Promoter Structure

A genetic and molecular analysis of two Hairy pair-rule stripes has been carried out in order to determine how gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. Regulatory sequences of hairy have been identified that are critical for the expression of h stripes 5 and 6. Fragments of 302 bp and 526 bp are required for stripe 5 and 6 activation, respectively. Posterior stripe boundaries are established by gap protein repressors unique to each stripe: hairy stripe 5 is repressed by the Giant protein on its posterior border and h stripe 6 is repressed by the Hunchback protein on its posterior border. Interestingly, Krüppel (Kr) limits the anterior expression limits of both stripes, the only gap gene to do so, indicating that stripes 5 and 6 may be coordinately positioned by the KR repressor.

In contrast to these very similar cases of spatial repression, stripes 5 and 6 appear to be activated by different mechanisms. Stripe 6 is critically dependent upon Knirps for activation, while stripe 5 likely requires a combination of activating proteins (gap and non-gap). To begin a mechanistic understanding of stripe formation, binding sites for the KR protein in both stripe enhancers were located. The stripe 6 enhancer contains higher affinity KR-binding sites than the stripe 5 enhancer, which may allow for the two stripes to be repressed at different KR protein concentration thresholds. The Knirps activator binds to the stripe 6 enhancer. Also, there is a for a competitive mechanism of KR repression of stripe 6 (Langeland, 1994).

The expression of the pair-rule gene hairy (h) in seven evenly spaced stripes along the longitudinal axis of the Drosophila blastoderm embryo is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene expression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing the trans-acting control of h stripe 7 expression as revealed by genetic analyses. The stripe seven enhancer is found in a minimal 932 bp region from a 1.5 kb DNA fragment of the h upstream region. The h7-element mediates position-dependent gene expression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators, Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Zygotic caudal expression is not required for activation. Caudal and Bicoid, which form complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h stripe-7 domain are brought about by the local activities of repressors that prevent activation. The spatial limit of h7 is significantly altered in the gap mutants tailless, knirps and kruppel, but not in embryos lacking either hunchback, giant or huckebein. There are seven binding sites for Bcd, twenty-three for caudal, five for Kruppel, fourteen for Knirps, eight for Hunchback and five for Tailless. In the absence of both cad and bcd, activation still occurs. Thus, a third activator, likely to be Kr, must function in such embryos. It is thought that Kr acts as both a repressor and an activator within the h7 element depending on its concentration. The posterior border is set in response to Tll activity under the control of the terminal maternal organizer system. The anterior border of the expression domain is due to repression in response to Kni. The results suggest that the gradients of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo (La Rosee, 1997).

The available in vivo evidence suggests that Kruppel acts as a transcriptional repressor; however, conclusive in vivo evidence demonstrating that Krüppel can additionally function as an activator of gene expression has been missing. Krüppel binds to the consensus sequence AAAAC/GGGGTTAA. The zinc finger domain of the Kr protein is framed by two evolutionarily conserved transrepressor domains [an N-terminal TR1 (transrepressor domain 1) and C64 (C-terminal repressor domain)] and a single, weak transactivator domain (TA1). C64 was initially identified when transferred to the DNA-binding domain of the yeast transcriptional activator GAL4: all three transacting domains, TR1, TA1 and C64, have been shown to confer their activities to the bacterial LacI protein. The two independent and transferable repressor domains of Krüppel have been shown to act to control expression of the pair-rule gene hairy, and the minimal cis-acting element of hairy stripe7 (h7) mediates either Krüppel- dependent activation or repression in different regions of the blastoderm embryo (La Rosee-Borggreve, 1999).

In Drosophila cultured cells, TA1 alone is incapable of acting as a weak transactivator domain. TA1 is however, activation-competent in the presence of the adjacent stretch of 51 amino acid residues of the Kr protein. This 51 amino acid region contains sequence motifs similar to those observed in the transactivation domains of CTF/NF1, Sp1 and Pit1, but since this sequence alone fails to mediate gene activation, it is referred to as the co-activating domain (CAD). Combined TA1 and CAD causes reporter gene activation even in the presence of TR1. Together, these two domains override the TR1-dependent transrepression activity. In contrast, when TA1 and CAD are directly fused with the C64 repressor domain of Kr, reporter expression is nullified. Therefore, the opposite regulatory activities of the TA1/CAD and C64 domains are extinguished when fused. Thus, it appears necessary that, as in the full-size Krüppel protein, these domains are separated in order to exert opposite regulatory functions on transcription (La Rosee-Borggreve, 1999).

The hairy stripe7 enhancer element, decodes the activity of three activators: the maternal homeodomain proteins Caudal and Bicoid, and the zinc finger protein Kruppel. Caudal and Krüppel activities are necessary, and sufficient, to activate h7-mediated lacZ reporter gene (h7-lacZ) expression but Bicoid activity is additionally required to achieve wildtype expression levels. Absence of Kr activity not only significantly reduces the level of h7-dependent reporter gene activation in the posterior region of the embryo, but also results in the appearance of a second and novel expression domain in a position corresponding to the highest levels of Krüppel in wildtype blastoderm embryos. Thus, h7 not only mediates gene expression in response to low levels of Krüppel in the posterior region of the blastoderm embryo, but it simultaneously prevents reporter gene expression at high concentrations in the central region of the embryo. To determine the ability of Kr to directly interact with the h7 element, DNaseI footprinting experiments were performed using bacterially produced Kr and subfragments of the h7 element. The h7 element has been shown to contain five in vitro Krüppel binding sites; this opens the possibility that Krüppel may act through multiple binding sites within the h7 element (La Rosee-Borggreve, 1999).

The C-terminal region of Krüppel that encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP (La Rosee-Borggreve, 1999).

In vitro experiments have shown that C64 provides a homodimerization surface that permits Krüppel homodimer formation at high protein concentrations. The homodimer acts exclusively as a transcriptional repressor, whereas the Krüppel monomer has been shown to function as a transcriptional activator both in vitro and in Drosophila tissue culture assays. Based on the in vitro results, it has been proposed that Krüppel acts as a transcriptional repressor in the central region of the blastoderm embryo and may function as an activator of target genes outside the central region where the concentration of Krüppel gradually decreases. The h7-mediated expression pattern in KrV mutant embryos is consistent with this proposal: the lack of the C-terminus, and hence the dimerization domain, does not affect Krüppel's ability to co-activate h7-mediated gene expression in a position of low Krüppel concentration in the embryo, but rather, strongly reduces its repressor function at high concentrations (La Rosee-Borggreve, 1999).

Two models have been proposed to explain Krüppel-mediated repression. One model suggests that Krüppel possesses two separate activities, one interfering with enhancer-bound activators by quenching, and the other directly inhibiting transcription by interacting with components of the basal transcription machinery. The second model proposes that Krüppel recruits a repressor complex that only functions locally. Some aspects of the results presented here fit with the first model, others with the second. For example, TR1 could be the repressor domain that acts through quenching. In this case, TR1 would interfere with Bicoid-dependent activation mediated by h7 in the central region of the embryo, but not with Caudal-dependent activation, which is predominant in the posterior region of the embryo. This assignment is consistent with the finding that repression of h7-mediated gene expression is strongly reduced in the central region of the KrV mutant embryo but no effect is observed in the posterior region of the embryo when Krüppel lacking the C-terminal region is expressed throughout the embryo (La Rosee-Borggreve, 1999).

Alternatively or additionally, C64 could act either by blocking activation via inhibiting basal transcription, or it may interfere with, and thereby extinguish, both Caudal and Bicoid activities directly. Direct inhibition of the basal transcription machinery would be consistent with in vitro data showing that C64 prevents transcription by interacting with the general transcription factor TFIIEbeta (Sauer, 1995a). This proposal would, however, be consistent with the recent finding that the C-terminal repression region of Krüppel inhibits certain activators only if the subset of affected activators would target TFIIEbeta to exert their function. The second model which explains transcriptional repression via a repressive complex formation is consistent with the observation that the C-terminal domain enables Kr to form heterodimer complexes with other transcription factors such as Knirps. A further possibility is that the C-terminal domain could serve to recruit more general co-repressors such as Groucho or CtBP to template DNA. A CtBP-binding motif has indeed been noted in the C- terminal repressor region of Krüppel. The Drosophila homolog, dCtBP, has been shown to interact in vitro with the gap gene product Knirps and gene-dosage interaction studies with dCtBP and knirps mutants have suggested that Knirps-dCtBP interactions are also able to occur in vivo (Nibu, 1998). The recruitment of dCtBP by short-range repressors, such as Knirps and Krüppel, may theoretically be able to alter the chromatin structure, its status of acetylation or the presence of transcriptional activators bound to a nearby site within the enhancer. Nevertheless, the weakest known knirps mutant, knirps14F, which lacks the dCtBP-interaction motif, develops an almost normal abdominal segment pattern with the exception that the abdominal segment 4 is consistently missing. This suggests that dCtBP may possibly be important for some specific but not all aspects of Knirps-dependent repressor function. The results shown here indicate that dCtBP is neither required for Krüppel-dependent repression of h7-mediated activation in the central region of the embryo, nor for Knirps-dependent repression of the expression domain in the posterior region of the embryo. Furthermore, dCtBP is also not required for repression of this expression domain in response to ubiquitously expressed Krüppel (La Rosee-Borggreve, 1999 and references therein).

The results shown here describe a previously missing piece of information surrounding Krüppel function; namely, that Krüppel possesses both activator and repressor function in vivo. The switch between activator and repressor functions is dependent on the concentration of Krüppel protein and is mediated by the C-terminus. The precise mechanism by which this mode of switching is regulated and potential cofactors of Krüppel are still unknown and need to be addressed by future studies (La Rosee-Borggreve, 1999).

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different 'strengths.' A combination of Bcd-site cluster analysis and evolutionary conservation has been used to predict Bcd-dependent CRMs. Tested were 14 predicted CRMs by in vivo reporter gene assays; 11 showed Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, no strong correlation is detected between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Krüppel, which may limit the posterior extent of activation by the Bcd gradient. It is proposed that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene (Ochoa-Espinosa, 2005).

In reporter gene assays, 11 of the 14 tested fragments directed expression patterns in wild-type embryos that recapitulate all or part of the endogenous patterns of the associated genes. These experiments identified several elements that control segmentation genes, including three new gap gene CRMs. Two CRMs were found in the genomic region that lies 5' of the gap gene gt. One CRM (gt23) is initially expressed in a broad anterior domain and then refines into two stripes. A second CRM (gt1) is expressed later in a small dorsal domain very near the anterior tip. Double stain experiments indicated that the timing and spatial regulation of both patterns are indistinguishable from the anterior expression domains of the endogenous gt gene. A CRM 3' of the gap gene tll was identified that drives expression similar to the anterior tll domain (Ochoa-Espinosa, 2005).

Four novel CRMs were identified near known pair rule genes. One CRM was detected in the 3' region of hairy and drives expression of a small anterior dorsal domain similar to the hairy 0 stripe of the endogenous gene. Another CRM is located 3' of the paired gene and directs expression of an early broad domain that coincides with the later position of the native paired stripes 1 and 2. Two more CRMs (slpA and slpB) were identified in the slp locus, which contains the two related genes, slp1 and slp2. Both slpA and slpB faithfully reproduce parts of the early slp1 and slp2 expression patterns (Ochoa-Espinosa, 2005).

Four other CRMs were identified near the genes bowl, CG9571, D/fsh, and bl/Mir7. In three cases (bowl, CG9571, and D/fsh), the newly identified CRMs direct patterns similar to their associated endogenous genes. The final CRM (bl/Mir7) is located in the sixth intron of the bl gene and directs a strong anterior domain of expression. However, the endogenous bl gene is expressed nearly ubiquitously , which makes it an unlikely target of regulation by this CRM. One potential target of this element is the microRNA gene (Mir7), which is located 7 kb downstream in the eighth intron of bl. Four of the CRMs reported here (gt1, gt23, slpA, and D/fsh) were also identified in a recent genome-wide search for new patterning elements based on clusters of combinations of different binding sites including Bcd. The fragments used in that study were significantly larger in size but show very similar patterns to those in this study (Ochoa-Espinosa, 2005).

Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene and the proneural genes achaete and scute. Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe) has been examined. These studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).

The D-h and V-h stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit. ac stripes are not expressed until 6 hours after puparium formation (APF). The flanking narrow D-h stripe is positioned a few cells anterior to the compartment boundary, allowing expression of two dorsal ac stripes in the anterior compartment. V-h, however, is expressed directly adjacent to the AP boundary so that there is only one ventral ac stripe in the anterior compartment. Expression of each h stripe in its proper register is essential for positioning of the ac stripes and consequently for sensory bristle patterning in the adult leg. Focus was placed on the mechanisms that lead to expression of the D-h stripe in its precise register near the AP boundary (Kwon, 2004).

