vestigial
Interactions between dorsal and ventral cells in the growing wing imaginal disc induce vestigial gene
expression along the dorsoventral boundary through a discrete, extraordinarily conserved imaginal disc-specific enhancer. The conserved region in intron 2 contains 118 identical base pairs comparing D. melanogaster and D. virilis. This region is incapable of forming strong secondary structures. Expression along the D/V boundary is symmetrically distributed in both dorsal and ventral cells. apterous, whose transcription is restricted to the dorsal compartment is required for vestigial expression at the boundary (Williams, 1993 and 1994), but it is unclear from this work whether the signal for vestigial is Notch, Wingless or another factor (see below: Doherty, 1996 and Blair, 1994).
Different signals activate separate enhancers to control vestigial expression: initially, in the dorsal/ventral organizer through the Notch pathway, and subsequently, in the developing wing blade by Decapentaplegic, and by a signal from the dorsal/ventral organizer. Signal integration must be a general feature of genes like vestigial, that regulate growth or patterning along more than one axis (Kim, 1996).
The spalt and optomotor-blind genes are expressed in a nested pattern in the wing imaginal disc, centered on and extending up to 20 cell diameters away from the stripe of decapentaplegic expression along the anteroposterior compartment boundary. The vestigial gene is even more broadly expressed and is required in all cells of the developing wing. Activation of vg through the quadrant enhancer is responsible for the broad expression of vg in the wing blade. The quadrant enhancer is found in an 806 bp DNA segment in the fourth intron of the vg gene. This enhancer is sufficient to drive expression in all of the developing wing blade. vg expression in developing wing-blade cells, beginning from the wing-blade primordium of the wing imaginal disc, requires a decapentaplegic signal from the A/P boundary and another signal from the D/V boundary. The term quadrant enhancer refers to the expression pattern, which is bisected by non-expressing DV boundary cells and becomes less intense along the A/P boundary in the late third instar, producing a quadrant-like pattern. Unlike the D/V boundary enhancer, which is regulated by the Notch pathway and is active in the second instar, the quadrant enhancer is not active until the early third instar. The quadrant enhancer is Suppressor of hairless independent, suggesting that Notch function is not required for formation of this part of the wing during the third instar. The temporal order of D/V boundary and quadrant enhancer activation suggests that D/V boundary cells must produce a signal (or signals) that organize the rest of the wing, including expression from the quadrant enhancer. Activation of the quadrant enhancer requires dpp, since thick veins mutants exhibit reduced Vg protein levels and have smaller wings and wing discs (Kim, 1996).
Because Mothers against Dpp is an intracellular signal transducer of Dpp receptors, the requirement of Mad activity for Vg expression in the wing imaginal disc was examined in mitotic clones with reduced Mad function. Mad has no effect on the Notch-dependent dorsoventral boundary enhancer, but the quadrant enhancer requires Dpp signaling and Mad function. The N-terminal MAD homology region 1 (MH1) plus the central less conserved proline-rich linker region bind DNA and protect a single interval within the quadrant enhancer. A 39 bp double stranded oligonucleotide of the vg quadrant enhancer contains the Mad-protected region. Mutating 12 base pairs within this region prevents Mad-directed expression. The C-terminal MH2 domain of Mad effectively inhibits DNA binding and suggests a mechanism that might contribute to inactivation of Mad in the absence of Dpp signaling. Examination of the Ultrabithorax midgut enhancer, for which the Dpp response element has been localized to the 95 bp DI-DII interval, and the labial endoderm enhancer reveals a Mad binding-site consensus of GCCGnCGC. The two sites of highest affinity match this consensus perfectly, and three lower-affinity sites contain mismatches in one or as many as three positions (Kim, 1997).
