Ultrabithorax
Proximal promoter 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).
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)
In Drosophila, two classes of genes, the
trithorax group and the Polycomb group, are
required in concert to maintain gene expression by regulating chromatin
structure. Trithorax protein (Trx) binding elements have been identified
within the bithorax complex. Within the
bxd/pbx regulatory region these elements are functionally
relevant for normal expression patterns in embryos and they confer Trx
binding in vivo. Trx binding elements have been localized to three closely situated sites
within a 3-kb chromatin maintenance unit with a modular structure.
Results of an in vivo analysis have shown that these DNA fragments (each
~400 bp) contain both Trx- and Polycomb-group response elements (TREs
and PREs) and that in the context of the endogenous
Ultrabithorax gene, all of these elements are essential for
proper maintenance of expression in embryos. Dissection of one of these
maintenance modules has shown that Trx- and Polycomb-group responsiveness
is conferred by neighboring but separable DNA sequences, suggesting
that independent protein complexes are formed at their respective
response elements. The activity of this
TRE requires a sequence (~90 bp) that maps to within several tens of
base pairs from the closest neighboring PRE and the PRE activity
in one of the elements may require a binding site for Pho, the protein
product of the Polycomb-group gene
pleiohomeotic. These results show that long-range maintenance
of Ultrabithorax expression requires a complex element
composed of cooperating modules, each capable of interacting with both
positive and negative chromatin regulators (Tillib, 1999).
A PCR-linked immunoprecipitation procedure has been developed in order to discover Trx specific DNA binding sites. This procedure involves PCR amplification of DNA fragments retained in a pellet after immunoprecipitation of TRX-DNA complexes from embryonic nuclear
extracts using Trx-specific antibody.
Trx protein
localizes to 10 discrete fragments of the BX-C. The identification of
TRX binding regions within the regulatory DNA of all three genes of the
BX-C, (Ubx, abdominal-A [abd-A], and
Abdominal-B [Abd-B]), is striking because trx is required to maintain the expression levels of all
three genes in embryos. Next,
high-resolution mapping of the Trx binding sites was carried out. Since the sequence of the
entire BX-C is now available, the identified Trx binding sites were mapped to DNA fragments from
200 bp to 2,000 bp in size. Trx binding sites are found in several regulatory regions of the BX-C: abx,
bxd/pbx, iab-2, iab-3,
iab-4, iab-7, and iab-8. A
number of studies have defined PREs and
Pc protein binding sites in the BX-C. Comparison of
the data in this study with those of previous studies has shown that six Trx binding sites in
bxd/pbx, iab-3, iab-4, and
iab-7 regulatory regions are either within or very close to minimal PREs or PC binding sites identified previously. Interestingly, several signals are detected in the bxd/pbx
region, suggesting that there are multiple TRX binding sequences within this regulatory region (Tillib, 1999).
Two criteria
identify TRE and PRE activities within the reporter constructs:
maintenance of lacZ reporter expression patterns in embryos,
and trx- and PcG-dependent maintenance of white gene expression in the eyes of adult flies, including
their effects on pairing-sensitive repression of white.