Expression of the endogenous D-h stripe is dependent on Hh signaling. In order to identify sequences that mediate Hh responsiveness, a dissection was undertaken of the D-h enhancer. The D-h enhancer maps to a 3.4 kb BamHI/EcoRI fragment located 32 kb 3' to the h structural gene. In third instar leg imaginal discs, this fragment directs lacZ expression in a dorsally restricted AP boundary-adjacent stripe. Two subfragments of the D-h enhancer were tested for the ability to drive reporter gene expression in leg imaginal discs. A 3' 2.4 kb HindIII/EcoRI subfragment of the D-h enhancer (REPE) directs no detectable reporter gene expression in leg imaginal discs. However, the complementary 5' 1.0 kb BamHI/HindIII fragment of the D-h enhancer drives expression in a stripe that is not dorsally restricted but rather traverses the entire length of the DV axis, suggesting it responds to Hh signaling in both dorsal and ventral leg cells. To determine whether Hh signals through the BamHI/HindIII fragment of the D-h enhancer, expression from a BamHI/HindIII-GFP transgene was assayed in leg clones lacking function of Smoothened (Smo), a transmembrane protein required for transduction of the Hh signal. Somatic clones lacking smo function were generated by FLP/FRT-mediated mitotic recombination. Cell-autonomous loss of GFP expression is lost in smo clones that overlap the GFP stripe. These observations imply that Hh signals through the BamHI/HindIII fragment, and therefore, this region is referred to as the D-h-Hh response element (HHRE) (Kwon, 2004).

Since the HHRE is Hh responsive, the element for the consensus binding site of the Hh pathway transcriptional effector, Ci was sought. Two potential Ci-binding sites (Ci-1 and Ci-2) were found, each of which matches the consensus, TGGG(A/T)GGTC, in a minimum of seven out of nine sites and binds the Ci zinc-finger domain (CiZn) in a electrophoretic mobility shift assay (EMSA). To determine whether the Ci-binding sites are required for HHRE-directed expression, point mutations were introduced into the Ci-1 and 2 sites. These mutations abolish Ci binding of the HHRE in vitro. Expression directed by the HHRE with a mutation in either the Ci-1 or Ci-2 sites is drastically compromised, and there is no detectable expression from an HHRE-lacZ transgene with both Ci sites mutated. Taken together, these studies indicate that D-h expression is activated primarily by the HHRE, through which Ci acts as an essential and direct transcriptional activator (Kwon, 2004).

Endogenous D-h expression is compromised in somatic clones lacking function of Mad, the transcriptional effector of Dpp signaling, and D-h-lacZ expression is severely decreased in leg imaginal discs with reduced dpp function. Furthermore, D-h-lacZ expression is ventrally expanded in wingless (wg) mutant legs, which have strong ventral dpp expression. These findings indicate a requirement for Dpp, in addition to Hh signal, for D-h expression. The most parsimonious model to explain how h integrates positive input from the Hh and Dpp signals, is that Mad acts synergistically with Ci through the D-h enhancer to activate D-h expression. However, Dpp is instead required to block REPE-mediated repression (Kwon, 2004).

A question is raised regarding the identity of the repressor(s) that acts through the REPE to refine HHRE-directed expression. A potential candidate, the transcriptional repressor of Dpp target genes, Brk, is suggested by evidence indicating that Dpp is required to override REPE function. In the wing and leg imaginal discs, brk expression is repressed by and is roughly reciprocal to Dpp signaling. Hence, in the leg disc, brk expression is lowest in dorsal-most leg cells. D-h-GFP is expressed within the region of low-level brk expression in leg discs. Furthermore, brk expression expands dorsally in dppd6/dppd12 legs, in which D-h expression is severely reduced (Kwon, 2004).

To determine whether Brk functions as a repressor of D-h expression, D-h-GFP expression was examined in clones lacking brk function. Loss of brk function results in ectopic expression of D-h-GFP on either side of the D-h-GFP stripe. Ectopic expression is observed in clones anterior to the D-h-GFP stripe. However, the expansion is confined to a region two or three cells wide, directly juxtaposed to D-h expression, which presumably corresponds to the HHRE-responsive zone. In addition, ectopic expression is observed in ventral clones. Overexpression of brk along the AP boundary drastically reduces D-h-GFP expression but does not affect HHRE-GFP expression, indicating that Brk acts through the REPE to repress D-h expression. Since D-h expression is activated primarily by the Hh-responsive HHRE, these observations identify Brk as repressor of Hh as well as Dpp target genes (Kwon, 2004).

Genetic data support a hypothesis in which Brk acts through the REPE of the D-h enhancer to modulate activity of the HHRE. If so, it might be expected that the REPE would contain one or more functional Brk-binding sites. Hence, the REPE was examined for the Brk consensus binding site, GGCG(C/T)(C/T), and a potential Brk binding site was identified that overlaps two sequences similar to a consensus binding sites for Mad: GCCGNCGC, and a sequence similar to a cAMP response element (CRE), TGACGTCA. The sequence of overlapping CRE, Brk and Mad sites was designated the CMB element. Site directed mutational studies are consistent with the hypotheses that Brk acts through the CMB to repress D-h expression (Kwon, 2004).

Thus, the D-h activation element, HHRE, has two consensus Ci-binding sites, which bind Ci in vitro, and are required for its activity. In addition, HHRE-GFP expression is abrogated in clones lacking function of smo, a transducer of the Hh signal. These observations suggest that Ci acts directly through the HHRE to activate D-h expression. h is one of a number of genes, including dpp, patched (ptc), knot and araucan/caup (ara/caup), that have been identified as targets of Hh signaling in imaginal discs. These genes are each expressed in a stripe along the AP compartment boundary, but curiously, stripe widths among the genes varies as does register relative to the AP boundary. This has been explained in terms of differential response of Hh-target genes to the repressor and activator forms of Ci (Ci-R and Ci-A, respectively) found in anterior compartment cells. ptc, for example, has been proposed to respond only to the maximal levels of Ci-A found in cells nearest the AP boundary, while dpp responds to lower levels of Ci-A and also to Ci-R. The broad AP boundary stripe of HHRE-directed expression suggests that the HHRE is highly responsive to Ci-A. Differential response to Ci-R and Ci-A is thought to be controlled by cis-regulatory elements outside the local context (within 100 bp) of Ci binding sites in Hh responsive enhancers. Consistent with this hypothesis, an element, the REPE, has been identified that appears to modulate the response of the HHRE to Ci-A (Kwon, 2004).

Although Ci-A is an essential and important activator, which acts directly through the HHRE, it is unlikely that Ci-A function is sufficient for HHRE activity. Several studies have suggested that signal response elements in enhancers are generally not sufficient to activate gene expression. Rather, the transcriptional effectors of signals must act cooperatively with other activators to direct robust expression of target genes. This phenomenon, which has been termed 'activator insufficiency,' presumably prevents promiscuous activation of potential target genes. It is likely then, that other sites in the HHRE are required in addition to the Ci sites for expression directed by this element. For example, since the HHRE drives reporter gene expression in the wing and antennal discs as well as the leg, it might be expected that a common factor expressed in all three discs acts through the HHRE in combination with Ci. Alternatively, the enhancer might harbor sites that respond to factors specific to each disc type (Kwon, 2004).

A short sequence in the REPE, the CMB, has been identified that functions to restrict HHRE expression to a narrow dorsal domain. In this study, evidence is provided for the hypothesis that the transcriptional repressor Brk acts through the CMB to repress D-h expression. Although previous studies have shown that brk expression is very low or undetectable in cells near the Dpp source, a genetic requirement has been demonstrated for brk in repression of D-h in this region. In addition, overexpression of brk results in a dramatic reduction of D-h-GFP expression, but only mildly affects expression from a D-h-GFP transgene with a compromised Brk binding site (Kwon, 2004).

Dpp acts through the REPE to block Brk-mediated repression. It is proposed that high-level Dpp signaling defines the domain of D-h expression within the HHRE-response zone. This idea is supported by the observations that D-h-GFP but not HHRE-GFP expression is dependent on Dpp, indicating that Dpp signals through the REPE, and that elevation of Dpp signaling results in expansion of D-h expression along the AP and DV axes, within the domain of HHRE activity. Current studies suggest that the function of Dpp in regulation of D-h expression may be limited to repression of brk. Yet, the presence of Mad-binding sites in the CMB suggests a potentially more direct role for activated Mad (act-Mad), the transcriptional mediator of Dpp signaling. Brk has been shown to be a potent competitor of Mad in vitro for binding to overlapping binding sites in Dpp target enhancers. Hence, a potential role for Mad would be to prevent Brk from binding the CMB, thereby blocking Brk repression in cells receiving high-level Dpp signaling. If this model is correct, one might have expected the Mad1/Mad2 (MM) mutation to compromise D-h expression, which was not the case. However, the destabilization of Brk binding to the MM mutant might have masked a requirement for the Mad sites in blocking Brk repression (Kwon, 2004).

It has recently been shown that an act-Mad/Shn complex represses brk expression by binding a silencer element. Therefore, since mutation of the Mad sites expands D-h expression, it is possible that Mad acts in concert with Brk through the CMB to repress D-h expression. This notion is not inconsistent with genetic evidence, indicating a requirement for Mad in D-h expression, since loss of Mad function elevates Brk levels, which can overcome the requirement for CMB-sequences other than the Brk site. However, if this were the case, a more severe expansion phenotype might be expected with the MM mutant, in which both Brk and Mad binding are compromised. Further analysis is required to determine the role, if any, of the CMB-Mad-binding sites in D-h expression (Kwon, 2004).

Given the genetic evidence that Brk represses D-h expression and that Brk binds the CMB element in vitro, the most straightforward hypothesis is that Brk acts directly through the CMB in vivo to repress D-h expression. However, since mutation of the CRE also causes loss of repression, it is formally possible that the CRE rather than the Brk site is important for repression. A potential explanation for this observation is that mutation of the CRE lowers the affinity of this element for binding to Brk, even though the Brk binding site is intact in the CMB-C mutant. Because the levels of Brk in the dorsal leg are limiting, altered affinity could have a significant effect on the level of Brk occupancy of the CMB. However, through EMSA analysis it has been observed that the CRE mutant CMB binds Brk with an affinity greater than that of the wild-type element (Kwon, 2004).

Since it was not possible to mutate the Brk site without affecting the CRE, the CRE was altered in the BM2 and MBM mutants such that it more closely resembles a canonical CRE. Nevertheless, this change in the CRE may have affected its function. If so, this would be consistent with a model in which the CRE mediates repression of D-h expression, and Brk acts indirectly through the CRE rather than the Brk site. However, the finding that Brk overexpression drastically reduces D-h-C-GFP but not D-h-CB-GFP expression suggests that Brk can act directly through the Brk site, independent of the CRE (Kwon, 2004).

The requirement for CMB-sequences outside the Brk binding site suggests that the context of the Brk site within the CMB is important for repression. A plausible explanation for the requirement of the CRE is that it is bound by a factor, X, which functions to facilitate recruitment of Brk under conditions where Brk levels are limiting, such as in the dorsal leg. Consistent with this hypothesis is the observation that overexpression of Brk greatly reduces D-h-C-GFP expression, suggesting that the requirement for the CRE can be bypassed if the levels of Brk are high enough. However, when Brk levels are limiting, the CRE might contribute more to D-h repression than the Brk site. For example, in the dorsal leg, Factor X might bind the CMB and then form a complex with Brk, relieving the necessity for Brk to bind the CMB directly. This model could explain why D-h expression appears to be significantly more sensitive to Brk-mediated repression than other Brk targets in imaginal discs, such as vestigial (vg) and optomotor-blind (omb). vg and omb are each expressed in broad domains across the center of the wing disc and are repressed by higher levels of Brk than is D-h. Perhaps, the CRE and/or other sequences in the REPE mediate heightened response to Brk. It will be of interest to determine whether other Brk-target genes, such as spalt, which are also repressed by very low levels of Brk, are similarly regulated (Kwon, 2004).

A second potential function for a CRE-binding factor X is to act in concert with Brk to mediate D-h repression. Several lines of evidence suggest that Brk is a versatile repressor, which can inhibit transcription by competing with activators for binding to a common site or by active repression. Active repressors can act either at short range, by inhibiting activity of activators bound to nearby elements (150 bp away or less), or at long range by interfering with activators bound at a greater distances. Brk can mediate active repression, and binds the co-repressors dCtBP and Groucho (Gro), which mediate short- and long-range repression, respectively. Brk requires Gro and/or dCtBP function for repression of a subset of its target genes, whereas neither is required for repression of others. In the D-h enhancer, the CMB is positioned about 1 kb from the HHRE, suggesting that CMB-binding repressor(s) act at long range to repress HHRE-directed expression. Although Brk directly binds Gro, factor X could facilitate recruitment of Gro or other co-factors required for long-range repression (Kwon, 2004).

This study has identified a novel function for Brk as repressor of Hh-target gene expression. Brk was originally identified as a repressor of Dpp-target genes and a recent study indicates that Brk can block Wg-mediated transcription as well. Brk was shown to antagonize function of a Wg-responsive element in the midgut enhancer of the Ultrabithorax (Ubx). The Ubx midgut enhancer drives Ubx expression in parasegment (ps) 7 of the embryonic midgut. Two elements, one of which is Wg responsive (the WRS) and another Dpp responsive (the DRS), function synergistically to activate Ubx expression in ps 7 expression. In the adjacent ps8, however, Brk binds to the DRS and blocks the activity of the WRS. Curiously, the D-h-CMB and the Ubx-DRS are similarly organized in that each consists of overlapping CRE/Mad and Brk sites. The Ubx-DRS appears to mediate two modes of signal integration which involve: (1) synergistic activation, in which Mad/Med and dTCF act together to activate expression; and (2) activation and refinement, in which there is Wg mediated activation combined with Brk repression, which is blocked by Dpp. In the D-h enhancer, however, the CMB appears to be a component of a dedicated repression element, which appears to mediate only the second mode of signal integration: activation and refinement. The similar organization of the CMB and DRS suggests that it may be possible to predict the structure of enhancers known to be Brk responsive and which integrate Dpp and a second signal (Kwon, 2004).

