Interactive Fly, Drosophila

Even-skipped contains domains that inhibit transcriptional activators present at the Ultrabithorax proximal promoter when bound up to 1.5 kb away from these activators. Three adjacent regions of EVE contribute to silencing. Repression in vitro correlates with binding of EVE protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other EVE molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA; the intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the Zeste transcription factor. Binding of EVE protein is shown to exclude binding by Zeste protein (TenHarmsel, 1993).

It is not clear how transcription factors bind at distal enhancer, nor how proximal promoter sequences cooperate to stimulate transcription in vivo. To distinguish between different models for the action of enhancer elements, DNA binding of the Drosophila activator Zeste was measured by in vivo UV crosslinking. Experiments in Drosophila embryos show that binding of Zeste protein to either the proximal promoter of the Ultrabithorax gene or to a Ubx enhancer element does not require the presence of the other element. However, significant transcription is observed only when both elements are present and bound by Zeste. The results indicate that stimulation by an enhancer can occur by a mechanism other than increasing the occupancy of an activator to binding sites near the start site of transcription, and suggest that the enhancer acts not by recruiting RNA polymerase to the promoter, but instead by activating an already bound polymerase (Laney, 1997).

A double-bromodomain protein, FSH-S, cooperates with Zeste to activate the homeotic gene Ultrabithorax through a critical promoter-proximal region

More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic [fs(1)h] gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. One of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. Three aspects are noted: (1) overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation; (3) FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner; (3) purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, FSH-S is involved in phosphorylation of itself and other regulatory factors. It is suggested that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers (Chang, 2007).

Drosophila HOX genes control development of body segments via highly restricted expression domains. These domains are first established by transiently expressed segmentation genes in early embryos and then maintained in an epigenetically heritable manner by the Polycomb group (PcG) of repressors, and the trithorax group (trxG) of activators. Like mammalian promoters that are regulated by distant elements, transcriptional regulation of HOX genes also requires coordinated long-range interactions between the basal transcription machinery assembled around the initiation sites and factors recruited at distant regulatory elements. How the epigenetic inheritance imposed by PcG and trxG is integrated into the general framework of such long-range interactions remains unclear. Its elucidation should provide an important model for understanding the regulatory mechanisms of genes under strict developmental control (Chang, 2007).

PcG repressors form at least two types of multimeric complexes that are targeted by sequence-specific binding proteins to a core PcG response element located ~25 kb upstream of the homeotic gene Ultrabithorax (Ubx). These complexes may block the access of the regulatory elements or modify chromatin by associated histone deacetylase and histone methyltransferase activities. In contrast to the highly targeted activities of PcG repressors, trxG activators appear to employ diverse mechanisms for chromatin remodeling and long-range interactions. For example, trithorax (trx) and absent, small or homeotic discs 1 (ash1) encode histone methyltransferases that are targeted to PcG response elements, promoters, and transcribed regions. In addition to these targeted activities, brahma, moira, and osa encode subunits of an ATP-dependent chromatin remodeling complex that can modulate the nucleosome fluidity to provide an open access of regulatory sequences. Moreover, kohtalo and skuld encode subunits of the Mediator coactivator complex that can facilitate interactions between distal factors and basal transcription machinery (Chang, 2007).

How signals provided by distal elements are integrated at the Ubx basal promoter remains unclear. The Ubx proximal region has several unique features. Instead of the consensus TATA box in the −30 region, Ubx contains the initiator around +1 and the downstream promoter element around +30, which are frequently found in genes lacking the TATA box in Drosophila and mammals. The ability of these elements to support Ubx transcription in vitro and in vivo indicates that they represent an authentic basal promoter. However, this basal promoter fails to integrate regulatory signals from distant elements without a proximal region from −200 to −32, revealing a critical requirement for this region in mediating long-range interactions (Chang, 2007).

Interestingly, this critical proximal region (CPR) contains multiple binding sites for Zeste and Trithorax-like (Trl) proteins. The Zeste sites appear to be particularly important, since CPR activity can be substantially replaced by tandem Zeste sites. Consistent with the transactivating role, zeste was initially identified as required for Ubx expression through transvection, a pairing-dependent effect believed to facilitate the transutilization of the regulatory elements on one chromosome by the promoter on homologous chromosome. Zeste protein can stimulate Ubx transcription in vitro and is necessary for the expression of Ubx transgenes containing subsets of regulatory sequences. Paradoxically, zeste is not essential for normal development or for expression of the endogenous Ubx promoter or a Ubx transgene with more complete regulatory sequences. The role of zeste is further complicated by the finding that zeste may be involved in Ubx repression. Clearly, other factors must be required for the activating effect of the Zeste sites in the CPR (Chang, 2007).

The maternal-effect gene female sterile (1) homeotic [fs(1)h] was identified as a transactivator of Ubx by its strong genetic interactions with Ubx, trx, and ash1 mutations. However, its direct role in homeotic gene activation has been obscured by complex phenotypes in mutant embryos. Sequence analysis indicates that fs(1)h encodes two putative proteins of approximately 120 and 210 kDa. The small isoform FSH-S, containing two widely spaced bromodomains and the extra terminal (ET) domain at its C terminus, is identical to the N-terminal half of the large isoform FSH-L. Bromodomains can bind acetylated lysine or histones and are frequently found in transcription or chromatin modification factors, whereas ET domains are found in a small family of double-bromodomain proteins (BET proteins) with no designated function. Several interesting properties have been shown for mammalian BET proteins. For example, human RING3 (or BRD2) is a growth-stimulated nuclear kinase acting on serine and threonine. Mouse BRD2-like protein can be copurified with the Mediator transcriptional coactivator complex. Recently, mouse BRD4 has been shown to be involved in the recruitment of positive transcription elongation factor b (Chang, 2007 and references therein).

This report provides several lines of evidence to support a direct role of fs(1)h in homeotic gene activation and the idea that FSH-S is primarily responsible for this function. Furthermore, it is shown that FSH-S acts directly on the Zeste site of the CPR. These results support a critical role for FSH-S in integrating signals from distal factors (Chang, 2007).

