ci that Sxl
stabilizes through C84S-Hh must not be the form responsible for Hh
patterning (Horabin, 2005).
The data presented here show that when Sxl is present, the Hh signal is
augmented. This is seen as an increase in full-length Ci in whole-mount tissue, and in Western blots which give a more quantitative sense of protein levels. In addition to elevating the levels of full-length Ci, several of the Hh downstream targets, including ptc, dpp and some of the downstream targets of Dpp, show an increase in expression. Conversely, removal of Sxl in female cells shows a
reduction in the strength of the Hh signal (Horabin, 2005).
Sxl also enhances the nuclear entry rate of Ci, with either endogenous Hh or Hh that has only the cholesterol modification. In females, when Sxl is co-expressed with Hh with only the cholesterol modification, the amount of LMB required to detect nuclear Ci is reduced (by almost sixfold), further supporting the idea that Sxl affects Ci nuclear entry rate on Hh signaling (Horabin, 2005).
Hh enhancement of Sxl nuclear entry also depends on the cholesterol and not
the palmitoyl modification. Given that Ci and Sxl are in a complex in the
cytoplasm and both respond to the Hh cholesterol modification, it is tempting
to speculate, although the data presented does not address this issue, that
the two proteins may also enter the nucleus as a complex. This may be the
method by which Sxl stabilizes Ci, diverting it from rapid proteolysis,
particularly the highly activated form that is functionally detectable
but has not been identified biochemically (Horabin, 2005).
Stabilization of full-length Ci by Hh with only the cholesterol
modification in females is in contrast to what occurs in males. this form of Hh can destabilize Ci as well as compromise the Hh signal, but only in males. The effect of the cholesterol moiety contrasts with the palmitoyl that potentiates Hh in activating Ci for patterning. This is generally also true in vertebrates,
where the cholesterol modification appears to have less of a role in
patterning and a more significant role in the release and extracellular
transport of the Hh ligand (Horabin, 2005).
In both sexes, ectopic expression of Sxl shows an increase in intensity of
ptc expression, indicating it is possible to further elevate the Hh
response. Other than en, which was difficult to score in these
experiments, ptc requires the highest levels of Ci activation for its
transcription (Horabin, 2005).
In females, the ectopic Sxl elevates ptc expression in the cells
near the AP boundary, but the depth of the cells showing this highest level of
Ci activation is reduced. A reduction in the number of cells transcribing
ptc, when compared with the wider but less intense width of
ptc transcription in the control half of the disc, suggests a
restriction in Hh diffusion. Elevated ptc transcription is expected
to produce more Ptc at the membrane, which should sequester more Hh close to
the AP boundary. This result shows that Sxl can both enhance the Hh response
and effectively alter the Hh gradient (Horabin, 2005).
In males, the increase in ptc transcription induced by Sxl both
intensifies and widens the ptc expression zone. This suggests that
the activation of Ci is at a lower peak in males than in females, and its
enhancement by ectopic Sxl does not reach the same maximum that additional Sxl
in females produces (Horabin, 2005).
Ectopic expression of Sxl in the dpp expression zone has
been shown to adversely affect female wing development, narrowing
the region between veins L3 and L4. This defect was taken to suggest that the
relative concentrations of both Ci and Sxl are important for their normal
function (Horabin, 2003). The data presented in this study support this conclusion while providing an explanation for the apparent decrease in effectiveness of the Hh signal. When additional Sxl is expressed, the slope of the
Hh gradient becomes steeper. Since Hh directly patterns the L3 to L4 wing vein
region, a steeper gradient of Hh will reduce the area patterned because the
normal Hh patterning minimum is reached more rapidly. The L3 to L4 intervein
region should correspondingly become narrower. No adult males expressing Sxl
were recovered (presumably because of upsets in dosage compensation) so their
wings could not be scored (Horabin, 2005).