Despite the similarities, there are important distinctions between the D-h and Ubx-midgut enhancers, suggesting that the mechanisms of Brk-mediated repression might differ in each case. In the Ubx-midgut enhancer, the DRS and WRS are separated by 10 bp, suggesting that Brk acts at short range to inhibit WRS activity. In the D-h enhancer, however, the CMB is positioned at least 1 kb from the HHRE, implying a long-range effect for this element. Furthermore, Brk repression of the WRS depends on Teashirt (Tsh), which binds Brk and acts as a co-repressor. Tsh is unlikely to be required for D-h repression because it is only expressed in proximal leg segments. The current studies suggest the requirement for a second DNA-bound factor, which binds the CRE, in addition to Brk for repression. The DRS-CRE, however, is required in addition to the Mad-binding sites for activation of Ubx in ps 7 (Kwon, 2004).

Together, these observations are consistent with a model in which Ci, acting through the HHRE, activates D-h expression. The domain of HHRE activity can be divided into two zones, 1 and 2. The HHRE has the potential to direct expression in both zones 1 and 2, but its activity is restricted to zone 1 by Brk and perhaps a second factor, X, which binds the CRE. In zone 2 cells, Brk would bind to the CMB and repress HHRE-directed expression. It is proposed that zone 1 is defined by the overlap of Hh and high-level Dpp signaling. Dpp promotes D-h expression by repressing brk expression in zone 1. However, the presence of Mad-binding sites in the CMB suggests the potential for a more direct role for Mad in D-h regulation, perhaps in competing with Brk for binding to the CMB, or in directly mediating repression. Confirmation of a role for the Mad sites awaits further analysis of the D-h enhancer (Kwon, 2004).

Establishment of D-h expression in a defined domain is a complex process. This may be explained in part by the observation that morphological elements such as leg bristle rows are remarkably invariant in position from one individual to the next in Drosophila melanogaster. Hence, precise expression of genes such as h, the function of which is so crucial for positioning of elements such as the leg sensory bristles, is essential. The organization of the D-h enhancer is reminiscent of that observed in another recently described enhancer, which is necessary for the development of a specific morphological element of an adult appendage. The knirps (kni) second longitudinal wing vein (L2) enhancer drives expression of kni, which is required to initiate L2 development, in a narrow stripe within the L2 primordia. As observed with D-h, localized expression of kn in the L2 primordia is established by an enhancer consisting of discrete activation and repression elements. The activation element directs broad expression, which is refined by the repression element. The structure of the kn repression element appears complex in that it is thought to bind a number of repressors, including perhaps Brk. Although the CMB element is important and essential for D-h repression, it is likely that there is a greater degree of complexity in the D-h-REPE, as well. Further analysis of the HHRE and REPE should provide mechanistic insight into how activation and repression elements function coordinately to establish precisely defined gene expression (Kwon, 2004).

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation.

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, a comprehensive systems-level examination was conducted of the regulatory architecture underlying pair-rule stripe formation. runt (run), fushi tarazu (ftz) and odd skipped (odd) were found to establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, long-range cis-regulation was observed across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, this study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo (Schroeder, 2011).

The transition from non-periodic to periodic gene expression patterns is a key step in the establishment of the segmented body plan of the Drosophila embryo. The pair-rule genes that lie at the heart of this process have been the subject of much investigation, but important questions, in particular regarding the organization of cis-regulation, have remained unresolved. We have revisited the issue in a comprehensive fashion by combining computational and experimental cis-dissection with mutant analysis and a detailed characterization of expression dynamics, both of endogenous genes and of cis-regulatory elements. This systems-level analysis under uniform conditions reveals important insights and gives rise to a refined and in some respects substantially revised view of how periodic patterns are generated (Schroeder, 2011).

The most important finding of this study is that ftz and odd, which had been regarded as secondary pair-rule genes, closely resemble eve, h and run in nearly all respects and should thus be co-classified with them as primary pair-rule genes. Expression dynamics clearly subdivide the pair-rule genes into two distinct groups, with eve, h, run, ftz and odd all showing an early and non-synchronous appearance of most stripes, whereas the 7-stripe patterns of prd and slp1 arise late and synchronously. The early expression of stripes is associated with the existence of stripe-specific cis-elements that respond to positional cues provided by the maternal and gap genes. Unlike previously thought, maternal and gap input is used pervasively in the initial patterning not only of eve, h and run, but also of ftz and odd. Aided by computational predictions, eight new stripe-specific cis-elements were identifed for ftz, odd, and run. Although molecular epistasis experiments reveal a stronger role for eve, h and run in pair-rule gene cross-regulation, odd clearly affects the blastoderm patterning of other primary factors as well and ftz affects the patterning of odd (Schroeder, 2011).

The revised grouping brings the classification of pair-rule genes in line with their role in transmitting positional information to the subsequent tiers in the segmentation hierarchy. By the end of cellularization, the expression patterns of h, eve, run and ftz/odd are offset against each other to produce neatly tiled overlaps of four-nuclei-wide stripes. In combination, these patterns define a unique expression code for each nucleus within the two-segment unit that specifies both position and polarity in the segment and is read off by the segment-polarity genes. ftz (as an activator of odd) and odd together represent the fourth essential component of this positional code and are thus functionally equivalent to h, eve and run. Placing all components of this code under the same direct control of the preceding tier of regulators presumably increases both the speed and the robustness of the process (Schroeder, 2011).

Although the results support a subdivision between primary and secondary pair-rule genes, they also reveal surprising complexities in the regulatory architecture that are not captured by any rigid dichotomy. The five primary pair-rule genes all have different cis-regulatory repertoires: stripe formation in h appears to rely solely on stripe-specific cis-elements, whereas eve employs a handoff from stripe-specific elements to a late-acting 7-stripe element. In run, ftz and odd, by contrast, the emergence of the full 7-stripe pattern is the result of joint action by stripe-specific elements and early-acting 7-stripe elements, with ftz and odd requiring the 7-stripe element to generate a subset of stripes. This difference in the importance of 7-stripe elements in stripe formation is supported by the results of rescue experiments: whereas the 7-stripe elements of both run and ftz provide partial rescue of the blastoderm expression pattern when the stripe-specific elements are missing, this is not the case for eve. Interestingly, run appears to occupy a unique and particularly crucial position among the five genes: it has both a full complement of stripe-specific elements and an early-acting 7-stripe element, and thus serves as an early integration point of maternal/gap input and pair-rule cross-regulation. Its regulatory region is particularly large and complex, with cis-elements acting over long distances across intervening genes and with partial redundancy between elements. Moreover, the removal of run has the most severe effects on pair-rule stripe positioning among the five genes. Another complexity lies in the fact that the early anterior expression of slp1 and prd, which otherwise exhibit all the characteristics of secondary pair-rule genes, has a clear role in patterning the anteriormost stripes of the primary pair-rule genes. In fact, evidence is provided that the most severe defect observed in the primary pair-rule gene mutants, namely the loss of pair-rule gene stripes 1 or 2 under eve loss-of-function conditions, is an indirect effect attributable to the regulation of these stripes by slp1 (Schroeder, 2011).

This investigation provides important new insight regarding the relative roles of maternal/gap input and pair-rule gene cross-regulation in stripe formation. As described, nearly all stripes of the primary pair-rule genes are initially formed through stripe-specific cis-elements. Cross-regulatory interactions between the pair-rule genes then refine these patterns by ensuring the proper spacing, width and intensity of stripes, as revealed by mutant analysis. However, these two aspects or layers of regulation are not as clearly separated as has often been thought. For example, 7-stripe elements have been regarded primarily as conduits of pair-rule cross-regulation; by contrast, this study found that the 7-stripe elements of run, ftz and odd are activefrom phase 1 onwards and contain significant input from the maternal and gap genes, resulting in modulated or partial 7-stripe expression early. In the case of the KR binding sites predicted within the run 7-stripe element, mutational analysis showed that this input is indeed functional. Conversely, stripe-specific cis-elements receive significant regulatory input from pair-rule genes. This is particularly clear in the case of eve and h: expression dynamics indicate that their 7-stripe patterns become properly resolved without the involvement of 7-stripe elements, and the defects in the h and eve expression patterns that were observe in pair-rule gene mutants occur at a time when only stripe-specific elements are active. In a few cases, pair-rule input into stripe-specific elements has been demonstrated directly, but the comparative analysis of expression dynamics underscores that it is indeed a pervasive feature of pair-rule cis-regulation (Schroeder, 2011).

An important question arising from these findings is how the different types of regulatory input are integrated, both at the level of individual cis-elements and at the level of the locus as a whole. Binding sites for maternal and gap factors typically form tight clusters, supporting a modular organization of cis-regulation. Such modularity is crucial for the 'regional' expression of individual stripes, which can be achieved only if repressive gap inputs can be properly separated between cis-elements. By contrast, binding sites for the pair-rule factors appear to be more dispersed across the cis-regulatory regions and not tightly co-clustered with the maternal and gap inputs. Unlike the gap factors, which are thought to act as short-range repressors, with a range of roughly 100 bp, most pair-rule genes act as long-range repressors, with an effective range of at least 2 kb. Pair-rule factor inputs may therefore influence expression outcomes at greater distances along the DNA, which would allow a single cluster of binding sites to affect multiple cis-elements. Remarkably, the 7-stripe elements of ftz, run, eve, prd and odd all combine inputs from promoter-proximal and more distal sequence over distances of at least 5 kb, and, is seen in the case of odd, the combination can involve non-additive effects. Thus, the refinement of expression patterns through pair-rule cross-regulation might rely on interactions over greater distances, consistent with the 'global' role of pair-rule genes as regulators of the entire 7-stripe pattern. How such interactions are realized at the molecular level and how the inputs from stripe-specific and 7-stripe elements are combined to produce a defined transcriptional outcome is currently unknown (Schroeder, 2011).

Taken together with previous studies, these data support a coherent and conceptually straightforward model of how stripes are made in theDrosophila blastoderm. In the trunk region of the embryo, the maternal activators BCD and CAD form two overlapping but anti-correlated gradients; they coarsely specify the expression domains of region-specific gap genes, which become refined by cross-repressive interactions among the gap factors. The result is a tiled array of overlapping gap factor gradients, centered around the bilaterally symmetric domains of KR and KNI, which are flanked on either side by the bimodal domains of GT and HB. The same basic principles are used again in the next step: following the initial positioning of stripes by maternal and gap factor input, cross-repressive interactions among the primary pair-rule genes serve to refine the pattern and ensure the uniform spacing and width of expression domains. The significant correlations that were observe between the strength of KR/KNI input and the position of the resulting stripes relative to the respective gradients supports the notion that these factors function as repressive morphogens in defining the proximal borders of the nascent pair-rule stripes. Importantly, owing to the symmetry of the KR and KNI gradients, combined with the largely symmetric positioning of the flanking GT and HB gradients, the same regulatory input can be used to specify two distinct positions, one on either slope of the gradient; this is exploited in a systematic fashion by dual-stripe cis-elements, which account for the majority of stripe-generating elements. Thus, the key to stripe formation is the translation of transcription factor gradients into an array of narrower, partially overlapping expression domains that are stabilized by cross-repressive interactions; the iteration of this process, combined with the duplication of position through bilateral gradient symmetry, creates a periodic array of stripes that is sufficient to impart a unique identity to each nucleus in the trunk region of the embryo (Schroeder, 2011).

With the exception of the anteriormost pair-rule gene stripes, which are subject to more complex regulation (as evidenced by the crucial role of slp1), this model accounts for most of the stripe formation process. Particularly striking is the similarity between the central gap factors and the primary pair-rule factors with respect to regulatory interactions and expression domain positions. The offset arrangement of two pairs of mutually exclusive gap domains, KNI/HB and KR/GT, is mirrored at the pair-rule gene level, with the anti-correlated and mutually repressive stripes of HAIRY and RUN phase-shifted against the similarly anti-correlated expression patterns of EVE and ODD. The parallel suggests that this regulatory geometry is particularly suited to robustly specify a multiplicity of positions. However, such a circuitry of cross-repressive relationships is compatible with a range of potentially stable expression states and thus is insufficient by itself to uniquely define position along the anterior-posterior axis. Therefore, the initial priming of position by the preceding tier of the regulatory hierarchy is crucial. At the level of the pair-rule genes, the extensive repertoire of maternal/gap-driven cis-elements that initiate stripe expression for all four components of the array is thus necessary to ensure that the cross-regulatory dynamics will drive the correct overall pattern. Finally, to achieve a smooth transition between the tasks of transmitting spatial information from the preceding tier of the hierarchy and refinement/stabilization of the pattern, the two layers of regulation need to be closely integrated. This is likely to be facilitated by the fact that in each of the mutually repressive pairs, one gene generates stripes purely through stripe-specific elements (h, eve), whereas the other has an early-acting 7-stripe element that mediates pair-rule cross-repression and acts concurrently with stripe-specific elements (run, odd) (Schroeder, 2011).