Ultimately the results obtained with both reporter genes led to similar
conclusions, and the TRE and PRE activities of one module, fragment C of the Ubx promoter, were shown to
be conferred by neighboring but separable DNA sequences. An essential TRE and two distinct PREs were detected in this
central C module. Further analysis has shown that the TRE activity and TRX binding require a 90-bp region, which, based on its length, is
likely to bind more than just a single protein. This suggests that a
number of primary DNA proteins may be associated with the TRE in the C1
fragment. A gel-shift analysis of the protein binding properties of
this 90-bp TRE fragment suggests that this fragment contains two core
binding sequences that are required for apparent cooperative binding
by several nuclear proteins. These core sequences,
which are located on the boundary of the C1-B and C1-C fragments and in
the C1-D fragment appear to contribute to the formation of a large
multiprotein complex. [Note: The fragments referred to have the following locations in the bxd region of Ubx: C1 (nt 218,835 to 218,959); C2 (nt 218,960 to 219,088); C3 (nt 219,089 to 219,249). DeltaBC1-A, DeltaBC1-B, and DeltaBC1-C are deletions of nt 1 to 27, 28 to 61, and 86 to 122, respectively]. There is a direct correlation between
the sequences that are required to form this protein complex and those that are required for the Trx binding and TRE activity. Most
strikingly, the AACAA repeat in an addition fragment, the C1-D fragment, seems to be crucial
for forming this complex: it has been shown to be crucial for the TRE
activity, since complex formation is virtually abolished when the AC
residues are changed to TG. Since direct Trx
protein binding to DNA has not been established by using a number of
DNA binding assays and since the DNA binding protein complex in the C1
fragment does not contain Trx, it is suggested that Trx
binds to this TRE through interactions with a complex of primary DNA
binding proteins. At present, the identity of the
proteins that associate with these TREs is unknown. While it would not be
surprising to find that some are products of the trxG, it is
unlikely that the Gaga factor or the Zeste protein are the primary binding
proteins in this case, since the C1 element does not contain consensus binding sites for either of these proteins (Tillib, 1999).
This analysis suggested that the C2 and C3 fragments each contain a PRE.
Each of these elements, C2 and C3, is required to confer
pairing-sensitive repression on a white reporter gene. These
elements may be functionally different because their activities require
different sets of PcG proteins and because there is no
significant sequence similarity between them. Both PREs are apparently
also required, in concert, to provide full maintenance function in
embryos. This follows from the fact that while very strong anterior
overexpression occurs in embryos when the entire C fragment is deleted,
only moderate overexpression in PS5 results from the deletion of a
single PRE. In addition, one of these PREs, C3, may
contain a functionally important binding site for the PcG protein Pleiohomeotic (Pho), since deletion of this binding site abrogates both
pairing-sensitive repression and responsiveness of the white
reporter gene to three PcG mutations: pho,
Pcl, and Scm. Therefore, it is suggested
that the protein products of these three PcG genes may be
components of a putative PcG protein complex that is formed at the C3
PRE. In addition to the Pho binding sites, the C2 and C3 DNA fragments contain three consensus binding sites for the GAGA protein (Trithorax-like).
Further analysis is required to determine whether the deletion of GAGA
sites has an effect on PRE function. It is likely, however, that Pho
and GAGA are not the only primary DNA binding proteins in these PREs,
since the C2 PRE does not contain consensus Pho binding sites. These
proteins may be DNA-interacting components of particular subsets of PcG
complexes (Tillib, 1999).
The TREs and PREs in the bxd region of Ubx
are clustered in three closely situated maintenance modules, each
approximately 400 bp. Each module contains elements for both of these
opposing activities. The analysis has been focused on the Trx protein,
although it is possible that there are other positive maintenance
elements in this region that require the products of other
trxG genes. Similarly, since PRE function was analyzed only
in Trx binding regions, some PREs may have gone undetected. Despite
these limitations, several TRE- and PRE-containing modules were discovered in the bxd region of Ubx that are all
essential for proper Ubx expression, since deletion of any
one of the three modules leads to a significant loss of maintenance
activity in embryos. In the context of a natural Ubx
promoter and a large part of its regulatory region, these modules are
all essential in embryos with respect to PRE and TRE function. However,
when tested for effects on white gene expression, deletion
of individual elements does not completely abolish either eye color
variegation or the responsiveness to trx and PcG
mutations. These differences suggest that repression of
white expression in adults may not accurately reflect the
function of these elements in the context of the entire Ubx
gene. Thus, cooperative interactions among multiple PREs and TREs are
required for proper function of the Ubx gene, and these interactions may involve activities not reflected in assays with reporters unrelated to Ubx expression (Tillib, 1999).