While many trxG mutations were identified by their suppressing effects on specific homeotic phenotypes caused by PcG mutations, their contributions to regulation of individual HOX genes have not been systematically examined. To address this issue, the effect of mutations of 18 trxG genes was examined on homeotic phenotypes caused by reduced Ubx expression, i.e., transformation of the third to second thoracic segment in adult flies. Interestingly, only fs(1)h [i.e., Df(1)C128], trx, and ash1 mutations showed strong enhancement on Ubx¹³⁰ phenotypes (increased from <1% to ~10%). Other trxG mutations showed weak or no effects on Ubx¹³⁰ mutation, despite that many could suppress PcG phenotypes as strongly as trx mutations. Thus, Ubx activation appeared to be highly sensitive to the dosages of fs(1)h, trx, and ash1. This selective effect was further supported by genetic interactions between fs(1)h and other trxG mutations. Again, fs(1)h showed strong synergistic effects with trx (>20%) and ash1 mutations (~10%) on Ubx phenotypes. By contrast, it showed weaker or no interactions with other trxG mutations. These results strongly suggest that fs(1)h, trx, and ash1 share some common role in certain critical steps of Ubx activation (Chang, 2007).

Loss of fs(1)h function results in complex defects in early embryos, leading to severe body distortion and lethality. These defects hamper the analysis of the role of fs(1)h in Ubx activation. To circumvent these problems, fs(1)h function was inactivated by shifting heat-sensitive fs(1)h¹ mutant embryos from the permissive temperature (21°C) to the restrictive temperature (29°C) during the onset of gastrulation. In wild-type embryos, high levels of Ubx transcripts can be detected in the ventral nerve cord (VNC) in a domain encompassing parasegments (PS) 5 to 12. In mutant embryos, a marked reduction of Ubx transcripts was seen. By contrast, no change was observed for caudal (cad), a HOX gene controlling the development of most posterior segments. Thus, fs(1)h appeared to be required for a subset of HOX genes (Chang, 2007).

fs(1)h encodes two double-bromodomain proteins, FSH-S and FSH-L. To define their roles in HOX activation, the Gal4/UAS binary system was used to induce high levels of FSH-S or FSH-L and examine their effects on HOX expression. UAS transgenes containing epitope-tagged FSH-S or FSH-L were driven by dpp-Gal4 in small subsets of imaginal cells. Targeted expression of FSH-S caused striking defects in the adult. Frequently, adults heads lacked maxillary palpi, and their aristae were transformed into distal legs with claws. Severe defects were also found in thoracic legs, including bifurcation of tibial segments and deletion of tarsal segments. Surprisingly, no discernible defect was seen in adults with targeted FSH-L expression, suggesting that FSH-L and FSH-S act differently (Chang, 2007).

Antenna-to-leg transformations can be induced by ectopic expression of the HOX gene Antennapedia (Antp) in antennal discs. To determine whether extra legs induced by FSH-S might be related to ectopic Antp expression, eye-antennal discs from third instar larvae were stained with an anti-ANTP antibody. Whereas ANTP is normally not expressed in these discs, strong ANTP signals were seen in antennal discs of transgenic animals. Using an anti-Flag antibody to mark tagged FSH-S, extensive overlaps were found between FSH-S and ANTP signals, suggesting that FSH-S is directly involved in ANTP induction. By contrast, no ectopic ANTP was induced by FSH-L, which was consistent with the normal appearance of adult flies. These effects further distinguished the role of FSH-S and FSH-L in HOX activation. Curiously, very little ANTP expression was induced by FSH-S in eye discs, despite its comparable levels in antennal and eye discs. The nature of this tissue-dependent response is unclear. Furthermore, no ectopic Ubx signal was found in eye-antennal and other discs. To avoid problems caused by induction timing or tissue dependence, an en-Gal4 line was used to drive FSH-S expression. Under such conditions, most larvae died before the third instar, while rare adult escapers (less than 1%) showed partial deletion of thoracic segments. In second instar larvae, ectopic Ubx signals could be detected in ventral ganglions. In addition to the transverse rows normally found within PS5 to PS12, Ubx signals appeared in small clusters of cells near the lateral margins of PS4, PS3, and PS2 at anteriorly diminishing frequencies. Occasionally, ectopic Ubx was found in PS4 extending to PS2 on both sides of the ganglion. These results strongly suggested that FSH-S can induce HOX genes (Chang, 2007).

Previous analysis predicted that fs(1)h protein products might be membrane associated, implicating a role in signal transduction. To further characterize FSH-S, antibodies were raised against three regions (S1, S2, and S3) common to both FSH-S and FSH-L. Using affinity-purified antibodies, two common bands were detected in embryonic extracts. The sizes of these two bands were consistent with predicted sizes of FSH proteins (~210 and ~120 kDa). In addition, an antibody specific for FSH-L (i.e., L3) reacted only with the larger protein. The authenticity of these proteins was further confirmed by the analysis of a larval-lethal mutant, fs(1)h¹⁷, which results from an insertion of a copia element in the intron following the FSH-S coding sequences. Unlike many other fs(1)h mutations, this mutation did not cause homeotic effects. Interestingly, the larger protein was severely diminished in mutant larvae at third instar, while the small one was unaffected. These results indicate that these proteins represent the two specific FSH isoforms and, more importantly, that FSH-L is not essential for the homeotic effect (Chang, 2007).

The developmental profiles and subcellular localization of FSH proteins in embryos were analyzed by immunostaining with affinity-purified S1 antibody. Muclear staining was clearly seen in syncytial embryos. Although S1 antibody reacted with both FSH-S and FSH-L, this nuclear staining was attributed to FSH-S, since FSH-L is primarily a centrosomal protein at this stage. In addition, tagged FSH-S was localized in the nuclei in both transgenic lines. The staining intensity appeared to be uniform throughout all developmental stages except in those cells located near invaginating furrows or in VNC. To further confirm the distribution pattern of FSH-S, whole-mount in situ hybridization was performed using a probe from the 3' UTR of FSH-S mRNA, which is absent in FSH-L mRNA. Again, a ubiquitous distribution of FSH-S transcripts was observed (Chang, 2007).