Distal-less induces ventral appendage development in Drosophila. Lack of Dll function causes a change in the identity of ventral appendage cells (legs and antennae) that often results in the loss of the appendage. Ectopic Dll expression in the proximal region of ventral appendages induces nonautonomous duplication of legs and antennae by the activation of wingless and decapentaplegic. Ectopic Dll expression in dorsal appendages produces transformation into corresponding ventral appendages; wings and halteres develop ectopic legs and the head-eye region develops ectopic antennae. In the wing, the exogenous Dll product induces this transformation by activating the endogenous Dll gene and repressing the wing determinant gene vestigial. It is proposed that Dll induces the development of ventral appendages and also participates in a genetic address that specifies the identity of ventral appendages and discriminates the dorsal versus the ventral appendages in the adult. However, unlike other homeotic genes, Dll expression and function is not defined by a cell lineage border. Dll also performs a secondary and late function required for the normal patterning of the wing (Gorfinkiel, 1997). The expression of vestigial during wing development is regulated
through two enhancers: the second intron or boundary
enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect
the patterns of expression directed by these regulatory regions:
vgBE produces a thin stripe over the
prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to
the vgBE and which fill in the developing wing blade. Both
vgBE and vgQE act as integrators of signaling systems that
drive wing development and, in this manner, these regulatory
regions determine the tempo and the mode of wing
development (Klein, 1999 and references).
The vgBE is activated first during the
second instar. Its expression pattern is
very similar to that of the Vestigial protein
at this stage suggesting that, in these early
stages, the vgBE is responsible for the
complete pattern of vg expression. Deletion analysis of the
enhancer reveals two regions essential for
its activity: a binding site for Suppressor
of Hairless and sequences contained in the
first 80 base pairs of the enhancer.
In an attempt to map the nature and
timing of the inputs into this enhancer, the activity of the wild-type vgBE and of
deletions of the two essential regions have been compared during wing development.
At the end of the second instar, lacZ reporter
expression from the wild-type vgBE
outlines a horseshoe over the ventral
region of the wing disc, with weak
expression in the ventral anterior region
where it overlaps with the expression of
wingless. lacZ expression
increases in this region at the beginning of the third instar. This increase does not occur in Suppressor of Hairless [Su(H)] mutants or in wg mutants.
The activity of the enhancer deleted for the Su(H)-binding
site is different from the wild type. The activity of this enhancer
is never initiated over the ventral region of the disc, where the
wing primordium is established and remains absent during later
stages. This result demonstrates that the activity of
Notch is required not only for the maintenance, but also for the
initiation of the expression of vg through the vgBE.
The activity of the enhancer deleted from 0-80 is similar to
that of the wild-type enhancer early on, but it never
acquires the high levels of activity in the anterior ventral region.
As the wing blade develops, a line of faint activity can be
detected over the DV boundary, but it fades quickly
and, by the end of the third instar, in most discs there is no
activity over the developing wing blade. This enhancer
still shows some activity in the flanking notal regions of the wing
disc in wild-type and Su(H) mutant discs. This
indicates the existence of additional inputs in the regulation of
the vgBE in the notal region. These results suggest that the cells in which the expression of the vgBE is upregulated at the end of the second instar represent the anlage of the wing and require Notch/Su(H) signaling. These cells are located at the DV interface, on the domain of wg expression and overlap the expression of nubbin. The suggestion that these cells represent the primordium of the wing pouch can explain why a deletion of the vgBE results in the abolition of the development of the wing pouch; in such a mutant, the anlage would never be defined (Klein, 1999).
In addition to the activity of Notch, the activity of wg is required for the initiation of vg
expression because in the second instar larval discs of wg mutants, the
activity of the vgBE is absent over the region of the wing anlage. The possibility that Wingless
acts on the vgBE is supported by the observation that a dominant
negative version of DFrizzled2, a receptor for Wingless, reduces
the activity of the vgBE. Altogether these results raise the possibility that Wingless has a direct input
on the vgBE. Consistent with this the first 80 bp of the vgBE, which are required for the full activity of the enhancer, contain two putative TCF-1-binding sites associated with
Wingless signaling. In contrast with ectopic expression of wg, which never leads to
ectopic expression of the vgBE, ectopic expression
of Delta leads to ectopic expression of the vgBE, but only where the levels of ectopic expression of Delta are high: within the developing wing blade and near the AP compartment boundary. An effect of
Wingless on this enhancer can be detected when Wingless is
coexpressed ectopically with Delta. Although Wingless alone
has no effect on this enhancer, coexpression of Delta with
Wingless extends the realm of activity of the vgBE into
regions where expression of Delta on its own has no detectable
effect. These results suggest that the major effect of Wingless on
the vgBE is to collaborate with Notch/Su(H) signaling during
the early stages of wing development (Klein, 1999).