Stripe positioning is not always symmetric around the central gap factor gradients. In such cases, the conflicting needs for appropriate regulatory input into the relevant cis-elements appear to have driven the separation of ancestral dual-stripe elements into more specialized elements optimized for generating a single stripe. The co-localization of the cis-elements that generate stripes 1 and 5 within all pair-rule gene regulatory regions, be it in the form of dual-stripe elements or of adjacent but separable single-stripe elements, provides strong evidence that this is indeed a key mechanism underlying the emergence of single-stripe cis-elements. Given the evolutionary plasticity of regulatory sequence, it is not difficult to envision such a separation and subfunctionalization of cis-elements. How the crucial transition from a non-periodic to periodic pattern is achieved in other insects is a fascinating question. Intriguingly, most of the molecular players and general features of their expression patterns are well conserved beyond Diptera, and it is tempting to speculate that dual-stripe cis-regulation might have arisen through co-option as the blastoderm fate map shifted to include more posterior positions. It will be interesting to see to what extent the mechanisms and principles of stripe formation that we have outlined here apply in other species (Schroeder, 2011).

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated in this study, led to a hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. This study investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods deviations from the wild-type patterns of the anterior-most pair-rule stripes were detected in different genetic backgrounds, consistent with Hkb-mediated repression. Moreover, an image processing tool was developed that, for the most part, confirmed the assumptions. Using an hkb misexpression system, specific repression on anterior stripes was detected. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes (Andrioli, 2012).

The aim of this study was to understand the mechanisms underlying the regulation of the anterior pair-rule stripes. The model tested was first proposed for eve 2 regulation. Transcriptional activators do not give enough patterning information, and the presence of repressors is instructive for determining the precise positioning of a particular stripe. The hypothesis was that transcription repressors could be working in a combinatorial manner to determine the correct positioning of the anterior stripes and prevent, in a spatial and temporal manner, the expression of stripe CRMs in the more anterior regions of the embryo by counteracting the activity of activators. There is plenty of evidence supporting this hypothesis, which was further confirmed in this study (Andrioli, 2012).

Regarding activators, computational analysis predicted Bcd, Hb and Btd binding sites are part of significant clusters in the anterior-most stripe CRM. These predictions agree well with previous genetic data and in vivo DNA binding data from ChIP/chip experiments. Thus, Btd, and above all the widely spread maternal factors Bcd and Hb, might activate anterior stripe CRMs early in the anterior blastoderm. Alternatively, the early broad expression patterns of pair-rule genes could be under the control of dedicated CRMs, although no such elements have yet been reported. It is possible that other regulatory elements could contribute to the expression detected early in the anterior blastoderm, for instance, the CRM responsible for the expression of h head patch or the CRMs responsible for eve 3, eve 5 and h 5, which were proposed to be activated by the maternal factor DSTAT (Drosophila Signal Transducer and Activator of Transcription), which is ubiquitously expressed in the embryo (Andrioli, 2012).

The expression of several gap domains covering all of the anterior regions of the embryo ahead of the seven-striped patterns is consistent with the expected subsequent local repression of pair-rule CRMs activated in the head region. Of these gap domains, Slp1 is a common repressor for anterior pair-rule stripes, but other repressors besides Slp1 were predicted to be necessary for correctly determining the borders of the anterior-most stripes. This study investigated hkb, which, in addition to tll, is the other major gap gene target of the Torso signaling regulation in the terminal system. In the anterior region, hkb is required for the proper formation of the foregut and midgut. Its domain at the anterior tip coincides with the region where the diffused early expression patterns of pair-rule genes first fade. These observations are consistent with local repression roles of Hkb. However, it was not possible to detect derepression of pair-rule genes in the anterior pole of hkb- embryos. One possibility is that the progressive non-detection of the expression of pair-rule genes might correspond to a failure in activation. In fact, Bcd activation was shown to be down-regulated by the Torso-signaling cascade at the anterior tip. Nevertheless, other data suggest that the Torso pathway might induce a repression mechanism at the anterior tip that would be parallel and redundant with Torso-induced inhibition of Bcd. Thus, one might predict that another repressor might still able to act on Hkb targets in the absence of Hkb protein (Andrioli, 2012).

Although no pair-rule derepression was detected in the anterior pole, it was possible to detect subtle deviations in the positioning of eve 1 in hkb- embryos, which was confirmed by morphological measurements using the image processing tool. Enhanced derepression effects were also detected for all anterior-most stripes investigated in slp-;hkb- double-mutant embryos compared to the effects observed in slp- embryos; these results were statistically significant. With the hkb misexpression system, repression effects were detected for h 1, eve 1, run 1 and ftz 1. With the exception of gt repression, no other gap domain disruption was detected in these assays. These results strongly suggest direct repression by Hkb on the CRMs of these stripes. In vivo binding data confirms this possibility. Moreover, with the bioinformatics analysis it was verified that Hkb, along with putative activators, increased the already high significance values of predicted clusters for activators that match these stripe CRMs. Therefore, the combined data suggest that Hkb acts as a repressor for a specific group of anterior pair-rule stripes (Andrioli, 2012).

These data also suggest that there is another possible mechanism underlying the repression that involves the activity of repressors further away from their original sources. One example of this mechanism is expression detected for the ectopic hkb domain, demonstrating that target CRMs are sensitive to Hkb-mediated repression even in the presence of low expression levels of Hkb. The prediction is that low concentrations of Hkb that have diffused away from its endogenous domain could still repress these CRMs. For this mechanism, repressors could fulfill additive repression roles at different anterior subdomains or even contribute to the definition of the anterior borders of stripes that are distantly positioned from where gap domains are detected. Thus, the increased derepression observed in slp-;hkb- embryos would be expected if a combinatorial additive mechanism existed in which each repressor had a small contribution to the overall repression. Following the same rationale, one can predict that at least one other repressor is still responsible for setting anterior border stripes in slp-;hkb- embryos (Andrioli, 2012).

The complexity of the regulation of genes involved in early patterning was postulated to be a condition that is necessary for sensing relatively small differences in the concentrations and combinations of many regulatory factors, which is likely the environment found in the syncytial blastoderm. In agreement with that hypothesis, recent studies revealed that the protein gradients of factors such as Bcd and Dorsal alone are not sufficient to determine all of the spatial limits of target gene expression and that these gradients might combine with other factors to pattern the early embryo. In the head region, it has been suggested that Bcd and the terminal system-mediated activities interact at the level of the target CRMs to generate the proper patterning for the head region of the embryo. In contrast to these studies that focused on gap genes, the current data shed light on a mechanism that is involved in the regulation of the anterior stripe CRMs, with the putative participation of hkb (Andrioli, 2012).

The correct positioning of the anterior pair-rule stripes must be a critical issue in the early developmental patterning of the fly. Even a slightly incorrect positioning of the anterior stripes, for instance, results in the non-formation of the mandibular segment in the slp null mutant. Thus, a complex repression mechanism is necessary to shape the stripes and to avoid inappropriate expression of their CRMs. Therefore, Hkb, Slp1 and other repressors are likely involved in a combinatorial repressive activity in the CRMs of the anterior stripes. Other experiments are necessary to test this hypothesis further and to reveal the underlying molecular mechanisms involved in this regulation (Andrioli, 2012).

Transcriptional Regulation

Ectopic expression of the 69 kDa Tramtrack protein significantly represses even-skipped, odd-skipped, hairy and runt. The 88 kDa form does not similarly repress these genes (Read, 1993).

Transient over-expression of runt under the control of a Drosophila heat-shock promoter causes stripe-specific defects in the expression patterns of hairy and even-skipped. Expression of hairy stripes can be generated by a two-step mode involving regulatory interactions between so-called primary pair rule genes hairy and runt. Expression of H stripes 3 and 4 is directed by a common cis-acting element that results in an initial broad band of gene expression covering three stripe equivalents. Subsequently, this expression domain is split by repression in the forthcoming interstripe region, a process mediated by a separate cis-acting element that responds to runt activity (Hartmann, 1994 and Tsai, 1994).

Pair-rule gene expression is disrupted in Dichaete mutants. Expression of the gap genes Krüppel, knirps, and giant are normal, indicating that Dicaetae acts in parallel or downstream of these gap genes. the so-called primary pair-rule gene even-skipped, Hairy, and runt each show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Russell, 1996 and Nambu, 1996).

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to (1) the maternal concentration gradient of the anterior determinant Bicoid and (2) repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. The zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. To establish the potential of Cad and Kni to interact with the cis-acting DNA that mediates hairy stripe 6-like expression in the embryo, in vitro footprinting experiments were performed with the 532 bp hairy stripe 6-element DNA. Cad and Kni bind to thirty six in vitro binding sites, some of which overlap, throughout the element. The sequence of the Cad and Kni binding sites matches the consensus described for each of the two proteins. Most of the potential Cad and Kni binding sites are close to or overlapped by binding sites for Kruppel (eight sites), Hunchback (eight sites), and Tailless (five sites). Tests using fragments of the 532 bp enhancer and of another element, 284-HT, show that sequences mediating activation of reporter expression are not maintained within a minimal activation element but instead are dispersed throughout the enhancer (Hader, 1998).

The results suggest that activation and the expression level mediated by the hairy stripe 6-element depend on the number of activator binding sites; both activation and expression level are likely to involve additive rather than synergistic interactions. An identical transacting factor requirement is found for hairy stripe 6 and 7 expression. The arrangement of the corresponding binding sites for the common factors involved in the control of the two stripes share a high degree of similarity, but some of the factors exert opposite regulatory functions within the two enhancer elements (Hader, 1998).

The two opposing gradients of Bicoid and Cad provide a complementing transcriptional activator system along the entire axis of the preblastoderm embryo necessary for proper hairy stripe expression. The importance of Caudal as a posterior activator had been overlooked for some time. This is because Bicoid can partially compensate for the role of Cad as an activator of posterior gap genes. The activating role of Cad in the posterior region of the embryo, already suggested in the context of the fushi tarazu cis-acting control element, is substantiated by misexpression studies on hunchback, a regulator of zygotic caudal expression. The results of the misexpression studies imply that gene activation in response to Cad involves the combined action of maternal and zygotic caudal activities. These results indicate, however, that zygotic caudal activity cannot compensate for the lack of maternal caudal activity in the case of hairy. While Bcd is both necessary and sufficient for the activation of anterior genes and may require co-activations such as HB for establishing proper expression domains, maternal Cad seems to act by providing a basal level of activation on which other factors, such as Bcd, Kni or Kr, act to set the biologically relevant time and level of gene expression (Hader, 1998 and references).

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted (Saulier-Le Dréan, 1998).

Two of the seven pair-rule genes tested do not show significant changes in expression at the stages examined. These include the genes odd-paired (opa) and, surprisingly, ftz. In odd minus embryos, ftz stripes do not resolve properly, remaining about 3 cells wide until well into the process of germ band extension. This suggests that Odd may be a repressor of ftz. However ectopic Odd does not repress ftz expression. Also unexpected was the fact that ectopic Odd has effects on all three of the 'primary' pair-rule genes. These were previously thought not to be regulated by Odd. In stage 5 embryos, stripe 1 of hairy is efficiently repressed by ectopic Odd. The first stripe of eve is also repressed at this stage. Repression of h stripe 1 continues in older embryos and is accompanied by weaker repression of stripes 2-6. These effects of Odd on h correlate with what appears to be a modest broadening of h stripes in odd-minus embryos, particularly stripe 1. Early repression of the first stripes of h and eve likely accounts for the cuticular head defects that arise from early pulses of ectopic Odd expression. Interestingly, in odd-minus embryos, the entire 7-stripe pattern of h appears to expand, both anteriorly and posteriorly. This is also true of eve and runt stripes. These data provide no explanation for this, but it may explain the fairly consistent spacing of h stripes, despite their apparent broadening (Saulier-Le Dréan, 1998).

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

Hh, secreted by maturing photoreceptor cells, is normally responsible for inducing cells within and ahead of the morphogenetic furrow to initiate photoreceptor differentiation. Nevertheless, cells that lack Smoothened (Smo) function, and hence the ability to transduce Hh, can form normal ommatidia. These findings suggest that Hh can induce photoreceptor differentiation in Smo-deficient cells through the induction of other signaling molecules in neighboring wild-type tissue. As a first step toward identifying such secondary signals and analyzing their roles, the consequences of creating clones of cells homozygous for smo3, an amorphic mutation, have been examined on two early markers of retinal development, the expression of Ato and Hairy, which are expressed in adjacent dorso-ventral stripes within and anterior to the morphogenetic furrow. Ato expression has two prominent phases in the developing eye. In the first phase, Ato is expressed uniformly in a narrow dorso-ventral swath of cells that demarcates the anterior edge of the furrow. This uniform swath then breaks up into small clusters of Ato expressing cells and resolves into the second phase, a spaced pattern of single Ato expressing cells (the future R8 photoreceptor cells). The first phase of Ato expression is severely reduced or absent in clones of smo3 cells, similar to large clones that lack Hh. However, the second phase of expression still occurs, even though it is displaced posteriorly, indicating that it is delayed. This displacement is more severe in the middle of the clone than along the dorsal and ventral borders, producing a crescent shaped distortion of the line of spaced single cells that express Ato. It is concluded that cells within smo mutant clones can be induced to express Ato even though they cannot receive Hh, provided that they are located near to wild-type cells across the clone border. Equivalent effects have been observed for Hairy expression. Hairy is normally expressed at peak levels in a dorso-ventral stripe positioned immediately anterior to the Ato stripe, but is abruptly downregulated in more posteriorly situated cells. Clones of smo3 cells have only a modest effect on Hairy expression anterior to the furrow, causing a slight, but consistent, posterior displacement of the anterior edge of the stripe. However, they are associated with a pronounced failure to repress Hairy expression in some, but not all, posteriorly situated smo3 cells. As in the case of Ato expression, the exceptional mutant cells that retain the normal downregulation of Hairy are those positioned close to the lateral and posterior borders of the clones. Just within the lateral border, a line of cells is typically observed, one or two cell diameter lengths wide, where Hairy expression is repressed. Along the posterior border, the zone of mutant cells in which Hairy expression is repressed is usually wider (Greenwood, 1999).