The genetic interactions, induction of HOX gene expression, nuclear localization, and the presence of a double bromodomain raised a strong possibility that FSH-S might directly affect HOX promoters. To test this, the ability of FSH-S to stimulate reporter constructs containing various promoters was tested by cotransfection experiments in a Drosophila haploid cell line which was shown to recapitulate Ubx regulation by trx and Pc. Reporter activities from constructs containing the P1 or P2 promoters of Antp or promoters of Ubx and the Heat shock protein 70 (Hsp70) were assayed following cotransfection of an Act5C-FSH-S effector or an Act5C control vector. The activities of the Antp-P2 and Ubx promoters were stimulated approximately 10-fold and 20-fold, respectively, while the Antp-P1 and Hsp70 promoters were only weakly affected. Thus, the effect of FSH-S appeared to be highly selective (Chang, 2007).

Several trxG genes have been shown to act on regulatory sequences located about 20 kb upstream of the initiation site. Since the UC construct used in this study only contained sequences from −3142 to +360, FSH-S appeared to act via distinct sequences near the basal promoter. To identify the FSH-S response elements (FRE), the effect of FSH-S on a series of Ubx deletion mutants was analyzed. Sequential deletion of 5' sequences from −3142 to −1762 (5Δ1), −628 (5Δ2), or −226 (5Δ3) did not alter the ability of the Ubx promoter to respond to cotransfected FSH-S. Deletion from +360 to +161 (3Δ1) resulted in a general reduction of the promoter activity by about twofold, regardless of the presence or absence of cotransfected FSH-S. Since the stimulatory effect of FSH-S was not affected, this downstream region most likely contains a positive element that is unrelated to FRE. No further effect was observed when sequences from +161 to +36 (3Δ2) were deleted. These results indicated that the FRE is not present in the regions upstream of −226 or downstream of +36. Consistently, a construct containing sequences from −226 to + 36 (3Δ22) was sufficient to respond to FSH-S. Conversely, an internal deletion of sequence from −200 to −32 (InΔ1; In is initiator) almost completely abolished the promoter activity. Since the initiator (ACATTC from −2 to +4) and downstream promoter elements (GGATA from +23 to +27) were intact in InΔ1 construct, the inactivation of the Ubx promoter should reflect the removal of regulatory elements. These results led to the conclusion that the FRE is located between −200 and −32, which corresponds to the CPR determined previously. Further refinement of the boundaries of the FRE was unsuccessful, since deletions from −226 to −127 (3Δ23) or from −127 to −32 inactivated the promoter (Chang, 2007).

Whether any specific domain of FSH-S is required for transactivation was examined. Mutant constructs carrying deletions of the N-terminal half of the first bromodomain (Δ1), the entire second bromodomain (Δ2) and its flanking sequences (Δ3), or both bromodomains (Δ12) or the C-terminal sequences including the ET domain (Δ4-6) were tested in transfection assays. It appeared that deletion of the first bromodomain results in a complete inactivation of FSH-S, suggesting a critical requirement of this domain. However, the full activity of FSH-S was also dependent on the second bromodomain and ET domain, since deletion of these domains resulted in partial inactivation. Interestingly, although FSH-L contains the entire FSH-S sequence, it appeared to be much less active than FSH-S. These results are consistent with the observation that FSH-L could not induce HOX genes in imaginal tissues and support further that FSH-S is primarily, if not exclusively, responsible for the transactivation function of fs(1)h (Chang, 2007).

Next, whether FSH-S could bind any specific sequences in the CPR was examined. An inducible S2 cell line containing the metallothionein promoter-driven Flag-tagged FSH-S was established. Tagged FSH-S was purified by immunoaffinity chromatography from whole-cell extracts after (NH₄)₂SO₄ enrichment. In addition to the major band corresponding to FSH-S, several less abundant proteins were also copurified. Although fs(1)h mutant showed strong genetic interactions with trx or ash1 mutants, FSH-S was not copurified with these proteins or Osa. In addition, FSH-S was not associated with Zeste protein, which was shown to bind the CPR. The ability of purified FSH-S to bind specific sequences of the CPR was demonstrated by EMSAs. Upon addition of increasing amounts of FSH-S to labeled Ubx-5 probe, a slower-migrating band appeared near the top of 3.5% native polyacrylamide gels, indicating the formation of protein-DNA complexes. The exceedingly slow mobility of this band suggested that a multisubunit protein complex is involved. FSH-S is a constituent of this putative complex, since a small but significant supershift was observed when an antibody against FSH-S was briefly incubated with FSH-S protein. A supershift was not observed when an antibody to FSH-L was used instead. Furthermore, this binding was sequence specific, since it could be completely blocked by the addition of excess amounts of unlabeled Ubx-5 or Ubx-6 but not by a random DNA fragment. Similar results were also obtained when Ubx-6 was used as the probe. To further narrow the binding region, four smaller probes from the CPR were used for EMSA. Specific binding was observed with probes Ubx-5b (−167 to ~−94) and Ubx-6a (−104 to ~−35) but not Ubx-5a (−226 to ~−146) or Ubx-6b (−55 to ~+36), indicating that the FRE is located between −167 and −35 (Chang, 2007).

The CPR contains clusters of binding sites for Zeste, Trl (also known as GAGA factor), and NTF-1. To determine whether any of these sites might correspond to FRE, competition assays were performed with DNA fragments containing tandem repeats of Zeste, Trl, or NTF-1 binding sites. Interestingly, only Zeste repeats effectively blocked binding activity. To exclude the possibility that fortuitous binding sites might be generated by multimerizaton of these repeats, an oligonucleotide containing one consensus Zeste site (CGAGTG) was tested with different flanking sequences. This oligonucleotide also blocked the binding activity of FSH-S. Thus, the Zeste site should represent the core FRE (Chang, 2007).

For further analyses of DNA binding properties, FSH-S and recombinant Zeste proteins were compared by an in-gel chemical footprinting technique. The DNA-cleaving ions OP-Cu used in this study gain more access to unprotected sequences than DNase I and are thus capable of revealing detailed differences in binding properties. Similar to studies with DNase I, three sites (Z1 to Z3) were protected by Zeste or FSH-S proteins in the Ubx-6a fragment. Despite an overall similarity, several important differences between these patterns were noticed. For example, the regions unprotected by Zeste produced bands with intensities comparable to those from free probes. However, fainter intervening bands were produced by FSH-S, suggesting weak protection on flanking sequences. Two additional differences were found over the Z1 site. FSH-S appeared to protect more 5′ sequences than Zeste. However, Zeste produced several bands more intense than the control, suggesting DNA distortion in this region. The lack of detectable Zeste protein and the distinct DNA binding properties exhibited by FSH-S clearly support the involvement of a novel binding factor (Chang, 2007).