Notch signaling is also required for the initial activity of the Quadrant Enhancer (vgQE).
The activity of the vgQE can be detected first at the beginning of the third instar, several hours after the upregulation of the vgBE, when it closely outlines the realm of the growing wing. This enhancer is only expressed in the growing wing blade and thus provides a
unique and most specific marker for wing blade tissue. A variety of experiments have shown that the vgQE receives a negative input from Notch signaling and a positive one from
Dpp. The presence of an E(spl)-binding
site in the sequence of the vgQE has led to the suggestion
that this suppression by Notch is mediated by the E(spl)
protein. However, no strong suppression of the
activity of the vgQE is found if E(spl)-m8 is ectopically
expressed, suggesting that the
effect of Notch requires other mediators. Although the vgQE is suppressed in the domain
of Notch activity, Notch signaling plays a non-autonomous
role in its activation. For example, the vgQE is
never active in Serrate (Ser) mutants in which wing
development initiates normally but is aborted very early. Ectopic expression of Delta
rescues the wing
pouch and leads to the activation of the vgQE.
Interestingly, this activity arises in regions devoid of Notch
signaling. This result suggests that
Notch signaling influences the activity of the vgQE in two
ways: it represses the activity of the vgQE autonomously but
it is also required for its activity in a non-autonomous way (Klein, 1999).
Wingless is shown to act synergistically with Vestigial to
promote the activity of the vgQE.
It is clear that the activity of the vgBE is required for the
activation of the vgQE, but little is known about how this interaction
takes place. The observation that vgQE is activated in Ser
mutant discs in which vg is expressed ectopically
suggests that the activation of the vgQE is mediated by Notch
signaling through the activity of Vestigial on the vgQE. However, in this experiment, expression of wg is also
restored and this raises the possibility that activation
of the vgQE is mediated through the presumed organizing
activity of Wingless. This is probably not the case since ectopic
expression of wg alone does not lead to the activation of
the vgQE in Ser mutants. The inability of Wingless
to activate the vgQE in this experiment is not due to a general
insensitivity of the cells to Wingless signaling, since ectopic wg
is always capable of inducing hinge fate ectopically.
These results clearly demonstrate a requirement for vg in the
activation of the vgQE. However, clonal analysis has shown
that vg acts cell autonomously and therefore,
in the wild type, the non-autonomous effects of Notch on the
vgQE must be mediated by another, diffusible molecule(s),
which is under control of Notch signaling. A number of
studies suggest that Wingless has an influence on the
expression of vg in the wing pouch and that its expression at
the wing margin is under control of Notch signaling. Therefore, it is
possible that Wingless is mediating the non-autonomous effect
of Notch on the vgQE. It might be that Wingless acts by acting
on the vgQE to elevate and maintain the levels of vg expression
that had been induced by Vestigial through the vgBE. In
agreement with this proposal, it is found that the activity of the
vgQE is elevated in response to the ectopic expression of wg and that expression of a dominant negative Wingless molecule
suppresses the activity of the vgQE and reduces the size of the
wing pouch.
Altogether these results suggest that the upregulation of vg
expression in response to wg is mediated by the vgQE. This
conclusion is supported by the existence of several putative
TCF-1 binding sites in the vgQE. However,
the effects of Wingless are always restricted to the normal
domain of vg expression, in agreement
with the results presented above that Wingless alone is not
sufficient to initiate ectopic expression of vg through the vgQE.
These effects are likely to be mediated by Vestigial itself. The role of Wingless on this regulation is
to maintain and modulate the levels of activity of the vgQE.
Consistent with the conclusion that Wingless enhances the
effects of Vestigial, coexpression of Wingless and
Vestigial, which leads to the ectopic induction
of pouch and hinge fate in the notal regions, triggers a widespread and stable
expression of the vgQE throughout the wing disc (Klein, 1999).
The interactions between Dpp and Wingless during
wing development take place at the level of the
vgQE and require the vg gene product.
Ectopic expression of wg alone leads to ectopic
expression of the vgQE in the posterior region of the domain of
vgBE expression, over a region in which dpp is
normally expressed. Expression
of dpp under the same conditions leads to an enlargement of the
wing blade along the AP axis, with the vgBE as a reference but
without extending it into the notum.