Depending on the expression driver used, ectopic Sxl is not only lethal to
males but also females. This is perhaps not altogether surprising given that
Sxl can modulate the signal strength of a molecule crucial to the development
of numerous tissues. The in vivo concentration of Sxl is, most likely, tightly
controlled. It has been shown that Sxl negatively regulates translation
of its own mRNA. Combined with its positive autoregulatory splicing feedback
loop, which ensures that essentially all of the Sxl mRNA is spliced
in the productive female mode in females, this dual negative and positive
autoregulation implies a homeostasis that keeps the concentration of Sxl in a
predetermined fixed range. The potent effect of Sxl on the Hh signal makes the
requirement for this dual regulation more readily understood (Horabin, 2005).
Mutations in Sxl that produce sex transformed females generally
result in animals that are small and male-like in size. Females transformed by
mutations in tra appear as males but maintain the female size,
indicating that sexual dimorphic body size is controlled by Sxl (Horabin, 2005).
The enhanced levels of full-length Ci
suggest that Sxl promotes disc growth. Indeed, when ectopic Sxl is being
expressed in the dorsal half, many of the discs, both male and female, show an
overgrowth phenotype with the dorsal half of the wing pouch frequently
expanded and distorted. This growth effect is non autonomous, indicating that it
is affected by a system that signals beyond the cells expressing Sxl. This is
consistent with the idea that Hh signaling is augmented to result in the
overgrowth. The experiments described here do not rule out the possibility
that Sxl may additionally regulate growth autonomously (Horabin, 2005).
Hh with only the cholesterol modification has the greater impact on Sxl and
its stabilization of full-length Ci. However, the Ci that is stabilized does
not appear to accomplish Hh patterning. This raises the mechanistic question
of how Sxl achieves growth of the entire disc (Horabin, 2005).
Simply reducing the levels of the repressor form of Ci (which is
accomplished by increasing the levels of full-length Ci) should increase the
expression of the growth factor dpp. This is because dpp is
affected by Ci at two levels: absence of the Ci repressor ameliorates
repression to give low levels of dpp expression, while activated
full-length Ci further elevates dpp transcription. Indeed, while the
wing patterning defect caused by the ectopic expression of C84S-Hh narrows the
region between wing veins L3 and L4 equally in the two sexes (due to its
dominant-negative effect on endogenous Hh), the overall sexual dimorphic size
difference is maintained. Consistent with this idea, co-expressing Sxl and Hh
with only the cholesterol modification produces an overgrowth phenotype in
discs, indicating Sxl can promote disc growth through this form of Hh (Horabin, 2005).
The growth induced by Dpp has been described as 'balanced', involving both
mass accumulation as well as cell cycle progression. The net effect is that
cell size does not change, nor does the ploidy. This is in contrast to growth
induced by hyperactivation of Ras, Myc or Phosphoinositide 3 kinase, which
increase growth but do not induce a progression through the G2/M phase of the
cell cycle and, as a result, increase cell size (Horabin, 2005).
It is proposed that in the wild-type gradient of Hh with both its lipid
modifications, Sxl augments the overall Hh signal to increase both full-length
as well as activated full-length Ci. The two Hh targets (Ci and Sxl) respond
differentially to the various components of the pathway
(Horabin, 2003). Since Sxl
is able to alter signal strength, the final outcome of the Hh signal must
reflect the balance in activities of the components, modulated by the lipid
moieties recognized, the membrane proteins used (Ptc versus Smo) and the
proteins present in the Hh cytoplasmic complex. The studies reported here
provide a strong rationale for why Sxl resides within the Hh cytoplasmic
complex (Horabin, 2005).
Sxl not only elevates expression of dpp and its downstream targets
to induce growth, but is able to elevate ptc expression. Enhancing
ptc suggests that the Hh signal is 'corrected' for the enlarged
patterning field, since short-range patterning has to be controlled by Hh. By
enhancing dpp, Sxl indirectly also enhances the long-range patterning
system of the disc. Augmenting the Hh signal would thus appear an elegant
solution for increasing overall size without changing the basic body plan or
pattern. Since Sxl is expressed in all female tissues from very early in
development and this expression is maintained for the rest of the life cycle,
Sxl is constantly available to upregulate the Hh signal. This augmentation
must be kept within check, however, because, as argued above, too high an
increase can change the overall slope of the Hh gradient, effectively changing
the final patterning of the tissue (Horabin, 2005).