If the FRE indeed corresponds to the Zeste site, the function of the Zeste site might be inactivated by fs(1)h mutations. Therefore, the effects were examined of fs(1)h mutation on expression of Ubx-lacZ transgenes containing two distal regulatory domains (BXD and ABX) and ~3 kb of immediate upstream sequences in addition to a wild-type CPR (Uβ) or tandem Zeste sites (Uβ-Z). In the wild-type background, strong lacZ signals were observed from PS5 to more posterior parts of the VNC in Uβ embryos. In addition, there was weaker misexpression in anterior parts of the VNC. The misexpression was more pronounced in Uβ-Z embryos. More importantly, lacZ transcripts were severely reduced throughout the entire VNC in both Uβ and Uβ-Z embryos upon inactivation of fs(1)h, indicating a strict requirement of fs(1)h. These results strongly support the physiological relevance of FSH-S to the Zeste site (Chang, 2007).

To further demonstrate that FSH-S is indeed associated with CPR of the endogenous promoter in vivo, chromatin immunoprecipitation assays were performed with formaldehyde-fixed chromatin prepared from male fs(1)h¹⁷ mutant larvae, which contain normal levels of FSH-S but diminishing amounts of FSH-L. Using five pairs of primers to cover sequences of more than 2 kb around Ubx start sites, it was found that FSH-S is preferentially associated with a CPR-containing DNA fragment. Although the antibody used here could cross-react with FSH-L, the contribution of FSH-L to the binding is excluded, because only a minute amount of FSH-L was present in fs(1)h¹⁷ mutant larvae, and, more importantly, no FSH-L signal was detectable in the Ubx promoter. In addition, this association appeared to be promoter specific, since only background signal was detected in the cad promoter (Chang, 2007).

Human RING3 protein, a FSH-S-like protein, has been shown to be a novel nuclear Ser/Thr kinase with scrambled subdomains. However, subsequent studies failed to show this activity in the mouse counterpart, FSRG-1, despite more than 90% sequence identity. To determine whether FSH-S could act as a kinase, the kinase activity in FSH-S preparations was examined. Addition of [γ-³²P]ATP resulted in substantial phosphorylation of FSH-S and an additional protein of ~56 kDa. Because this smaller protein was consistently copurified, it will be referred to as FAP56 (FSH-associated protein of 56 kDa). Phosphoamino-acid analysis of in vitro phosphorylated proteins revealed that FSH-S was phosphorylated at the serine residue, while FAP56 was phosphorylated at both serine and threonine residues. Although FSH-S phosphorylation was readily detected by radioactive labeling, no mass increase was found upon incubation with 0.1 mM ATP. However, when treated with calf intestine phosphatase, the mass of FSH-S appeared to decrease slightly, indicating a limited phosphorylation of FSH-S (Chang, 2007).

The kinase activity of RING3 kinase could be restored by renaturation on nitrocellular filter after SDS-PAGE. Using this procedure, no FSH-S phosphorylation was detected in parallel experiments. However, it was reasoned that if FSH-S is a kinase, it must be able to bind ATP. An ATP analog, FSBA, has been used for affinity labeling of ATP binding proteins including kinases. Therefore, the reactivity of FSH-S toward FSBA was examined. Using an FSBA-specific antibody, it was found that FSH-S could indeed be covalently linked to FSBA. More importantly, the degree of cross-linking was substantially reduced by excessive ATP, indicating that FSH-S can bind ATP specifically (Chang, 2007).

Addition of FSH-S to cell extracts in which endogenous kinases were heat inactivated resulted in phosphorylation of many proteins, suggesting the presence of many kinase substrates. The clustering of multiple binding sites for FSH-S (or Zeste) and Trl in the CPR suggests that they might be spatially juxtaposed upon binding to the CPR, raising the possibility that Trl might be a potential kinase substrate. Using in vitro kinase assays, it was found that addition of FSH-S to purified recombinant Trl indeed resulted in its phosphorylation (Chang, 2007).

This report has provided several lines of evidence to support a direct role of FSH-S in HOX gene activation. Unlike other trxG proteins, FSH-S acts directly on the CPR of the Ubx promoter. The revelation of several interesting properties of FSH-S offers important mechanistic insights into the Ubx regulatory circuitry. Lack of functional fs(1)h is known to cause complex developmental defects including homeotic transformation and early embryonic lethality. The contribution of two different fs(1)h products to these effects had not been determined. Based on the following observations, it is suggested that FSH-S is primarily involved in HOX regulation. First, it was shown that FSH-S, but not FSH-L, can effectively activate homeotic promoters in imaginal discs and cultured cells. Second, FSH-S is a nuclear protein, while FSH-L is mainly found in centrosomes and is involved in organization of mitotic spindles in early embryos. Third, FSH-S can bind and function both in vitro and in vivo through a specific motif in the CPR. Lastly, no homeotic phenotype has been observed in an fs(1)h¹⁷ mutant lacking FSH-L. Thus, FSH-S is directly responsible for the homeotic effect of fs(1)h. Although FSH-L contains the entire sequence of FSH-S, these results clearly indicate that it does not play any significant role in the homeotic effect. The complex developmental functions of fs(1)h are very likely to be divided between different isoforms (Chang, 2007).

The abilities of FSH-S to bind a specific motif and to affect promoter activity through the CPR indicate that FSH-S plays an important role at the CPR for activation of the Ubx promoter. Among 18 trxG genes examined, fs(1)h, trx, and ash1 form a small but interesting subgroup that is most critical for Ubx activation and is known to act through specific regulatory sequences. Previous studies have shown that TRX and ASH1 act primarily through distal sequences that are essential for domain-specific Ubx expression. Recently, they have also been implicated in transcriptional elongation by their association with promoter and transcribed sequences. FSH-S is the only factor that functions primarily, if not entirely, on the CPR. Given the critical role of the CPR in promoter activity, FSH-S is very likely to play a key role in integration of activating signals from distal elements and factors. The strong synergistic effects reported in this study for fs(1)h, trx, and ash1 mutations indicate that they are involved in a critical step of Ubx promoter activation and that intimate functional relationships probably exist between these factors. Although they appear to exist in distinct protein complexes, it is highly likely that they interact directly or through associated factors. Such interactions may facilitate the action of TRX and ASH1 in the promoter and more downstream regions. An alternative (but not mutually exclusive) possibility is that FSH-S might be involved in attenuation of the repressing activity of PcG proteins. Since the distal response elements for PcG proteins and TRX/ASH1 are largely overlapping and their histone modification activities are functionally antagonistic, destabilization of PcG complexes could result in more efficient occupancy and/or more potent chromatin modification by TRX and ASH1. In either case, the activities of these distal factors might also be modulated by the kinase activity associated with FSH-S (Chang, 2007).