These results are interpreted to indicate that (1) Hh normally induces cells to express a secondary signal (or signals) that can activate Ato expression and repress Hairy expression; (2) this signal acts non-autonomously, allowing it to move from wild-type cells where it is induced by Hh to nearby smo3 cells where it regulates Ato and Hairy expression; and (3) the range of this signal is short, restricting its action to only one or two cells across the lateral borders of smo3 mutant clones. A somewhat greater range of action is apparent along the posterior borders of such clones, perhaps because the adjacent wild-type cells were induced by Hh to send this signal at an earlier time than those along the lateral (more anterior) borders of the clone, allowing the signal more time to accumulate to higher levels and to move deeper into mutant tissue (Greenwood, 1999).

To examine how the posterior displacements in Ato and Hairy regulation in smo3 clones influence subsequent ommatidial development, the expression of the protein Elav, a marker of photoreceptor differentiation was examined. Clones of smo3 cells are capable of differentiating as photoreceptors, in agreement with previous findings. However, there is a significant delay. In wild-type tissue, Elav expression initiates immediately posterior to the morphogenetic furrow with the specification of the R8 cell and continues as other photoreceptors are recruited into the ommatidial cluster. In clones of smo3 cells, there is a clear posterior displacement in the onset of photoreceptor differentiation in mutant cells: photoreceptor differentiation is first seen at the posterior, and occasionally lateral, edges of the clone, correlating with the effects of neighboring wild-type tissue on Hairy and Ato expression and indicating a general delay in photoreceptor differentiation. However, as seen in more posteriorly situated clones, most or all of the smo3 tissue eventually differentiates as normally patterned ommatidia. Thus, Hh signal transduction is not autonomously required for presumptive eye cells to express Ato, downregulate Hairy, or differentiate as photoreceptors. This is in contrast to the general requirement for Hh signaling revealed by experiments in which Hh signaling is blocked throughout the entire disc using temperature-sensitive hh mutations. In the latter case, loss of Hh signaling causes a rapid and complete block in photoreceptor differentiation and furrow progression. Hh signaling appears to induce at least two secondary signals that are essential for the normal recruitment of undifferentiated cells to form the R8 photoreceptors. One of them appears to be the short-range signal that can induce Ato expression and repress Hairy in clones of smo minus cells. The second, Decapentaplegic (Dpp), appears to act at longer range to prime cells to receive this short range signal (Greenwood, 1999).

One candidate for a secondary signal, which acts downstream of Hh in the developing retina, is the TGF-beta homolog Dpp. Dpp is induced by Hh just anterior to the morphogenetic furrow. Moreover, experiments in other discs have established that Dpp can act at long range from its source to mediate the organizing activity of Hh on more anteriorly situated tissue. However, previous studies have shown that Dpp signaling is not essential for either photoreceptor differentiation or propagation of the furrow once photoreceptor differentiation initiates at the posterior edge of the eye primordium. These findings challenge the notion that Dpp mediates the organizing activity of Hh in front of the furrow. To test whether Dpp has such an organizing role, two kinds of experiments were performed. In the first, Dpp or activated Thickveins (Tkv), a type I TGFbeta receptor required for all known Dpp activities, was ectopically expressed anterior to the furrow. In the second, Dpp expression or Tkv activity was blocked. The results of these experiments indicate that Dpp signaling is both necessary and sufficient to upregulate Hairy expression anterior to the furrow and to maintain the normal rate of furrow progression, but that it is neither necessary nor sufficient to activate Ato expression and initiate photoreceptor differentiation in more posterior cells (Greenwood, 1999).

The dppblk mutation is associated with a deletion of cis-acting regulatory sequences that are essential for Hh-dependent transcription of dpp in the eye. As a result, Dpp signaling in the eye disc is abolished or severely reduced anterior to the furrow, and the resulting eye is greatly reduced in size in both the dorsal-ventral and antero-posterior axis. Hairy expression in wild-type and dppblk disks were compared, using the upregulation of Cubitus interruptis (Ci), a protein that is stabilized in response to Hh signaling, as a marker of the position at which the furrow should normally form. In wild-type eye discs, Ci accumulates to peak levels in a dorso-ventral stripe of cells just posterior to the stripe of peak Hairy expression, consistent with the finding that Hairy expression is repressed in response to Hh signaling within the furrow, but is activated by Dpp signaling anterior to the furrow. In contrast, the stripe of maximal Hairy expression is displaced posteriorly in dppblk discs relative to the stripe of maximal Ci expression. Moreover, the furrow appears to have moved only a small distance from the posterior edge of the presumptive eye primordium, even in eye discs from mature third instar larvae, consistent with the 'small eye' phenotype observed in the adult. These results indicate that Dpp signaling is normally required to activate high level Hairy expression in a stripe positioned just anterior to the furrow. They also indicate that Dpp signaling is necessary to sustain the normal rate of furrow progression. Finally, they suggest that Dpp signaling influences the response of cells to peak levels of Hh signal transduction: Hairy expression is downregulated in these cells in wild-type discs, but not in dppblk discs (Greenwood, 1999).

It is envisaged that pre-proneural cells are metastable, having a latent proneural capacity that is actively held in check by proneural repressors such as Hairy and Emc. How does activation of Raf precipitate the transition to the proneural state? Because the simultaneous loss of both Hairy and Emc activities causes an expansion of Ato expression similar to that resulting from the expression of activated Raf, it has been suggested that Raf activation may normally induce transition to the proneural state by blocking the expression or activity of these repressors. Consistent with this possibility, Hairy contains potential phosphorylation sites for MAPK, a kinase downstream of Raf in the signaling pathway. Daughterless expression is also upregulated in the furrow and is necessary to maintain Ato expression. Moreover, Daughterless, like Hairy, contains phosphorylation sites for MAPK, raising the possibility that Raf activity may directly potentiate proneural activators at the same time that it downregulates the activities of their repressors. Similar events may also occur in mammalian neural differentiation, as NGF-induced differentiation of the mammalian neuronal cell line PC12 is mediated by the phosphorylation of HES-1, a Hairy related protein (Greenwood, 1999).

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Frizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression, and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during Drosophila development is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch (Wesley, 1999).

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001).

In mitotic clones of the Notch null allele N54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated (Baonza, 2001).

Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a ‘pre-proneural’ state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001).

Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001).

Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001).

It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001).

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax (hth), and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).

Anterior to the MF, at least three cell types can be distinguished by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal imaginal disc, expresses Hth, but not Tsh or Ey. In a slightly more posterior domain, all three of these factors are coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).

The definition of the PPN domain stems from the observation that the induction of neural cell fates in the eye disc requires at least two signals downstream of Hh. The first signal is Dpp, which creates a zone of cells ahead of the MF, termed the PPN domain, which is competent to receive a second, proneural-inducing signal. Cells in the PPN domain express high levels of hairy. Only cells that receive the Dpp signal are able to respond to the second, shorter-acting signal. This second signal is Dl, which is expressed by cells in and behind the furrow and is required for the down-regulation of hairy. In addition to Dl, neural induction, in particular the initiation of ato expression, may also require another signal that is transduced by the ser/thr kinase raf (Bessa, 2002).

hth has been linked to the PPN domain in three ways: (1) in wild-type eye discs, hth expression abuts hairy expression; (2) Hth represses hairy -- these data suggest that hth defines the anterior limit of hairy expression (3) Dpp is a repressor of hth. Together, these results suggest that the anterior limit of the PPN domain is defined by hth expression, and that, as the MF moves anteriorly, hth is repressed by Dpp, allowing the PPN domain and hairy expression to shift anteriorly. In these experiments, only some anterior hth- clones de-repressed hairy. This result is interpreted as suggesting that hairy expression is both activated by Dpp and repressed by hth. Consequently, hth- cells that do not receive enough Dpp would still be unable to express hairy (Bessa, 2002).

In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).

Hairy is downregulated redundantly by Hh and N signaling. Prolonged Hairy expression is not sufficient to block differentiation completely but it does antagonize it (e.g., in Su(H) ci clones). Downregulation of Hairy in response to Hh as well as N explains why both ci and Su(H) mutant clones can differentiate promptly, and why N enhances differentiation in response to Dpp but is not required for differentiation in response to Hh (Fu, 2003).

Targets of Activity

hairy, even-skipped and runt, the so called primary pair-rule genes, are involved in the refinement rather than establishment of fushi tarazu stripes. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy stripes, as would be expected if ftz stripes were generated by H repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, even-skipped or runt, with normal expression patterns decaying only after cellularization, in the absence of primary pair-rule genes (Yu, 1995). This work calls into question the distinction between primary and secondary pair-rule genes.

Runt and Hairy are required for the proper transcriptional regulation of fushi tarazu during the blastoderm stage of Drosophila embryogenesis. Runt and Hairy act on ftz through a common 32 base-pair element, designated fDE1. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by Runt and repression by Hairy. Examination of reporter genes with mutated fDE1 elements provides further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicate that the opposing effects of Runt and Hairy are not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to Runt and Hairy (Tsai. 1995).

Pair-rule genes even-skipped, runt and hairy, activate paired expression in stripes. With the exception of stripe 1, which is activated by even-skipped, and stripe 8, which depends upon runt, the primary pair-rule proteins are required for subsequent modulation rather than activation of the paired stripes (Gutjahr, 1993).

Hairy acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. achaete is a direct downstream target of H regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity H binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by H plays an essential role in pattern formation in the PNS (Van Doren, 1994).

Hairy binds to DNA and has novel DNA-binding activity. Hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete proneural gene blocks Hairy-mediated repression of ac transcription in cultured Drosophila cells. Moreover, the same CACGCG mutation in an ac minigene creates ectopic sensory hair organs like those seen in hairy mutants. Together these results indicate that Hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene (Ohsako, 1994) .

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. Although Hairy's embryonic patterning activity requires an intact basic (DNA binding) domain, none of the sequences in fushi tarazu promoter implicated in ftz repression by Hairy contain Hairy consensus binding sites. It is uncertain whether Runt acts primarily as a gene repressor or activator, as it behaves as a repressor of even-skipped and as an activator of fushi tarazu. 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 and the effects of such substitution were examined on presumed targets of Hairy and Runt. Expression of Hairy-Act during the blastoderm stage disrupts embryonic segmentation by driving ectopic expression of ftz, runt and odd-skipped. Activation depends on an intact basic domain, indicating that direct regulation occurs via sequence-specific binding to DNA. Expression of Runt-Act during the blastoderm stage likewise drives ectopic even-skipped, and shows that the normal apparent activation of fushi-tarazu by Runt is indirect, suggesting that Runt acts predominantly as a repressor. Hairy-Act has also been 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).

DPTP61F is a non-receptor protein tyrosine phosphatase that is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein which are targeted to different subcellular locations. The two transcripts differ in the C-termini. There is an alternate splice site in exon 7, which is spliced to exon 8 to generate the transcript encoding DPTP61Fn. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. The spatial and temporal expression of the two alternative transcripts of dptp61F has been examined during Drosophila embryogenesis. The two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression along the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation along the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes. In hairy mutants, the complement of DPTP62Fm stripes is reduced by half, to approximately eight wide stripes. It is presumed that odd numbered stripes have been deleted. Within embryos homozygous for a strong eve allele, odd stripes are absent except for stripe 1. In odd paired mutants every even stripe is decreased. In paired mutants odd numbered domains of expression are shifted anteriorly towards the even numbered domains. wingless, hedgehog, naked and patched are involved in refining the pattern of mRNA accumulation within each parasegment (Ursuliak, 1997).

Hairy transcriptional repression targets and cofactor recruitment in Drosophila

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. The chromatin profiling method DamID was used to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. DamID profiling was adapted such that tightly gated collections of embryos (2-6 h) could be used, and 20 Hairy targets related to early embryogenesis were found. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. Fifty-nine putative transcriptional targets of Hairy were identified. In addition to finding putative targets for Hairy in segmentation, groups of targets were found suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development (Bianchi-Frias, 2004).

The 59 putative Hairy targets identified correspond to bands of Hairy immunostaining on polytene chromosomes, suggesting that the polytene chromosome staining faithfully represents Hairy binding. Polytene chromosomes are functionally similar in transcriptional activity and display factor/cofactor binding properties similar to chromatin of diploid interphase cells, despite their DNA endoreplication (Bianchi-Frias, 2004).