The Hh pathway can also control body size in mammals. ptc1
mutations in mice provide an overgrowth phenotype with large body size, while
increasing ptc1 expression decreases body size.
Humans with basal cell nevus syndrome, an autosomal-dominant condition caused
by the inheritance of a mutant ptc allele, have been reported to have
multiple developmental abnormalities and, relevant to this study, larger body
size. Whether the mechanism described in this study is global to sexually dimorphic organisms that use Hh for patterning remains to be seen (Horabin, 2005).
The Drosophila master sex-switch protein Sex-lethal (Sxl) regulates the splicing and/or translation of three known targets to mediate somatic sexual differentiation. Genetic studies suggest that additional target(s) of Sxl exist, particularly in the female germline. Surprisingly, detailed molecular characterization of a new potential target of Sxl, enhancer of rudimentary [e(r)], reveals that Sxl regulates e(r) by a novel mechanism-polyadenylation switching-specifically in the female germline. Sxl binds to multiple Sxl-binding sites, which include the GU-rich poly(A) enhancer, and competes for the binding of Cleavage stimulation factor 64 kilodalton subunit (CstF64: involved in binding and processing mRNA for polyadenylation) in vitro. The Sxl-binding sites are able to confer sex-specific poly(A) switching onto an otherwise nonresponsive polyadenylation signal in vivo. The sex-specific poly(A) switching of e(r) provides a means for translational regulation in germ cells. A model is presented for the Sxl-dependent poly(A) site choice in the female germline (Gawande, 2006).
Since all known examples of Sxl regulation involve its binding to uridine-rich sequences, a search was performed of the entire Drosophila genome for potential high-affinity Sxl-binding sites. The Sxl consensus (UUUUUGUU(G/U)U(G/U)UUU(G/U)UU) was used used for the search; a search using a shorter Sxl-binding site (U8) yielded a significantly larger and experimentally unmanageable number of hits in the genome and therefore the search was not pursued further. Seven of the candidates showed sex-specific mRNA isoforms. This study describes the detailed characterization of one of the seven candidates, e(r). The GADFLY annotation database showed two e(r) transcripts (CT5770 and CT29800) resulting from an alternatively spliced exon and two alternative polyadenylation sites. Multiple potential Sxl-binding sites were found in e(r): one adjacent to the 3' splice site of exon 2 and three downstream of the proximal polyadenylation site (Gawande, 2006).
To determine whether alternative splicing of exon 2 was the basis for the sex-specific isoform of e(r), RNase protection analysis was performed. Exon 2 was found to be alternatively spliced, but was not spliced in a sex-specific manner. Next, an RNA blot was probed with the fs-UTR probe, which corresponds to the sequence between the two polyadenylation sites of e(r) and includes potential Sxl sites. This probe hybridized to the longer isoform that was present in females, but not to the shorter isoform present in both sexes. The shorter isoform is referred to as e(r)-non-sex-specific [e(r)-nss] and the longer isoform will be referred to as e(r)-female-specific (e(r)-fs). Presence of non-sex-specific and female-specific isoforms of e(r) is reminiscent of the known Sxl target, tra, although it is the sex-specific splicing regulation that contributes to the synthesis of the two isoforms of tra. It is concluded that use of two poly(A) sites, rather than alternative splicing, accounts for the sex-specific size difference of the e(r) transcripts (Gawande, 2006).
This study demonstrates that Sxl regulates e(r) expression in vivo by a novel mechanism-polyadenylation switching-which allows translation regulation in the female germline. A model that explains the Sxl-mediated regulation of e(r) must answer the following questions. First, what is the molecular basis for the default polyadenylation pattern in male flies? Second, how does Sxl mediate poly(A) switching in females? Third, why does the poly(A) switching of e(r) occur primarily in the female germline (Gawande, 2006)?