The stimulatory effects of FSH-S on the Ubx basal promoter also suggest that FSH-S may directly affect the basal transcription machinery. A closely related Saccharomyces cerevisiae protein, BDF1, has been shown to be a TFIID-associated factor, acting potentially as a functional substitute for TAF1 in higher organisms. Mouse BRD4 stimulates transcription by binding to positive transcription elongation factor b. Although it is unclear whether FSH-S possesses similar activities, the presence of structurally similar domains suggests that it may interact with these basal transcription factors. Therefore, it is speculated that FSH-S provides a dual interface for interactions with distal factors and basal transcriptional machinery for optimal Ubx transcription (Chang, 2007).

The sharing of the same target sequences between FSH-S and Zeste may help clarify a long-standing enigma about the role of Zeste in Ubx regulation. The function of zeste was revealed by a pairing-dependent phenomenon called transvection in which Ubx alleles with defective promoters can partially complement alleles with impaired regulatory sequences. Thus, zeste can facilitate the transutilization of the regulatory sequences on one chromosome by the Ubx promoter on a paired homologous chromosome. However, zeste is not required for expression of an intact endogenous Ubx gene or expression of a Ubx transgene containing more complete regulatory sequences (i.e., 35-kb sequences in 35UZ transgene), despite the fact that Zeste binds to the CPR and is required for expression of Ubx transgenes containing partial regulatory sequences. Moreover, zeste is dispensable for viability. These findings indicate that zeste is not essential for Ubx expression under normal genetic contexts. In contrast, FSH-S is indispensable for Ubx regulation and for development. The ability of FSH-S to bind the same target sequences indicates that FSH-S represents a critical component of a regulatory circuitry that utilizes regulatory signals present on the same chromosome to insure proper transcription of intact Ubx promoter. It is interesting that zeste-independent transvection has also been found that appears to employ the mechanisms that normally operate between the distal elements and the proximal promoter. It is speculated that FSH-S is also very likely to play a role in zeste-independent transvection (Chang, 2007).

The finding of DNA binding activity in FSH-S is surprising, since the double-bromodomain and the C-terminal ET domain, two prominent domains required for the function of FSH-S, are not known for DNA binding activity. It is possible that FSH-S may possess a novel DNA binding domain. Alternatively, the binding activity might be contributed by a factor that is associated with FSH-S, since several proteins were copurified with FSH-S and since recombinant FSH-S did not show the same activity. Further characterization of FSH-S and associated factors is necessary to resolve this question (Chang, 2007).

Another interesting feature of FSH-S is the kinase activity. The structural similarities to the RING3 nuclear kinase, the detection of a similar kinase activity, and the ATP binding activity are consistent with the notion that FSH-S contains a Ser/Thr kinase activity. However, the lack of kinase activity in bacterially expressed FSH-S suggests that posttranslational modification or an additional factor(s) is required for such an activity. It is interesting that, in addition to FSH-S and FAP56, many other proteins including Trl can be phosphorylated by FSH-S in vitro, suggesting a broad substrate specificity. Thus, it seems plausible that FSH-S may modulate the activities of factors that are brought into its proximity. Once it occupies the CPR, it is speculated that FSH-S may affect multiple factors that are in close contact with CPR by either short- or long-range interactions (Chang, 2007).

Upstream promoter regions (part 1/2)

There are three key control regions in the Ubx upstream promoter called PBX, ABX and BXD. Each of these confers an expression pattern mimicking certain aspects of Ubx expression. The PBX and ABX patterns are limited to the Ubx domain with anterior boundaries at parasegments 6 and 5. In contrast, the BXD pattern extends from head to tail. PBX or ABX expression boundaries are imposed on the BXD pattern, if either PBX or ABX is linked to BXD. These boundaries, although not the PBX and ABX expression limits themselves, are dependent on Polycomb function. PBX and ABX are recognized by repressors which act across large distances to suppress BXD activity. Stable and heritable Ubx expression boundaries are thus mediated through a process of long range repression (Muller, 1991).

The transcription factors Hunchback, Krüppel, Fushi tarazu, and Knirps combine to regulate the PBX element of the Ubx gene. Within the PBX enhancer, Hb and Kr act in the more 5' region to assure blastoderm expression and mesodermal expression at the germ band extention phase. Hb, Ftz, Kr and Kni function in a more 3' region to regulate expression in parasegmental stripes 6, 8, 10 and 12 (reviewed in Arnone, 1997).

Fushi tarazu and Even-skipped act through particular key control regions of Ubx to generate ftz- or eve-like stripe patterns. FTZ protein acts directly as a transcriptional activator of Ubx. Its activity outside the Ubx expression domain is suppressed by Hunchback, a repressor of Ubx. Some DNA binding sites for FTZ protein are adjacent to binding sites for HB protein, while others overlap. FTZ protein competes with HB protein for DNA binding and/or for transcriptional activation. This competition mechanism results in a sharp anterior expression boundary (Muller, 1992).

hunchback a segmentation gene, acts as a direct repressor or "silencer" of Ultrabithorax and thus prevents ectopic activity of this gene. HB protein binding sites are capable of repressing at a distance the activity of an embryonic Ubx enhancer outside the Ubx expression domain. This silencing activity is observed at advanced embryonic stages, at a time when the hb gene product is no longer detectable or required, and is dependent on the function of Polycomb (Pc). In a "hit-and-run" fashion HB protein may effect stable and heritable silencing of the Ubx gene throughout advanced stages of development, thus mediating repression of this homeotic gene outside its realm of function (Zhang, 1992).

Appropriate Ubx transcription requires a long upstream control region (UCR) defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. 35 kb of UCR DNA confers an expression pattern that closely parallels normal Ubx expression throughout development. The severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in mutants. With 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development (Irvine, 1991).