Interestingly, trx function is found to be required for
Ubx expression not only in its normal domain of expression
in the posterior region of the embryo but also in the anterior region, when Ubx is overexpressed due to a deletion of PREs. There
are clear differences between the anterior and posterior regions of the
embryo with respect to both the effect of a trx mutation and the requirements of TREs for the expression patterns of these transgenes. (1) In trx mutants, the loss of expression in the
anterior is much more severe than it is in the posterior. (2)
Anterior expression is very strong when one of the three TREs is
deleted, and only a simultaneous deletion of two TREs leads to a
decrease of this expression that is comparable with the effect of a
trxB11 null mutation. In the posterior, however,
deletion of only one element mimics an almost complete loss of
trx-related activity, and deletion of two elements has no
further effect. This might be explained by a different mode of
functioning in the anterior versus posterior regions of the embryo.
Such a functional difference is also suggested by the
observation that different trx protein products, which
result from alternatively spliced mRNAs, may be required for the
maintenance of expression of the ANT-C genes (in the anterior region)
and not for maintenance of BX-C gene expression (in the posterior
regions). This is based on an analysis of the effects of different
trx alleles on the two homeotic complexes and on the finding
that the expression of one of the early trx RNAs is
spatially restricted to the posterior region where the BX-C genes are
expressed. Based on these observations, it has been concluded
that there are quite complex requirements for the activities of the
three maintenance modules in different cells. Functionally similar
maintenance units may regulate other genes in the BX-C as well, since
the other Trx binding regions detected are associated with either
PRE function, Pc protein binding sites, or both (Tillib, 1999).
The finding of discrete TRE and PRE sequences
argues against a direct competition between the proteins of these
opposing groups for binding sites on DNA, although the question of
whether they normally occupy their response elements simultaneously
within a given module remains open. In addition, some data suggest that the association of trxG and PcG proteins with a particular gene depends
on its transcriptional status: (1) the strength of Trx binding to
salivary gland polytene chromosomes at the site of a transcriptionally
active gene, such as fork head, is much higher than it is at
the location of the BX-C, which is silent in the salivary glands; (2) immunoprecipitation of Pc protein from
Drosophila cells has been found to be more abundant at silenced
genes than at activated genes of the BX-C; (3) when
transcription of a reporter gene is induced by GAL4, several PcG
proteins are displaced from the chromosomal site of insertion of a
Fab-7 transgene. Although this is not
directly related to trxG functioning, it indicates that PcG proteins
are not bound abundantly to actively transcribed genes, suggesting that
there might be quantitative or qualitative differences in bound trxG
and PcG protein complexes depending on the transcriptional activity of
a particular gene. This work suggests that the occupancy of TREs and
PREs may be independent rather than mutually exclusive. Since the
formation of functional activating or silencing complexes may depend on
and in turn lead to the maintenance of the on-off state of
Ubx expression in a particular tissue, it is suggested that the
occupancy of the TRE by a functional trxG complex alters, directly or
indirectly, the composition of nearby PRE complexes without necessarily
abolishing binding by PcG proteins (Tillib, 1999).
How do active trxG and PcG protein complexes function? There have as
yet been no specific biochemical activities associated with PcG
proteins. Most of the PcG proteins are associated with chromosomes, and
it is assumed that they act by forming repressive multiprotein
complexes that prevent active transcription. One of the functions of
trxG proteins may be simply to counteract the formation of these
repressive PcG complexes and thus to increase the accessibility of
enhancers to the neighboring regions of DNA. However, there is growing
evidence that the trxG represents a heterogeneous family of proteins
with diverse functions. Some of them, such as Trx, Ash1, Ash2, Gaga,
and Zeste, are associated with particular sites on polytene chromosomes, while others, such as Brm and Snr1,
are found in chromatin remodeling complexes that may not be associated
with specific chromosomal regions. There is some evidence that one of
the functions of trxG proteins may be to recruit chromatin remodeling
complexes to DNA. Gaga factor is required for the function of one
chromatin remodeling complex, the Drosophila NURF complex, and Trx has been shown to physically interact with Snr1, a component of the Drosophila SWI/SNF complex.