Since the microarray chips used contained roughly half of Drosophila cDNAs, the actual number of Hairy targets was estimaed to be approximately twice that number (i.e., 118 targets). This predicted number of Hairy targets is close to the approximately 120 strongly staining sites observed on polytene chromosomes. Of the 59 putative Hairy targets identified in both the Kc cell and embryo DamID experiments, 58 correspond to bands of Hairy staining on the polytene chromosomes, suggesting that polytene chromosome staining is representing Hairy binding sites without regard to tissue specificity. It is not yet clear what is limiting Hairy accessibility in different tissues or why Hairy's access does not appear to be limited in salivary glands. It may be that polytene chromosome organization necessitates a looser chromatin structure or that the large number of factors that seem to be endogenously expressed in salivary glands affects accessibility. Ultimately, additional confirmation of the DamID and polytene staining correspondence will require microarray tiling chips containing overlapping genomic DNA fragments; however, such genomic DNA tiling chips are currently unavailable (Bianchi-Frias, 2004).

DNA methylation by tethered Dam has been shown to spread up to a few kilobases from the point where it is brought to the DNA. It was of concern in the beginning that Hairy targets might be missed if the DNA fragments of 2.5 kb or less that were recovered for probes were far away from the start of the transcribed region, especially since the Drosophila microarray chip used was generated using full-length cDNAs. Indeed, Hairy has been described as a long-range repressor; it is likely to bind at a distance from the transcription start site. However, the targets identified by DamID in both Kc cells and in embryos correspond closely to the Hairy staining pattern on polytene chromosomes. As is the case for Hairy, the distribution of DamID-identified loci that recruit the long-range repression-mediating Groucho corepressor corresponds well with the distribution of Groucho binding sites on polytene chromosomes. These results suggest that there is a higher-order structure to the promoter that is allowing factors that bind far upstream of the transcription start site to have physical access to the transcribed region (i.e., DNA looping) or that Hairy does not bind as far away from the transcription start site as it had been proposed to do (Bianchi-Frias, 2004).

Hairy is needed at multiple times during development, where it has primarily been associated with the regulation of cell fate decisions. During embryonic segmentation, ftz has long been thought to be a direct Hairy target. However, the order of appearance of ftz stripes is not inversely correlated with those of Hairy, as would be expected if ftz stripes are generated by Hairy repression. While it was not possible to assess ftz as a direct Hairy target using DamID, no evidence was found for ftz being a direct Hairy target based on the association of Hairy with polytene chromosomes. Indeed, the evidence suggesting that ftz is a direct target of Hairy is based on timing, i.e., that there is not enough time for another factor to be involved. Since the half-life of the pair-rule gene products is very short (less than 5 min), it is possible that additional factors could be acting and that the interaction between Hairy and ftz is indirect (Bianchi-Frias, 2004).

Interestingly, one of the Hairy targets identified in embryos is the homeobox-containing transcriptional regulator, prd. Pair-rule genes have been split into two groups: primary pair-rule genes mediate the transition from nonperiodic to reiterated patterns via positional cues received directly from the gap genes, whereas secondary pair-rule genes take their patterning cues from the primary pair-rule genes and in turn regulate the segment polarity and homeotic gene expression. The transcriptional regulator prd was originally categorized as a secondary pair-rule gene since its expression is affected by mutations in all other known pair-rule genes. However, prd stripes were subsequently shown to require gap gene products for their establishment, and the prd locus has the modular promoter structure associated with primary pair-rule genes. Thus prd has properties of both primary and secondary pair-rule genes and is a good candidate to directly mediate Hairy's effects on segmentation. Hairy can specifically bind to C-box sequences in the prd promoter and interacts genetically with prd. Further experiments will be required to determine if Paired in turn binds to the ftz promoter, such that the order of regulation would be Hairy > prd > ftz (Bianchi-Frias, 2004).

In addition to identifying potential targets for Hairy in segmentation, targets were identified that implicate Hairy in other processes including cell cycle, cell growth, and morphogenesis. The group of targets implicating Hairy in the regulation of morphogenesis includes: concertina, a G-alpha protein involved in regulating cell shape changes during gastrulation; kayak, the Drosophila Fos homolog involved in morphogenetic processes such as follicle cell migration, dorsal closure, and wound healing; pointed and mae, both of which function in the ras signaling pathway to control aspects of epithelial morphogenesis; egh, a novel, putative secreted or transmembrane protein proposed to play a role in epithelial morphogenesis, and Mipp1, a phosphatase required for proper tracheal development (Bianchi-Frias, 2004).

Hairy has been thought to be involved mostly in the regulation of cell fate decisions. However, mosaic experiments in the eye imaginal disc have suggested that Hairy may also play a role in the regulation of cell cycle or cell growth. Consistent with this, another group of Hairy targets implicates Hairy in the regulation of cell cycle or cell growth; this group includes stg, the Drosophila Cdc25 homolog; dacapo, a cyclin-dependent kinase inhibitor related to mammalian p27kip1/p21waf1; IDGF2, a member of a newly identified family of growth-promoting glycoproteins, and ImpL2, a steroid-responsive gene of the secreted immunoglobulin superfamily that functions as a negative regulator of insulin signaling. Consistent with a role for Hairy in growth signaling, mammalian HES family proteins have been linked to insulin signaling (Bianchi-Frias, 2004).

Since cells that are dividing or proliferating cannot simultaneously undergo the cell shape changes and cell migrations required for morphogenetic movements, Hairy may be required to transiently pause the cell cycle in a spatially and temporally defined manner, thereby allowing the cell fate decisions regulated by the transcription cascade to be completed. Since Hairy is itself spatially and temporally expressed, Hairy must be only one of several genes necessary to orchestrate these processes. While much progress has been made in understanding the regulatory networks governing pattern formation, cell proliferation, and morphogenesis, and while it is clear that they must be integrated, the details surrounding their coordination have not yet been elucidated. Thus, the putative Hairy targets identified are consistent with known processes involving Hairy and suggest that in addition to regulating pattern formation, Hairy plays a role in transiently repressing other events, perhaps in order to coordinate cell cycle events with the segmentation cascade. Further experiments will be needed to determine how these different roles for Hairy fit together (Bianchi-Frias, 2004).

The numbers of loci that recruit Groucho, dCtBP, and dSir2 cofactors are consistent with the breadth of interaction they have been shown to exhibit. One hundred and fifth-five loci were identified that recruit Groucho and, as expected, roughly twice as many sites were found on polytene chromosomes. Although Groucho was the first Hairy cofactor identified and its interaction site is often described as Hairy's 'major' repression motif, Groucho is associated with only a minority of Hairy targets in Kc cells. Groucho's dominance as a cofactor during segmentation may reflect a preference for Groucho in the reporter assays used previously to assess corepressor activity, or it may be more heavily recruited to Hairy's targets during segmentation. In the future it will be interesting to determine the loci that recruit Groucho in early embryos and, because Groucho binds a number of other repressors, which, if any, of these factors recruits Groucho as its major cofactor (Bianchi-Frias, 2004).

CtBP was identified as a repressive co-factor, first on the basis of its binding to the C-terminal region of E1A, and in Drosophila by its association with the developmental repressors Hairy and Knirps. CtBP is an integral component in a variety of multiprotein transcriptional complexes. It has been shown to function as a context-dependent cofactor, having both positive and negative effects on transcriptional repression depending upon the repressor to which it is recruited. More than 40 different repressors have been shown to recruit CtBP. Consistent with this wide recruitment of CtBP, 496 loci that recruit dCtBP were found by DamID profiling and roughly twice that many sites on polytene chromosomes. A global protein-protein interaction study has shown that the binding partners for Groucho and dCtBP are largely nonoverlapping. This, along with the near exclusivity of Groucho and dCtBP binding as assayed by DamID and polytene chromosome staining, makes it unlikely that both cofactors work together as a general rule and strengthens the possibility that the binding of each of these factors assembles different protein complexes that are, for the most part, mutually exclusive (Bianchi-Frias, 2004).

dSir2 was only very recently identified as a corepressor for Hairy and other HES family members. 107 loci were identified by DamID profiling that recruit dSir2 and roughly twice that many sites on polytene chromosomes. Surprisingly, the distribution of loci recruiting dSir2 identified by DamID profiling, as well as dSir2's staining on polytene chromosomes, shows regional binding specificity. This binding specificity may be a reflection of the different nuclear compartments in which these regions of the chromosomes are found. Sir2 has been described mostly as a protein involved in heterochromatic silencing rather than in euchromatic repression. The number of dSir2 euchromatic sites observed is similar to that of Groucho, suggesting that euchromatic repressors (in addition to HES family members) are likely to recruit Sir2. Consistent with this, a recent report has described a role for mammalian Sir2 in repressing the muscle cell differentiation program. The region-specific binding of dSir2 might reflect a difference in the types of factors it can associate with, or the association of dSir2 with particular chromosomal regions or nuclear domains (Bianchi-Frias, 2004).

Interestingly, dCtBP and dSir2 recruitment are largely overlapping, and this association continues outside of those loci where Hairy binds: 90% of dSir2-recruiting loci also recruit dCtBP. dCtBP and dSir2 are unique among transcriptional coregulators in that they both encode NAD+-dependent enzymatic activities. As NAD and NADH levels within the cell exist in closely regulated equilibrium, it is possible that dCtBP and dSir2 function as NAD/NADH redox sensors. In this way, the cell could use coenzyme metabolites to coordinate the transcriptional activity of differentiation-specific genes with the cellular redox state (Bianchi-Frias, 2004).

Delta and Hairy establish a periodic prepattern that positions sensory bristles in Drosophila legs

In vertebrates and invertebrates, spatially defined proneural gene expression is an early and essential event in neuronal patterning. In this study, the mechanisms involved in establishing proneural gene expression were investigated in the primordia of a group of small mechanosensory bristles (microchaetae), which on the legs of the Drosophila adult are arranged in a series of longitudinal rows along the leg circumference. In prepupal legs, the proneural gene achaete (ac) is expressed in longitudinal stripes, which comprise the leg microchaete primordia. Periodic ac expression is partially established by the prepattern gene, hairy, which represses ac expression in four of eight interstripe domains. This study identifies Delta (Dl), which encodes a Notch (N) ligand, as a second leg prepattern gene. Hairy and Dl function concertedly and nonredundantly to define periodic ac expression. The regulation of periodic hairy expression was explored. In prior studies, it was found that expression of two hairy stripes along the D/V axis is induced in response to the Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) morphogens. This study shows that expression of two other hairy stripes along the orthogonal A/P axis is established through a distinct mechanism which involves uniform activation combined with repressive influences from Dpp and Wg. These findings allow formulation of a general model for generation of periodic pattern in the adult leg. This process involves broad and late activation of ac expression combined with refinement in response to a prepattern of repression, established by Hairy and Dl, which unfolds progressively during larval and early prepupal stages (Joshi, 2006).

Patterning of the leg imaginal disc along its circumference axis is controlled by the Hh, Dpp and Wg morphogens. This study sought to elucidate the molecular mechanisms through which these signals give rise to specific morphological features of the leg, the mechanosensory microchaetae. Patterning of leg mechanosensory microchaetae was shown to requires spatially defined expression of the proneural gene ac and its repressor Hairy. Expression of hairy in two pairs of longitudinal stripes, the D/V-hairy and A/P-hairy stripes, is directed by separate enhancers that are Hh-, Dpp- and Wg-responsive. The D/V-hairy and A/P-hairy stripes are differentially regulated by Dpp and Wg and distinct mechanisms are utilized to control hairy expression along the A/P and D/V axes. D/V-hairy expression is locally induced near the A/P compartment boundary by Hh signaling. In addition, Dpp and Wg positively influence expression of the dorsal and ventral components of the D/V-hairy stripes, respectively, by acting together with Hh to define the register of these stripes relative to the compartment boundary. In contrast, the A/P-hairy stripes, which are expressed orthogonal to the D/V-hairy stripes and A/P compartment boundary, are not activated via local induction. Rather, it appears that they are broadly activated along the leg circumference and repressed by Dpp dorsally and Wg ventrally to define their dorsal and ventral boundaries. This model for A/P-hairy regulation is supported by the observations that hairy is ectopically expressed in dorsal, but not ventral, clones lacking tkv or Mad function and that A/P-hairy expression is compromised by elevation of Dpp signaling. Furthermore, ventral, but not dorsal, clones lacking dsh function also ectopically express hairy and high-level Wg signaling results in loss of A/P-hairy expression (Joshi, 2006).

A potential caveat to this model for regulation of A/P-hairy expression is that conclusions were drawn from analysis of endogenous hairy expression rather than by examining expression directed by isolated A/P-hairy enhancer(s). Hence, it is possible that the ectopic hairy expression seen in tkv, Mad and dsh mutant clones is a result of expansion of D/V-hairy rather than A/P-hairy expression. However, several lines of evidence argue against this interpretation. First, through genetic and molecular analyses of D/V-hairy enhancer function, it has been demonstrated that Dpp and Wg positively regulate D/V-hairy expression, an observation that is inconsistent with the suggestion that D/V-hairy is ectopically expressed in clones unable to respond to Dpp or Wg signaling. Furthermore, in 3rd instar and early prepupal leg discs, stages at which the A/P-hairy stripes are not expressed, ectopic hairy expression is not observed in tkv mutant clones. Second, it was found that the D/V-hairy stripes can only be expressed in anterior compartment cells near the A/P boundary, which are the cells that receive and respond to Hh signal. Thus, it is unlikely that ectopic hairy expression observed in clones at distance from the compartment boundary, which receive little or no Hh signal, and in the posterior compartment, in which cells do not respond to Hh signal, corresponds to D/V-hairy expression. Finally, it was found that elevation of Dpp or Wg signaling specifically disrupts A/P-hairy but not D/V-hairy expression. Taken together, these findings are consistent with the conclusion that A/P-hairy rather than D/V-hairy is expressed in clones compromised in their response to Dpp and Wg signaling (Joshi, 2006).