The Ubx gene is required to specify the third thoracic and first abdominal segments. Mutations in the bithoraxoid (BXD) region, a 40 kb DNA stretch upstream of the Ubx promoter, affect cis-regulatory elements responsible for the ectodermal expression of the Ubx gene in the posterior compartment of the third thoracic segment and anterior compartment of the first abdominal segment. Genetic combinations involving mutations affecting the bxd region show that (1) redundant or cooperatively acting sequences are required for Ubx gene expression in the anterior compartment of the first abdominal segment, and (2) the expression of Ubx in the posterior compartment of the third thoracic segment is modulated by positive and negative cis-regulatory elements (Castelli-Gair, 1992a).

An upstream control region (a BXD fragment) from Ubx confers a Ubx-like expression pattern in the embryonic ectoderm. There are several distinct enhancer elements spread through the whole BXD fragment each of which is active in transformed embryos, mediating a different pattern of beta-galactosidase expression in the ventral nerve cord. The strongest of these patterns mimics Ubx expression within the Ubx domain. This pattern is strictly dependent on Ubx function. Thus, the BXD control region contains a Ubx response element, suggesting that positive autoregulation of Ubx may occur in the central nervous system of the developing embryo (Christen, 1992).

A 500 bp DNA fragment, approximately 30 kb away from the structural gene, contains one of the distant Ubx regulatory elements known as BRE. During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). The HB protein binds to the BRE element. Such binding is essential for HB repression in vivo. HB protein also binds to DNA fragments from two other regulatory regions. HB represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps (Qian, 1991).

A 14.5-kb fragment from the postbithorax/bithoraxoid region of Ultrabithorax exhibited proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. The integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation (Chang, 1995).

The Ultrabithorax gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Although the function of much of the Ubx DNA is unknown, it is clear that many elements required for the normal Ubx expression pattern lie distant from the Ubx promoter. The anterobithorax (abx) and bithorax (bx) mutations, located as much as 35 kb downstream of the Ubx promoter, show loss of the PS5 pattern of expression in the embyronic central nervous system and produce PS5 transformations in the adult (e.g., anterior haltere to wing). Likewise, bithoraxoid (bxd) or postbithorax (pbx) mutations, which lie as far as 45 kb upstream of the Ubx promoter present a reduction in Ubx expression in PS6 or in the posterior region of the haltere discs. These two mutations transform (respectively) the embryonic cuticle of PS6 into PS5 and the posterior haltere into the posterior wing. These distant regulatory regions probably influence the promoter by looping. Most models of looping would posit a target site close to the promoter with which the distant enhancers would interact (Casares, 1997 and references).

The distribution of Polycomb protein has been mapped at high resolution on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Sheared chromatin was immunoprecipitated and amplified by linker-modified PCR, before using as a probe on a Southern of the entire PX-C walk. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Among the identified sites are peak F (the bxd PRE) and peak G (the bx PRE), both of which contain GAGA consensus sequences. Three other sites, E, D and C correspond to iab2, iab3 and iab4. No PC binding is seen in the regulatory domains iab6, iab7 or iab8, indicating that these domains positively regulate Abd-B expression. These results suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. The Abd-B sites include the iab7/iab8 regulatory region, and the Fab-7 PRE. The Fab-7 PRE does not bind Polycomb. It is proposed that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs (silencing elements) and as trithorax group response elements. It is suggested that a GAGA site in the Antennapedia promoter is both a PRE (binding PC protein) and a TRE (binding GAGA) factor (Strutt, 1997).

Two P-element "enhancer traps" have been recovered within Ubx that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lacZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome. Transvection depends on the proximity of the regulatory sequences on one chromosome to the promoter of the homolog (the second chromosome), since rearrangements that separate them eliminate or reduce transvection. It is concluded that the looping model is not adequate to describe the function of distal enhancers nor the roles of proximal promoters. The Ubx promoter and nearby sequences are not required to establish a normal late embryonic pattern, and the cloned enhancer regions function autonomously to direct site specific expression (Casares, 1997).

To test for a chromatin structure involved in Polycomb group repression, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax gene, which is regulated by PcG proteins. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bithorax region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins (McCall, 1996).

Mutations in zeste do dot affect the cis-regulation of endogenous Ubx, but expression of small Ubx promoter constructs are strongly dependent on zeste. This difference is due to redundant cis-regulatory elements in the Ubx gene, which presumably contain binding sites for factors that overlap in function with Zeste. (Laney, 1996).

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7') dominantly transforms parasegment 11 into parasegment 12. This chromosomal region contains both a boundary element and a silencer. Previous studies have suggested that removal of a domain boundary element on the proximal side of Fab-7' is responsible for dominantly transforming gain-of-function phenotype. The Fab-7 boundary element maps to two nuclease hypersensitive sites, HS1 and HS2. This article demonstrates that the Fab-7' deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment (the HS3 site) and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of an artificial BXD, Ubx/lacZ reporter transgene. Both mini-white and Ubx/lacZ silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste' allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans the HS3 nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. It is suggested that PRE-PRE cooperation, either in trans or in cis, may be an important feature of the silencing process within the BX-C and that boundaries may limit PRE-PRE cooperation (Hagstrom, 1997).

The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodeling and human cancers. The role of the POZ domain of the GAGA transcription factor (Trithorax-like) in promoter recognition has been examined. Natural target promoters for GAGA factor typically contain multiple GAGA-binding elements. The POZ domain mediates strong co-operative binding to multiple sites but inhibits binding to single sites. Promoters regulated by GAGA have been identified by in vivo as well as in vitro studies. The Ultrabithorax (Ubx), fushi tarazu (ftz), hsp70 and evenskipped (eve) promoters were used to compare the binding of GAGA polypeptides. All these promoters are characterized by the presence of multiple GAGA-binding sites. DNase I footprinting experiments reveal a dramatic difference in DNA-binding properties between full-length GAGA and the polypeptides lacking the POZ domain. The GAGA elements on the natural promoters are bound efficiently by full-length GAGA but not by equal molar amounts of either deltaPOZ (lacking the POZ domain) or a construct possessing only the DNA binding domain (DBD). The amount of GAGA required to bind the multiple promoter elements is significantly lower (>4- to 12-fold, depending on the promoter) than that required to bind a single site, indicative of co-operative DNA binding. The spacing of the GAGA elements in these different promoters varies considerably. However, GAGA appears to be quite flexible and able to bind co-operatively to GAGA sites located at variable distances from each other. The hsp70 promoter is generally GA rich and, at increasing GAGA concentrations, the footprints start to spread and most of the promoter DNA is protected against digestion (Katsani, 1999).