This study shows that three key factors account for the default poly(A) pattern of e(r) in male flies: differences in the binding affinities of CstF-64 for the two alternative GU-rich elements; order of the two polyadenylation sites; and cis competition between the two poly(A) signals. First, the longer GU-rich enhancer element downstream of the proximal cleavage/polyadenylation site provides at least in part a basis for the increased apparent binding affinity for CstF-64, most likely by providing multiple registers for binding, and thus confers competitive advantage to the proximal poly(A) site (Gawande, 2006).
Second, the order of the two poly(A) sites is also important. Contrary to expectation that the proximal site is inherently stronger than the distal polyadenylation (DP) site, DP males exclusively use the DA site. This demonstrates that the arrangement of the two sites is important for the default poly(A) site choice and excludes the possibility that the distal site is inherently weak. The usage of the DA site is consistent with the known RNA-binding properties of CstF-64 and sequences of natural poly(A) sites. Moreover, in this system, the promoter-proximal site is always used by default in males. Both transcription and splicing influence polyadenylation. Since an EGFP reporter, unlike the endogenous transcript, lacks an intron, coupling between the transcription and polyadenylation machineries best explains why the promoter-proximal site is always preferred in males (Gawande, 2006).
Third, mutation of the Sxl-binding site or the proximal GU-rich element compromises CstF64 binding and activates the distal site in males. This is what would be expected if the Sxl-binding site is also a polyadenylation element (or the CstF64-binding site) and if Sxl blocks the proximal poly(A) site, which is consistent with the Sxl blockage model proposed for splicing regulation. Since CstF-64 binds directly to the GU-rich enhancer element to facilitate recruitment of the polyadenylation machinery to the poly(A) signal, reduced affinity of CstF-64 to the mutant proximal GU-rich element provides a basis for the activation of the distal site in males. This shows that the longer GU-rich element associated with the proximal site is also important for the default poly(A) site choice. It is possible that the non-canonical poly(A) element (UAUAAA instead of AAUAAA) and its sequence context confer increased dependence on the long GU-rich element for polyadenylation at the proximal site. This feature could make the proximal GU-rich element and CstF-64 binding an attractive target for Sxl regulation (Gawande, 2006).
The simplest explanation for the combined results is that in the female germline Sxl competes for the binding of CstF-64 to the proximal GU-rich element and represses polyadenylation at the proximal site, leading to activation of the distal site; Sxl does not bind to the distal GU-rich element. The proximal GU-rich element is much longer than is necessary for polyadenylation or CstF-64 binding, most likely to confer competitive advantage (by providing multiple registers for CstF-64 binding to the proximal site in males and to accommodate Sxl binding in females. Moreover, Sxl and CstF-64 have similar sequence preference (GU-rich sequences devoid of cytidines) . Therefore, it is not surprising that the sequence requirement of the proximal GU-rich element for polyadenylation in males and for poly(A) switching in females could not be uncoupled, as was performed using a three nucleotide U-to-C substitution for Sxl-mediated splicing regulation of tra, which involves competition between Sxl and U2AF; U2AF prefers uridine- and cytidine-rich sequences devoid of guanosines. Specificity of Sxl for the proximal but not the distal GU-rich element and the ability to convert a poly(A) site that is nonresponsive to poly(A) switching to a site that supports female-specific switching provide an explanation for why switching occurs in wt and D123D' females, but not in DP and DD' females. Moreover, Sxl-dependent poly(A) site switching of e(r) as well as splice site switching of Sxl and msl2 transcripts displays a need for multiple Sxl-binding sites for efficient regulation. The ability of Sxl to discriminate between alternative GU-rich sequences is the crux of the sex-specific poly(A) switching. GU-rich sequences belong to a class of regulatory RNA motifs that are defined by base composition rather than an exact sequence. Such simple repetitive sequences offer several advantages for gene regulation: for example, binding affinity but not specificity for RNA-binding proteins can change as a function of the length of the sequence; two proteins (e.g., Sxl and CstF-64) with different binding site length requirements can recognize the same sequence; a regulatory protein such as Sxl can regulate multiple processes, polyadenylation and splicing, by antagonizing CstF-64 and U2AF65, respectively, because of the possibility of overlapping binding specificities; and single nucleotide changes are less disruptive (Gawande, 2006).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Sex lethal:
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.