This study identified Dl as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence are are presented that support this conclusion. First, it was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. Second, it is shown that ac expression is expanded in legs with reduced Dl function. Third, it was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. Finally, activation of N signaling was observed within the hairy-OFF interstripes (ac interstripes that do not express hairy), in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields (Joshi and Orenic, unpublished cited in Joshi, 2006). By generating rescue and reporter constructs, an enhancer has been identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization of the cis-regulatory elements that control expression of ac stripes in different regions of the leg is observed. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).

hairy and Dl function to repress ac expression in complementary domains. hairy encodes a transcriptional repressor which has been previously shown to directly repress ac expression in the wing by binding a specific site in the ac promoter. It is likely that Hairy acts through a similar site to repress ac expression in the leg. Dl represses ac expression via a different mechanism: presumably, cells of the microchaete proneural fields, which express high levels of Dl, signal to adjacent cells to activate N. This suggestion is supported by the observation that expression of two N-responsive reporters is specifically activated in cells corresponding to the hairy-OFF interstripes. One of the reporters used in this study, E(spl)mβ-CD2, and other similar reporters recapitulate endogenous E(spl)mβ-CD2 expression in wing and leg imaginal discs. E(spl)mβ is one of seven genes in the E(spl)-C that encode bHLH repressors related to Hairy. Hence, it appears that ac expression in the leg microchaete proneural fields may be established by a prepattern of periodically expressed bHLH repressors (Joshi, 2006).

N signaling is not activated within ac-expressing cells, even though these cells express high levels of Dl. This could be explained by a dominant-negative effect of Notch ligands on N signaling, which has been previously observed in the wing. In the wing, it has been shown that N signaling is not activated within cells expressing high levels of Dl and Ser but, rather, that these cells signal to adjacent cells to activate N signaling within the wing margin. Consistent with the hypothesis of a potential dominant-negative function for Dl in the leg microchaete proneural fields is the observation that over-expression of Dl along the leg circumference results in expansion of ac expression into the hairy-OFF interstripes, which would be expected if N signaling was disabled. Over-expression of N ligand expression has been shown to exert a similar effect in other tissues (Joshi, 2006).

A curious observation of this study is that, as suggested by genetic evidence and the expression of two N-responsive reporters, N signaling, with one exception, is not activated within the hairy-ON interstripes, even though each Hairy stripe is straddled on either side by a Dl stripe. This suggests either that Dl signals asymmetrically or that there is an asymmetric response to N signaling and raises questions regarding the underlying mechanism of asymmetric activation of N-target gene expression. A potential mechanism for asymmetric signaling by Dl is suggested by studies in the notum, in which it has been shown that the N receptor is distributed in a pattern complementary to Dl. If N levels were higher within the hairy-OFF vs. the hairy-ON interstripes in the leg, this could allow for preferential signaling within these domains. However, N expression was assayed in prepupal legs and it was found that N appears to be uniformly distributed along the leg circumference. Hence, either there is an asymmetric response to N or alternative mechanisms are responsible for establishing the directionality of Dl signaling in the leg, such as post-translational modification N signaling pathway components. For example, glycosylation of N by the Fringe glycosyltransferase influences its interactions with its ligands (Joshi, 2006).

Another intriguing finding is the overlap of N signaling with the V-Hairy stripe. This result was surprising because it would suggest redundancy between hairy and Dl/N signaling in this region. However, an absolute requirement was observed for hairy function in the ventral leg. An explanation for this puzzling finding is suggested by the specific loss of the V-Gbe+Su(H)m8-lacZ stripe in hairy mutant legs, which indicates that Dl/N signaling or responsiveness in the ventral leg is dependent on hairy function. The specific loss of N signaling in the ventral leg could be a result of the expansion of Dl expression in hairy mutant legs, which as explained earlier might have a dominant-negative effect on N signaling. This proposal is corroborated by the expansion of ac expression along the circumference of legs ectopically expressing Dl throughout the tarsus. The overlap of hairy and Dl/N signaling in the ventral leg raises questions regarding the function of Dl/N signaling in this domain. It was observed that V-hairy and Gbe+Su(H)m8-lacZ expression overlap only partially, suggesting that combined function of Dl and Hairy in the ventral leg could serve to establish a broader domain of repression in this region in comparison to other interstripe domains. This idea is supported by the morphology of the adult leg tarsus in which the spacing of bristles is most pronounced along the ventral midline. However, the function of N in the ventral leg is not as yet clear. It is plausible that there is a role for Dl/N signaling in the ventral leg that is unrelated to regulation of ac expression (Joshi, 2006).

The potential function of Dl as a regulator of proneural ac expression in the leg was suggested by studies in the notum, on which mechanosensory microchaetae are also organized in longitudinal rows. In the notum, Dl/Notch signaling, rather than Hairy, regulates periodic ac expression. The current studies suggest a distinct mechanism for leg microchaete patterning in which Hairy and Dl act together and nonredundantly to define periodic ac expression. In both the leg and notum, Dl signals to adjacent cells to repress ac expression. However, whereas in the notum Dl activates N signaling in cells on either side of each Dl/Ac stripe, in the leg, N signaling is activated (with one exception) only within the hairy-OFF interstripes. Although the pattern of mechanosensory bristles on the leg and notum is overtly similar, the bristle rows are more precisely aligned in the leg. The more organized pattern on the leg may be a consequence of the combined function of Hairy and Dl which might more precisely define the domains of proneural gene expression (Joshi, 2006).

Dl function is essential for proper patterning of ac expression and it is suggested that accurate positioning of the Dl stripes is necessary for activation of Notch signaling within appropriate domains. Hence, regulation of Dl expression is an important aspect of leg microchaete patterning. In legs lacking hairy function, Dl expression expands into four broad domains and ectopic hairy expression greatly reduces Dl expression, indicating that periodic expression of Dl is regulated in part by hairy. Concomitant with the expansion of Dl expression, there is loss of N signaling in the ventral leg, suggesting that hairy functions to create an apposition of cells expressing high levels of Dl to cells expressing low levels of Dl, which allows for activation of N signaling in the ventral leg. Regulation of Dl expression in proneural fields is not understood. A plausible hypothesis is that, like hairy, Dl expression is established in response to the morphogens that control pattern formation during leg development (Joshi, 2006).

This and previous studies suggest an outline of general genetic pathway for the regulation of ac expression in the leg microchaete proneural fields. This process involves broad and late activation, by an unknown factor, of ac expression along the leg circumference combined with refinement in response to a prepattern of repressors, which is established during larval and early prepupal stages. Hairy and Dl have been identified as the primary prepattern factors that regulate ac expression along the leg circumference. Position-specific expression of both hairy and Dl in longitudinal stripes is essential for proper ac expression. The longitudinal stripes of hairy are established in direct response to the Hh, Dpp and Wg signals, which globally pattern the leg, indicating that hairy acts as an interface between ac and these morphogens. Dl expression is regulated by Hairy, but its regulation is otherwise poorly understood. In addition to elucidating a pathway for establishment of periodic ac expression during leg development, these studies also provide insight into the mechanisms through which morphogens function to generate leg morphology (Joshi, 2006).

Periodic ac expression is established progressively. The first evidence of periodicity is expression of the longitudinal stripes of hairy expression. The D/V-hairy stripes are expressed first in the early 3rd instar leg disc followed by the A/P-stripes between 3 and 4 h APF. Between 4 and 6 h APF, Dl expression within the mechanosensory microchaete primordia is established. Then, ac expression is activated uniformly along the leg circumference. By the time that ac expression is activated, the interstripe domains have been defined by the four Hairy stripes and Dl/N signaling (Joshi, 2006).

The delay of ac expression in the microchaete proneural fields until mid-prepupal stages is likely due to the requirement of ac function for formation of all leg sensory organs. Leg sensory bristles can be grouped into two broad categories based on their time of specification: one group includes the early-specified mechanosensory macrochaetae (large bristles) and chemosensory microchaetae, and the second group includes the more numerous late-specified mechanosensory microchaetae. During the 3rd instar and early prepupal stages, ac is expressed in small clusters of cells that define the primordia of early-specified bristles, while expression of ac in the mechanosensory microchaete primordia is activated later in the mid-prepupal stage. This late expression of ac is activated broadly along the leg circumference and is presumably delayed to allow for expression of the hairy and Dl stripes during earlier stages. Premature expression of this normally late ac expression would likely lead to disturbances in sensory organ patterning, suggesting that temporal control of ac expression is an important aspect of its regulation (Joshi, 2006).

SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation

Body pattern formation during early embryogenesis of Drosophila relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. This study identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation (Matyash, 2009).

This study provides evidence that Kr exerts transcriptional repression not only by association with the corepressor dCtBP but also by site-specific deacetylation of histones, a mechanism that involves an interaction between Kr and dSAP18. The dual mode of Kr-dependent repression might explain earlier studies showing that Kr represses eve stripe 2 expression, but not h stripe 7 expression, in a dCtBP-dependent manner. Consistent with these observations, a mutant Kr protein that lacks dCtBP-binding sites still associates with dSAP18, which in turn interacts with the Sin3A-HDAC1 repressor complex (Drosophila HDAC1 is Rpd3). dSAP18 was also shown to bind the homeodomain transcription factor Bicoid, causing repression of anterior gap genes such as hunchback in the late Drosophila blastoderm embryo. SAP18-dependent repression involves histone deacetylase both in flies and mammals, and SAP18 that links the HDAC1 complex with sequence-specific transcriptional repressors bound to chromatin is also found in plants. These results are consistent with such a SAP18-dependent mode of Kr-dependent repression that provides target gene-specific repression. Because both dCtBP and SAP18 are uniformly distributed in the embryo, it will be important to learn how the eve stripe 2 and the h stripe 7 enhancer distinguish between the dCtBP- or SAP18-dependent modes of repression. One possibility is that differential packing of the enhancer DNA into nucleosomes might account for the difference in susceptibility to the SAP18/HDAC1-mediated repression (Matyash, 2009).

dSAP18 binds to three distinct regions of Kr, including the 42-amino acid-long repressor region, which is conserved in Kr homologs of all Drosophila species. However, as observed for dCtBP, dSAP18 alone cannot account for Kr-dependent repression of h7-lacZ, because prolonged expression of Kr is able to overcome the lack of dSAP18 activity as observed for the h7 element in dSAP18 mutants. Therefore, it is likely that the full spectrum of Kr-dependent repression is mediated redundantly, employing at least two different corepressors that involve different modes of repression (Matyash, 2009). In vitro, dSAP18 binds to the sequence motif 344RRRHHL349 of Kr and to a similar motif (143RRRRHKI149) of Bicoid; the latter is consistent with the results reported by Zhu (2001). In both proteins, the dSAP18-binding sites are localized in the C-terminal portion of their DNA-binding domains. Thus, when acting from weak binding sites in vivo, transcription factors might be able to form strong complexes with dSAP18. In fact, Bicoid-dependent repression of hunchback, which depends on both SAP18 and HDAC1 (Singh, 2005), occurs only at the very anterior tip of blastoderm embryos where the Bicoid concentration is highest and the target gene enhancers contain multiple weak Bicoid-binding sites (Matyash, 2009).

dSAP18 also interacts with the histone-specific H3K27 methyl-transferase E(z) (Enhancer of zeste) (Wang, 2002), a component of the polycomb group protein complex, and with the GAGA factor, a transcription factor of the trxG (trithorax group) protein complex. Thus, dSAP18 is capable of interacting with two regulatory protein complexes that have antagonistic functions in gene regulation. Whereas the polycomb group complex acts as a repressor of homeotic genes in ectopic locations, the trxG complex is required for activation and maintenance of their transcription. However, this clear-cut distinction between polycomb group and trxG functions has been questioned, because polycomb group and trxG group members were shown to act both as context-dependent repressors and activators of transcription, and factors with such dual functions include both the E(z) and GAGA factor proteins. In fact, interactions between dSAP18 and GAGA factor at the iab-6 element of the bithorax complex, for example, were shown to cause transcriptional activation and not repression (Matyash, 2009).

This study suggests that Kr mediates repression through at least two pathways involving either dCtBP or SAP18. dCtBP-dependent and -independent repression of the transcription factors Knirps and Hairless exert quantitative effects, whereas Kr distinguishes dCtBP and dSAP18 recruitment at different enhancers. It was observed, however, that the loss of SAP18 activity does not affect the pattern of eve stripe expression and that prolonged Kr can suppress h7-lacZ expression in the absence of dSAP18. Thus, although both dSAP18 and dCtBP act independently from each other, the two corepressors, or other yet unknown corepressors, can functionally substitute for each other under forced conditions. However, their mode of repression appears to involve different mechanisms. One mechanism is exemplified by the dCtBP-dependent repression of eve-stripe 2 and not yet established at the molecular level. dCtBP-dependent repression does not act via unleashing local heterochromatization, does not require dHDAC1 activity, and is insensitive to the HDAC inhibitor TSA. Consistently, coimmunoprecipitation studies failed to detect HDAC activity in the dCtBP immunoprecipitates, histone H3 remained acetylated in dCtBP-deficient embryos, and transcription was not repressed. Other studies, however, implied an association of dCtBP with HDACs. Thus, the mechanism of the dCtBP mode of repression is not yet fully understood (Matyash, 2009).