In contrast to full-length GAGA, equal molar amounts of the deltaPOZ or DBD polypeptides fail to bind the GAGA target promoters significantly. On the Ubx, ftz and eve promoters, protection of a single GAGA site by deltaPOZ and DBD can be observed. As expected, these sites are the ones that most closely resemble the optimal GAGA-binding sequence. In these experiments, deltaPOZ and DBD fail to bind to the weaker GAGA sites. This indicates that POZ-mediated co-operativity increases the binding affinity for these sites by at least one order of magnitude. Together, these DNase I footprinting experiments demonstrate that efficient binding of GAGA to its natural target promoters depends critically on the presence of the POZ domain, in addition to the DBD (Katsani, 1999).

Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy reveals that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. These results indicate a novel DNA binding mode by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. A model is proposed in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation. Since the GAGA DBD clamps almost one turn of the DNA, GAGA binding to multiple sites within a nucleosome repeat length is expected to severely compromise histone-DNA contacts. These contacts might be hampered further by DNA bending and wrapping around a GAGA oligomer. However, it is not clear whether GAGA binding leads to complete displacement of the histone core or whether some histone-DNA contacts are preserved. In summary, after transient chromatin remodelling by NURF to allow for GAGA binding, GAGA may function as an architectural factor that reorganizes the promoter DNA and maintains it in an open conformation (Katsani, 1999).

The Ultrabithorax gene includes two functionally distinguishable regions. One is the Ubx transcription unit, which gives rise by alternative splicing to a family of morphogenetic UBX proteins. The other is its upstream bithoraxoid (bxd) region. On the basis of genetic and molecular studies, it is generally assumed that the Ubx transcription unit contains internal positively acting cis-regulatory elements controlling Ubx expression in the T3a compartment of the body of Drosophila, while the bxd region contains positive cis-regulatory elements controlling UBX expression in the T3p and A1a compartments. A genetic analysis has been performed of bx bxd cis double mutant chromosomes containing one mutation (bx alleles) affecting the Ubx unit, and a second (bxd alleles) affecting the bxd region of the Ubx gene. Different bx bxd/bx combinations show that bxd alleles partially rescue the adult mutant phenotypes of bx alleles, which suggests that the bxd region contains a negative cis-regulatory element involved in the control of the activity of the Ubx gene in the T3a compartment (Martinez-Laborda, 1996).

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. The ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ubx PRE has been tested. The gypsy element and the scs element interposed between PRE and the miniwhite gene protect miniwhite against silencing but the scs element is only weakly effective. Blocking the action of gypsy requires su(Hw). When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, the construct can still establish efficient silencing in some lines but not others. This silencing can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action. Thus blocking elements can prevent communication along a chromatin fiber, and enhance silencing of PREs in trans (Sigrist, 1997).

The suppressor of Hairy-wing [su(Hw)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the su(Hw) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. These studies investigate whether repression resulting from the Polycomb group genes (derived from a gypsy element) can be blocked by the su(Hw) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group response element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. The su(Hw) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome (Mallina, 1998).

Polycomb response elements (PREs) in several genes contain conserved sequence motifs. One of these motifs is the binding site for the protein coded for by the recently cloned gene polyhomeotic (pho), the Drosophila homolog of mammalian YY1. The conserved sequence extends beyond the YY1 core consensus sequence suggesting that parts of Pho may impose additional DNA sequence requirements. In this respect and unlike YY1, PHO has an additional 45 amino acids following the fourth zinc finger. It is also possible that Pho may bind to PREs together with another protein in order to fully exploit the conserved sequence. The conserved sequence motif CNGCCATNDNND, includes the YY1 core consensus CCATNWY. Eight consensus sites have been identified in 6 PREs of the bithorax complex (BX-C): bxd, iab-2, Mcp, iab-6, iab-7 and iab-8. The bxd PRE harbors all three characteristics used to define PREs (maintenance of expression of a lacZ reporter assay throughout development; pairing-sensitive repression of a mini-white reporter, and creation of an additional chromosomal binding site of the PcG-repressing complex in a salivary gland assay). The iab-2 PRE contains two homology boxes (a and b) and has been identifed in the maintenance and pairing-sensitive assays. The Mcp and iab-6 PREs have been characterized in the pairing-sensitive assay. The iab-7 PRE contains two homology motifs, a and b. This PRE has been characterized in all three assays. The iab-8 PRE has been identified in the maintenance assay. The conserved sequence motif is found in three PREs from Sexcombs reduced regulatory regions, and has been identified in the pairing-sensitive assay. The sequence motif found in two PREs from the engrailed regulatory region has been characterized in the pairing-sensitive assay. The sequence motif is also found in polyhomeotic, and has been identified in the pairing-sensitive and salivary gland assays (Mihaly, 1998)

A functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila bithorax complex (BX-C) has been undertaken. Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an ~230-bp chromatin-specific nuclease-hypersensitive region called HS3. HS3 has been shown to be capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. GAGA and Pho interact with these sequences in vitro, and the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo (Mishra, 2001).

The iab-7 PRE was initially identified in transgene assays using fragments from the iab-6 to -7 region of BX-C. These studies showed that an 860-bp iab-7 fragment can establish and maintain Pc-G-dependent silencing complexes in two different assays: the pairing-sensitive silencing of mini-white and the maintenance of parasegmentally restricted patterns of Ubx:LacZ expression. At the proximal end of this 860-bp fragment is the ~230-bp nuclease-hypersensitive region, HS3. Since Pc-G-dependent silencing is generally believed to involve a marked reduction in DNA accessibility, not enhanced accessibility, it is important to determine whether this nucleosome-free region of chromatin plays any role in the silencing activity of the iab-7 PRE. Two lines of evidence argue that sequences in HS3 are critical for silencing activity: (1) it has been shown that a small 260-bp fragment spanning HS3 is sufficient to mediate Pc-G-dependent silencing activity in the mini-white assay; (2) site-directed mutagenesis experiments indicate that sequences essential for silencing activity map to HS3 (Mishra, 2001).