Coexpression of wg and dpp leads to a pronounced extension
of the wing blade toward the notum. In some
instances, the development of up to eight wing
blades occurs, all with a common origin over a point ventral to the
notum. All these wings express the vgQE and each
of them has a defined margin. It is possible that these
multiple wings are produced by the splitting of an initial primordium.
In the wild type, the activity of the vgQE is initiated at the
intersection of wg and dpp expression and radiates from this
focus. The results presented
here indicate that this overlap determines important parameters
of the morphogenesis of the wing. For example, it is possible that the distance from this focus -- perhaps defined by the range
of diffusion of the two molecules -- defines a threshold that
contributes significantly to the determination of the size and
shape of the wing blade. By tampering with these overlaps, one
can alter the shape of the wing or, by
generating a
series of them, trigger the development of multiple wings from
one primordium (Klein, 1999).
Vestigial contributes Distal-less gene expression in the wing blade.
In the wild-type wing, Dll is expressed in the wing pouch in
a circle of cells that lies within an area defined by the expression of vestigial. Ectopic expression of vg elevates the levels of Dll expression within the developing wing
blade and triggers ectopic expression of Dll outside this area. This differs from the effects of ectopic expression of wg, which are always restricted to the developing wing blade
and never induce ectopic expression of Dll. Coexpression of
both, wg and vg, results in synergistic effects similar to those
described before for other targets and an increase in the area
of ectopic expression. The effects of vg are independent of wg, since they
are achieved even in apterous mutants, where wg is never expressed
along the DV boundary. These results indicate that Vestigial can indeed regulate gene
expression in the wing blade. In addition, the correlation
between levels suggests that different concentrations of
Vestigial elicit different effects and that, in some instances,
these effects seem to be independent of the levels of Wingless.
Thus Vestigial might act in concert with Wingless, and not
simply as an effector of wg, in the regulation of gene
expression. Very high levels of Vestigial expression, or long-term exposure to vestigial
expression, results in the cessation of proliferation, loss of gene expression and, eventually, in cell death. This might account for the small wings that are often visible when vg is overexpressed in the
developing wing and would correlate with the zone of non-proliferation that appears at the wing margin, where the levels of vg expression are elevated (Klein, 1999).
The contributions of the DV and AP axes to the patterning
of the wing are becoming clear. However, little is still known
about what triggers the foci of proliferation that drive the
growth of the wing blade or how the interactions described here control the final size of the wing. The results presented here suggest a more indirect role of wg and vg in cell
proliferation than previously suggested. Although the loss of
function of each of these genes has a significant influence on
the cell proliferation in the wing, the overexpression of both
does not lead to the increase of the wing pouch of the late third instar. This suggests a more
permissive role for the two genes in cell proliferation, probably
to maintain the identity of the wing pouch. Studies that suggest that the proliferation is induced by local cues and is a cell autonomous property support this conclusion (Klein, 1999 and references).
The Drosophila Vestigial protein has been shown to play an
essential role in the regulation of cell proliferation and
differentiation within the developing wing imaginal disc.
Cell-specific expression of vg is controlled by two separate
transcriptional enhancers. The boundary enhancer
controls expression in cells near the dorsoventral (DV)
boundary and is regulated by the Notch signal transduction
pathway, while the quadrant enhancer responds to the
Decapentaplegic and Wingless morphogen gradients
emanating from cells near the anteroposterior (AP) and DV
boundaries, respectively. MAD-dependent activation of the
vestigial quadrant enhancer results in broad expression
throughout the wing pouch but is excluded from cells near
the DV boundary. This has previously been thought to be
due to direct repression by a signal from the DV boundary;
however, this exclusion of quadrant enhancer-dependent
expression from the DV boundary has been shown to be due to the
absence of an additional essential activator in those cells.