The results of this study showing a lack of H3 deacetylation at the eve stripe 2 enhancer in response to Kr repression are consistent with the argument that eve stripe 2-mediated repression involves the corepressor CtBP. The second, dCtBP-independent mode of Kr-dependent repression, as exemplified by the h stripe 7 element (and possibly also eve stripes 1, 3, and 4) does require both dSAP18 and HDAC1 activities. In support of this mode of repression, the following phenomena were observed in Kr-overexpressing embryos (1) a dSAP18-dependent loss of K9,14H3 acetylation on the h stripe 7 element, (2) an increased resistance of the h7 enhancer DNA to sonication, and (3) SAP18-dependent repression of the h7 reporter gene in response to Kr activity. These Kr-dependent effects were dependent on HDAC1 enzymatic activity as revealed by experiments using the HDAC1 inhibitor, TSA. These results therefore suggest that dSAP18-dependent repression by Kr involves structural changes of chromatin, such as compaction or condensation, likely to be caused by site-specific heterochromatization in response to enhancer-specific HDAC1 activity (Matyash, 2009).

Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes

Transcriptional repression is essential for establishing precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on their ranges of action, acting either locally to quench adjacent activators or broadly to silence an entire locus. Paradoxically, these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two distinct modes of repression, chromatin analysis was performed using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal that Knirps causes local changes of histone density and acetylation, and the inhibition of activator recruitment, without affecting the recruitment of basal transcriptional machinery. In contrast, Hairy induces widespread histone deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. This study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner (Li, 2011).

To directly compare functional aspects of Hairy- and Knirps- mediated repression in the Drosophila embryo, these proteinsÂ’ interactions were studied with two segmentally expressed pair-rule genes. Hairy directly represses fushi tarazu (ftz), a secondary pair-rule gene expressed in the blastoderm embryo in a seven-stripe pattern. ftz is regulated by both regionally acting gap genes and the segmentally expressed hairy pair-rule gene. Chromatin immunoprecipitation (ChIP) experiments have revealed dense clusters of peaks around the ftz gene for key transcription factors active in the blastoderm embryo, including Caudal, Hunchback, Knirps, Giant, Huckebein, Krüppel, and Tailless. These transcription factors bind to the promoter-proximal Zebra element, the stripe 1+5 enhancer located 3' of ftz, and a presumptive 5' regulatory region located between 23 kbp and 28 kbp. Hairy has been found to bind in vivo to all of these regions. This repressor is expressed in a striped pattern in the blastoderm embryo; therefore, the ftz gene is active in some nuclei and repressed in others. In order to obtain a homogeneous population of nuclei for chromatin studies, Hairy protein was overexpressed in embryos using a heat-shock driver, which results in complete repression of ftz. This repression requires the recruitment of the Groucho corepressor, because a mutant version of Hairy that does not bind to Groucho fails to repress ftz (Li, 2011).

Interestingly, a titration of heat-shock induction resulted in a nonuniform, progressive loss of specific ftz stripes, with stripe 4 being the most sensitive and stripe 1+5 the least. This result points to the intriguing possibility that Hairy can act locally on specific enhancers, at least very transiently, although the end result of Hairy repression is complete silencing of all enhancer elements. The asynchronous repression of the ftz locus also suggests that Hairy-mediated long-range repression does not act solely by direct targeting the basal promoter, as suggested by a previous model for this class of repressor, because this mechanism should cause uniform inhibition of stripe elements. Similar to ftz, the pair-rule gene even skipped (eve) is also expressed in a seven-stripe pattern and is regulated by multiple modular enhancers. eve is a well-characterized target of the short-range repressor Knirps, which sets posterior boundaries of eve stripe 3 and 4 and anterior borders of eve stripe 6 and 7. After substantial overexpression of Knirps (20 min heat-shock induction), the repressor is able to repress all of the eve stripe enhancers except for the stripe 5 enhancer. When the induction is titrated, Knirps represses individual enhancers in a stepwise manner, with the most sensitive enhancers downregulated earliest, at a low dose of Knirps. Together, these experiments indicate that Hairy can initially act locally but ultimately acts in a globally dominant fashion, whereas Knirps acts in a restricted manner (Li, 2011).

To compare the effects of repression by Hairy and Knirps, chromatin changes associated with repression of ftz and eve were studied via ChIP. No significant change of histone H3 occupancy were detected at regions sampled throughout the ftz locus after Hairy overexpression (although some regions showed modest differences. In contrast, Knirps repression of eve resulted in significantly increased histone H3 density, particularly in two of the three regions corresponding to the Knirps-sensitive enhancers, namely stripe 4+6 and stripe 2. Little change was noted in the promoter region, transcribed region, or the stripe 1 and 5 enhancers, which are not readily repressed by Knirps. An apparent increase in histone H3 density on the repressed stripe 3+7 enhancer, although of low statistical significance, correlates with other alterations common to repressed enhancers, noted below (Li, 2011).

To provide a more detailed picture of chromatin structure, a micrococcal nuclease (MNase) mapping protocol used in yeast and cultured cells was adapted for Drosophila embryos. MNase mapping showed that Hairy repression had little effect on chromatin accessibility throughout the ftz locus, whereas Knirps induced a significant increase in MNase insensitivity specifically at the eve stripe 3+7, 2, and 4+6 enhancers and a minor increase in stripe 1 protection. The promoter and the eve stripe 5 enhancer were little changed, mirroring the patterns noted for overall histone H3 occupancy. The changes noted for the eve locus appear to be specific, because Knirps did not induce any change of a nontargeted intergenic site on the third chromosome. Hairy also had no effect at this locus. The similar results from overall histone H3 density and MNase mapping suggest that Hairy-mediated long-range repression does not involve a general compaction of chromatin on the ftz locus. In contrast, repression by Knirps is associated with an increase in the histone density of targeted enhancer regions, which may result either from Knirps recruitment of factors that mediate chromatin condensation or the blocking of proteins responsible for loosening of chromatin. Recruitment of Groucho by other repressor proteins is also associated with distinct effects: Runt-dependent repression of slp1 does not involve changes in H3 density, but Brinker repression of the vgQ enhancer does. The distance dependence of these repressors has not been established, but in light of the current results, it is apparent that the Groucho corepressor can be involved in distinct effects depending on the context of recruitment (Li, 2011).

Histone acetylation is dynamically regulated on transcribed genes in eukaryotes, with histone acetylation generally correlated with active loci. The histone deacetylase Rpd3 is a component of both Hairy and Knirps corepressor complexes; therefore, histone acetylation levels were assayed across the eve and ftz genes before and after repression. Hairy repression resulted in widespread histone H4 deacetylation throughout the ftz locus. The ectopically expressed Hairy protein itself was not observed to spread but remained restricted to regions of the gene previously observed to bind endogenous Hairy. Using anti-H3-acetylation antibodies, similar widespread H3 deacetylation was also noted. This distributed effect on the ftz locus correlates with prior observations that Hairy-mediated long-range repression might involve a Groucho-mediated 'spreading' mechanism. By this means, Rpd3 may be delivered to extensive areas of a gene. To test whether a spreading of histone deacetylation might correlate with the successive inhibition of ftz enhancers, histone acetylation levels were investigated across ftz after a brief 5 min heat shock followed by immediate fixing, before the entire complement of enhancers can be repressed. In this setting, deacetylation was mostly concentrated around the stripe 1+5 enhancer and the immediate 5' regulatory region, areas that show Hairy occupancy in vivo. More distal 5' regulatory regions and the transcription unit itself showed little initial change, consistent with a spreading action of this repressor during the more extensive repression period (Li, 2011).

A different picture emerged from studies of Knirps acting on eve. Here, repression led to selective decreases in H3 and H4 acetylation levels, concentrated over the eve stripe 4+6 and stripe 2 enhancers, with lesser decreases noted at stripe 3+7 and stripe 1 enhancers. A local change in acetylation was also noted near the transcriptional initiation site, but not immediately 5' and 3' of this area. The reductions in histone acetylation levels seen on both eve and ftz are consistent with Hairy and Knirps recruiting deacetylases to their target genes. However, it is striking that the broad deacetylation mediated by Hairy on ftz is not associated with dramatic changes in histone density or resistance to nuclease accessibility, whereas increased histone density and resistance to nuclease digestion are associated with Knirps repression on eve. It is possible that in addition to inducing deacetylation, Knirps triggers additional histone modifications or interacts with nucleosome-remodeling complexes to further alter chromatin at the enhancers. H3 lysine 27 methylation is one chromatin signature associated with silenced genes; however, no significant change in this modification was noted at ftz or eve upon repression (Li, 2011).

Previous studies indicated that Hairy can effectively repress a reporter gene without displacing the activators. Attempts were made to test whether this was the case on an endogenous gene, ftz, by examining occupancy by Caudal, a transcription factor that also activates eve. Caudal activates the posterior stripes of both ftz and eve, and it was found that Caudal binds the ftz 5' regulatory region and the promoter-proximal Zebra element. Repression of the locus by Hairy did not affect the Caudal binding pattern, similar to the results obtained with a Hairy-regulated reporter gene. In contrast, Knirps repression decreased Caudal occupancy specifically at the eve 3+7 and 4+6 enhancers, bringing overall protein occupancy down to near baseline levels. This decrease is not an effect of global decrease of Caudal occupancy, because the Caudal binding peak at the eve promoter was not affected. A similar decrease in Caudal occupancy was also observed on a hunchback enhancer after repression by Knirps. Interestingly, Bicoid occupancy of the eve stripe 2 and stripe 1 enhancers was not altered by Knirps, although these enhancers were repressed. Clearly, loss of transcription factor occupancy is not required for short-range repression of a cis-regulatory element. It is possible that different transcriptional activators exhibit differential sensitivity to chromatin changes induced during repression (Li, 2011).

New insights have suggested that many developmental genes, including those regulated by short-range repressors such as Snail, feature RNA polymerase paused in the promoter region even in their inactive state, suggesting postrecruitment levels of regulation. Components of the core machinery were analyzed before and after repression by Hairy and Knirps. Upon Hairy repression, a marked decrease of RNA polymerase II (Pol II) occupancy was observed at the ftz locus. The same trend was observed for the preinitiation, initiation, and elongation forms of Pol II. These results suggest that Hairy directly or indirectly blocks recruitment of Pol II. Similar decreases were noted with levels of TATA box-binding protein (TBP) at the promoter (Li, 2011).

In contrast, induction of Knirps did not change Pol II occupancy at the eve transcription unit, even under condition where most enhancers were repressed. (Under conditions tested in this study, over three-quarters of the embryos had shut down expression of all but stripe 1 and/or 5.) Similarly, TBP occupancy remained at a comparable level before and after Knirps repression. The constant level of RNA polymerase on the eve transcription unit was a surprise in light of the sharp reduction in mRNA production as measured by in situ hybridization. However, there is precedence for this effect: Runt repression of slp1 appears to act through elongation control, which causes no change of the concentration of Pol II on slp1. Knirps may produce a similar effect by inducing a slower transit rate of Pol II on the repressed eve locus. Similar observations have been made at the hsp70 gene upon depletion of elongation factors such as Spt6 or Paf1 (Li, 2011).

The differential distance dependence of short- and long- range repressors such as Hairy and Knirps has been observed in many contexts. However, the mechanisms by which these proteins function have been poorly understood. With the recent demonstration that transcriptional factors considered to be short- and long-range repressors utilize shared cofactors, namely CtBP and Groucho, there has been a question of whether long-range repression is actually functionally distinct from short-range repression (Payankaulam, 2009). The current study provides evidence that the chromatin states associated with long- and short-range repressors are distinct in several ways. It is not yet knowm whether the effects seen on ftz are observed for all Hairy targets, although the similarity of changes observed on the lacZ reporter subject to Hairy repression suggests that they are conserved (Martinez, 2008). Similarly, the reproducibility of Knirps-induced changes at different eve enhancers indicates that this protein can effect related chromatin changes on cis-regulatory modules bound by different activators. Snail, another short-range repressor, also appears to mediate localized deacetylation and activator displacement; thus, this mechanism may be a common feature of this entire class of repressors (Qi, 2009; Y. Nibu, personal communication to Li, 2011). It will be interesting to determine how general are the observations made in this study for long- and short-range repression, a question that can be approached using genome-wide methods. In any event, the highly divergent activities of Knirps and Hairy demonstrated in this study not only underscore the fact that these proteins can mediate biochemically divergent events but also raise interesting questions about how similar cofactors can participate in such distinct effects in a context-dependent manner. It is possible that the corepressors adopt distinct conformations when recruited by different repressors, or the corepressor may form distinct complexes with unique activities. In addition to determining how cis- and trans-acting factors affect repression pathways, these mechanistic insights will provide important contextual information for interpretation of genome-wide transcription factor binding and chromatin modifications and will inform quantitative modeling of cis-regulatory elements for the aim of understanding the activity and evolution of enhancers (Li, 2011).

Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function

Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. This study shows that the utilization of dual, or 'shadow', enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P; Scute, for example) proteins and basic helix-loop-helix (bHLH) repressor (R; Hairy/Enhancer of split (Hes) class) factors (a 'P+R' regulatory code), NP-specific enhancer modules were identified associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, P sites in these modules were shown to mediate activation by proneural factors in 'proneural cluster' territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. These results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), it was demonstrated that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event (Miller, 2014).

hairy: Biological Overview | Evolutionary Homologs | Protein interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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