An attractive hypothesis is that HS3 provides accessible target sequences for one or more sequence-specific DNA binding proteins. In this model, these DNA binding proteins would interact with their cognate sequences in HS3 and nucleate the assembly of Pc-G silencing complexes by recruiting Pc-G proteins. It seems likely that nucleosome-free regions of chromatin play a similar role in the functioning of other PREs. For example, the three other known PREs in the Abd-B cis-regulatory region, the iab-8 PRE, the iab-6 PRE, and Mcp, all map to small DNA fragments that contain one or more prominent nuclease-hypersensitive sites. Of these, the Mcp PRE has been characterized in the most detail. Like the iab-7 PRE, the nuclease-hypersensitive region of Mcp is essential for its silencing activity. However, it is not sufficient on its own to direct the assembly of functional silencing complexes, and adjacent proximal or distal flanking sequences are required. The chromatin structure of the Mcp element at ectopic sites has also been examined. (A ftz-LacZ transgene was used in this analysis. Unfortunately, the mini-white transgenes are not suitable for examining the chromatin structure of the iab-7 PRE fragments.) The transgene Mcp element has a nuclease-hypersensitive region of approximately the same size and position as that of the endogenous element.

These experiments also indicate that two DNA binding proteins, the GAGA factor and Pho, interact with target sites in HS3 and play a critical role in the silencing activity of the iab-7 PRE. The GAGA factor was initially identified as a potent activator of transcription in nuclear extracts and has generally been thought to be involved in the activation rather than the repression of gene expression. The stimulatory activity of the GAGA factor appears to be due to its ability to prevent histones and other repressive proteins from associating with promoters that have GAGA binding sites. In in vitro chromatin assembly experiments the GAGA factor facilitates the formation of a nucleosome-free region of chromatin across the hsp70 promoter. In vivo, mutations in the GAGA binding sites of heat shock promoters reduce promoter accessibility and suppress transcription. Further support for a role in transcriptional activation comes from genetic studies on mutations in Trl, the gene encoding the GAGA protein. Trl mutations exhibit genetic interactions with homeotic genes in BX-C that are hallmarks of the trx-G genes, not the Pc-G genes. Additionally, the expression of several pair rule genes that have GAGA binding sites in their promoters is severely reduced in embryos from Trl mutant mothers (Mishra, 2001).

Although it is now well established that the GAGA factor promotes the transcription of many different genes, the results argue that this protein must also play an essential role in the silencing activity of the iab-7 PRE. Several lines of evidence support this conclusion: (1) the silencing activity of the iab-7 PRE is impaired by Trl mutations; (2) the GAGA protein binds to the iab-7 PRE both in vivo and in vitro; (3) mutations in the GAGA binding sites of the iab-7 PRE eliminate GAGA protein binding in nuclear extracts and abrogate silencing activity in vivo (Mishra, 2001).

What role does the GAGA factor play in the silencing activity of the iab-7 PRE? At this point the most plausible hypothesis is that the GAGA factor is required to generate a nucleosome-free region over HS3. In this view, the function of the GAGA factor would be analogous to its presumed role in gene activation, namely, to ensure that sequences in HS3 are accessible for the assembly of large multicomponent protein complexes. When the GAGA protein is reduced as in Trl mutants or when the GAGA binding sites are mutant, it is suggested that the HS3 nucleosome-free region will not be formed properly. As a consequence, target sequences for the DNA binding proteins (such as possibly Pho) that are actually responsible for recruiting the large Pc-G silencing complexes to the PRE would be unavailable. While this hypothesis is consistent with the well-documented activities of the GAGA factor at promoters both in vitro and in vivo, the possibility that GAGA is not only required for the formation of HS3 but also plays a more active role in recruiting Pc-G proteins to the iab-7 PRE cannot be excluded. Supporting this hypothesis, it has been shown that GAGA binding is required for the in vitro association of Pc-G complexes with fragments from the bxd PRE (Mishra, 2001).

The other protein that is critical for the silencing activity of the iab-7 PRE is Pho. Like the GAGA factor, Pho appears to function by directly interacting with target sequences in HS3. Several lines of evidence support this conclusion: (1) the silencing activity of the iab-7 PRE in vivo depends on pho function and is eliminated by mutations in the pho gene; (2) the Pho protein binds to two conserved target sequences in the iab-7 PRE; (3) mutations in these two sites not only eliminate binding in vitro but also compromise silencing activity in vivo. Pho has also been directly implicated in the silencing activity of three other PREs, one from the en gene and two from BX-C. The Pho protein has been shown to bind to these PREs in vitro, while mutations in either the Pho binding sites or in the pho gene itself reduce or eliminate silencing (Mishra, 2001).

Unlike that of Trl, the phenotypes of pho mutants are similar to those seen for other Pc-G genes. Animals homozygous for loss-of-function alleles die at the pupal stage and exhibit homeotic transformations of legs and abdomen. The late lethal phase is due to a substantial maternal contribution, and mutant embryos lacking a maternal source of wild-type Pho die with severe homeotic transformations and other developmental defects. The homeotic transformations evident in mutant animals indicate that pho is likely to have a direct role in Pc-G silencing. For the iab-7 PRE, the results argue that silencing activity depends on the binding of the Pho protein to the two target sites in HS3. Both sites seem to be important, since silencing activity is compromised when one site is deleted. Whereas it is supposed that the major function of the GAGA factor is to ensure that sequences in HS3 are accessible to other proteins, the phenotypic effects of pho mutations suggest that it plays a more active role in silencing. A plausible hypothesis is that it functions (perhaps together with as yet unidentified factors) to recruit components of the silencing machinery to the PRE, such as Polycomb or Sex Combs Midleg, which do not appear to interact directly with DNA. Supporting the possibility that other factors besides Pho play a critical role in recruiting Polycomb group complexes, a PRE fragment from iab-2, which contains Pho binding sites and which is able to silence mini-white, has been shown to be insufficient to confer full Pc-G maintenance activity. Moreover, mutations in the two Pho binding sites have only a minor effect on the maintenance activity of the 860-bp iab-7 PRE fragment in an iab-7 Ubx-LacZ assay system. Clearly it will be of interest to identify these other factors (Mishra, 2001).

Ubx regulation: Table of contents

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