The Drosophila POU domain transcriptional regulator,
Drifter, is expressed in all cells within the wing pouch
expressing a vgQ-lacZ transgene and is also excluded from
the DV boundary. Viable drifter hypomorphic mutations
cause defects in cell proliferation and wing vein patterning
correlated with decreased quadrant enhancer-dependent
expression. Drifter misexpression at the DV boundary
using the GAL4/UAS system causes ectopic outgrowths at
the distal wing tip due to induction of aberrant Vestigial
expression, while a dominant-negative Drifter isoform
represses expression of vgQ-lacZ and causes severe
notching of the adult wing. In addition, an essential evolutionarily conserved sequence element
bound by the Drifter protein with high affinity has been identified and it has been located adjacent to the MAD binding site within the quadrant
enhancer. These results demonstrate that Drifter functions
along with MAD as a direct activator of Vestigial expression
in the wing pouch (Certel, 2000).
Signaling by Dpp activates targets such as vestigial indirectly through negative regulation of brinker. The Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity is found in an amino-terminal region containing a putative homeodomain. Brk binds to a Dpp
response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for Mad. Furthermore, Brk is able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs from the Smad3/Smad4 binding site consensus. The Dpp response element from Ubx is more sensitive to repression by Brk than is the vg quadrant enhancer. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Evidence suggests that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad (Kirkpatrick, 2001).
Binding of Brk to the Ubx and vg probes generates multiple bands, possibly indicating that Brk binds to more than one site. The Ubx element contains an inverted repeat of GGCGCT that overlaps a
previously identified Mad binding site. Whereas the Mad site embedded in this repeat resembles the vg Mad site, the repeat as a whole is only matched at
7 of 12 positions in vg. Brk was tested for the ability to bind one copy of this sequence in a DNA probe that was otherwise divergent in sequence from the
Ubx element. Brk binds to the GGCGCT probe with affinity that is similar to its affinity for the Ubx probe and
yields a single major shifted band at about the same position as the lower most band observed with the Ubx probe. Although two weak upper bands are also
observed with the GGCGCT probe, overall, these results are consistent with high affinity interaction of Brk with just one site in the GGCGCT probe (Kirkpatrick, 2001).
Polycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position. The phiC31 site-specific integration tool was adapted to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw) reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. The Hox PRE Frontabdominal-7 (Fab-7) was compared with a PRE from the vestigial (vg) gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, the phiC31 tool was used to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT) that is essential for silencing. This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of phiC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design (Okulski, 2011).
Though operationally defined as cis-regulatory elements, enhancers can also communicate with promoters on a separate homolog in trans, a mechanism that has been suggested to account for the ability of certain alleles of the same gene to complement one another in a process otherwise known as transvection. This homolog-pairing dependent process is facilitated in Drosophila by chromatin-associated pairing proteins, many of which remain unknown and their mechanism of action uncharacterized. This study tested the role of the gypsy chromatin insulator in facilitating pairing and communication between enhancers and promoters in trans using a transgenic eGFP reporter system engineered to allow for targeted deletions in the vestigial Boundary Enhancer (vgBE) and the hsp70 minimal promoter, along with one or two flanking gypsy elements. A modest 2.5-3x increase was found in eGFP reporter levels from homozygotes carrying an intact copy of the reporter on each homolog compared to unpaired hemizygotes, although this behavior was independent of gypsy. However, detectable levels of GFP protein along the DV wing boundary in trans-heterozygotes lacking a single enhancer and promoter was only observed in the presence of two flanking gypsy elements. These results demonstrate that gypsy can stimulate enhancer-promoter communication in trans throughout the genome in a context-dependent manner, likely through modulation of local chromatin dynamics once pairing has been established by other elements and highlights chromatin structure as the master regulator of this phenomenon (Schoborg, 2013).
Why do two flanking gypsy insulators, but not a single upstream gypsy insulator, support transvection? One might assume that if gypsy contributes to homolog pairing, then a single insulator located just upstream of the enhancer and promoter would still be capable of ensuring that those two elements remain in close proximity in trans. However, it is argued that the most critical determinant is chromatin structure itself-it is widely accepted as a key regulator of transcription in cis, so the same principles would also apply in trans. Even if pairing were to bring enhancers and promoters in close proximity, the underlying chromatin must still be permissible in order for transcription to occur. Insulators were originally identified based on their ability to buffer the effects of surrounding chromatin influences (i.e., position effects) on transgene expression, and regardless of the mechanism by which insulators accomplish this task (chromatin looping, etc.) it is likely that the transvection observed is due to the flanking insulators establishing a permissive chromatin environment favorable for transcription. The single gypsy insulator, on the other hand would not be able to establish the same environment and therefore even if pairing were established by other elements, transcription would still be unlikely to occur given the lack of a suitable chromatin landscape. su(Hw) mutant data supports this hypothesis, as significant reductions in GFP expression were observed in both homozygotes and hemizygotes, highlighting the importance of chromatin structure on transgene expression regardless of pairing influences. These findings, along with a number of other studies linking chromatin proteins to transvectionand the failure of other studies to observe transvection except when their reporters were located in defined PhiC31 genomic sites that are highly permissible to transcription, suggests that chromatin itself is the master regulator of this phenomenon (Schoborg, 2013).
Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. This study shows that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. It is proposed that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription (Herzog, 2014).
By analysis of endogenous transcripts and by using ectopic overexpression strategies in vivo it was demonstrated that transcripts from opposite strands of the vg PRE/TRE have opposite effects on PRE/TRE status. However, in vitro, both noncoding RNAs have equivalent inhibitory effects on the HMTase activity of PRC2. Taking the in vitro and in vivo data together, it is proposed that the specificity of the noncoding RNA interaction with PcG proteins in vivo is not a result of inherently different affinities of PRC2 for different noncoding RNAs but of the availability of a given noncoding RNA (regulated by interactions of that RNA with other molecules) to interact with PRC2 and inhibit its enzymatic activity. It is proposed that the forward-strand noncoding RNA promotes silencing by facilitating pairing between PRE/TREs. PRE/TRE pairing has been shown to be essential for maximum silencing by the vg PRE/TRE, and this silencing is genetically dependent on PcG. Thus, it is proposed that forward strand-induced pairing might facilitate or stabilize PcG-mediated pairing-dependent silencing. E(Z) is detected at the vg PRE/TRE in ChIP analyses but does not interact with the forward strand. Thus, it is proposed that E(Z) binds at the silenced PRE/TRE independently of RNA. The forward-strand noncoding RNA might facilitate or stabilize pairing by binding to additional bridging proteins. These or other proteins might also prevent binding and inhibition of E(Z) by the RNA (Herzog, 2014).
Upon switching to the active state, transcription of the reverse PRE/TRE strand would be incompatible with forward-strand transcription because the reverse transcript runs through the forward-strand promoter. Reverse-strand transcription might thus destabilize pairing, enabling the activation of the PRE/TRE. In addition, the reverse strand binds E(Z) and, upon binding, would inhibit E(Z) HMTase activity, and it might also remove E(Z) from the locus. In this way, multiple self-reinforcing events could contribute to the stable switching of the PRE/TRE into an active state. The vg PRE/TRE responds to TrxG mutations by loss of activation. Whether TrxG-dependent activation acts via the noncoding RNAs will be a key question for future studies (Herzog, 2014).
Genome-wide analysis identifies many sites that might share functional features with the vgPRE/TRE. It is proposed that these elements can exist in a 'neutral' state, in which neither they nor their associated genes are transcribed (this is consistent with the observation that the vg PRE/TRE is not transcribed in tissues or at embryonic stages that do not express vg mRNA). However, in tissues that have the potential to transcribe the gene, the element might be switched either to the forward or reverse mode, thereby boosting either silencing or activation. This might serve to sharpen spatial expression boundaries, to stabilize gene expression states or to accelerate the kinetics of activation or repression (Herzog, 2014).
In conclusion, this work provides a new paradigm linking forward and reverse noncoding transcription to dynamic and developmentally regulated switching of PRE/TRE properties and thus to the maintenance of cell identities during development. Furthermore, the demonstration that any RNA is a potent inhibitor of PRC2 enzymatic activity in vitro but that only specific RNAs are able to bind and inhibit PRC2 in vivo strongly implies that specific RNAs are masked in vivo from interacting with PRC2. This provides an enormous potential for the regulated and reversible RNA-mediated inhibition of local PRC2 activity (Herzog, 2014).
Continued: vestigial Transcriptional Regulation part 2/3
| part 3/3
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
vestigial:
Biological Overview
| Evolutionary Homologs
| Targets of Activity and Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.