However, there is no evidence thus far that these interactions are
mediated through particular TREs. In addition, there is evidence that
Trx and its human homolog, ALL-1/HRX, may be involved directly in the
activation of promoters, since both of these proteins possess
transactivation activity in cells. Therefore, it
is likely that trxG proteins not only can counteract formation of
PcG-mediated repressive chromatin structure but may also play a more
direct role in maintaining transcription (Tillib, 1999 and references).
In conclusion, three discrete TRE/PRE modules have been identified in the Ubx regulatory
region. These modules are contained within a complex, 3-kb maintenance unit in which each detected element
is essential with respect to both PRE and TRE function. Furthermore, it has been found that Trx binds sequences in other regulatory regions of the BX-C
that are consistently associated with either PRE function, Pc protein
binding, or both; this suggests the possibility that similar maintenance
units are employed for regulation of other genes in the complex.
Functional dissection of one of these modules has shown that the TRE and
PRE activities can be ascribed to separable DNA elements, even though
they are located within tens of nucleotides of one another. This
proximity suggests that there may be some direct interaction between
protein complexes formed at these elements. In addition, the TREs and
PREs that have been identified do not contain extensive sequence
similarities, suggesting that they are bound by protein complexes of
different composition (Tillib, 1999).
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).
P elements containing a 7 kb DNA fragment from the
middle of the Drosophila bithorax complex insert
preferentially into the bithorax complex or into the
adjacent chromosome regions. The 7 kb fragment does not contain any known
promoter, but it acts as a boundary element separating
adjacent segmental domains. An enhancer-trap P element
was constructed with the homing fragment and the
selectable marker flanked by FRT sites. P insertions can be
trimmed down by Flp-mediated recombination to just the
lacZ reporter, so that the beta-galactosidase pattern is not
influenced by sequences inside the P element. Twenty
insertions into the bithorax complex express beta-galactosidase
in segmentally limited patterns, reflecting the
segmental domains of the bithorax complex where the
elements reside. The mapping of segmental domains has
now been revised, with enlargement of the abx/bx, bxd/pbx,
and the iab-3 domains. The FRT sites in the P elements
permit recombination between pairs of elements on
opposite chromosomes, to generate duplications or
deletions of the DNA between the two insertion sites. Using
this technique, the length of the Ultrabithorax transcription
unit was varied from 37 to 138 kb, but there was
surprisingly little effect on Ultrabithorax function (Bender, 2000).
The segmental domains of the BX-C can now be mapped with
precision, allowing for investigation of the roles of parasegment
enhancers, Polycomb response elements, and boundary
elements in the creation of these domains. The enhancer traps
reported here redefine several segmental domains. The most
proximal group (HC71-1, HC179, and HC16-1)
show that the PS5 domain extends at least 20 kb more proximal
than the proximal-most bx mutant lesion, almost to the final exon of the Ubx transcription unit.
Likewise, the HC184B line extends the limit of the bxd region,
mapping 15 kb distal to the most distal bxd mutation so far
reported.
The most surprising observation is that both the HCJ192B and
HCJ200 insertions give lacZ patterns with the anterior edge at
PS8, thus extending the iab-3 domain. Two rearrangement breaks
proximal to the HCJ200 site had been called iab-4 lesions in a
prior analysis. These
should now be called iab-3 alleles. There is no contradiction
implied by the redefinition, because breaks in the iab-3 domain
also lack iab-4 function: they cut the iab-4 domain away from
the abd-A gene it is meant to regulate. The iab-4 domain may
begin just distal to the HCJ200 site; another P element insertion
less than 2 kb from HCJ200 shows lacZ expression with a PS9
anterior boundary (Bender, 2000).
Models for P element homing generally invoke a homotypic
interaction between proteins bound to the homing fragment and
identical proteins bound to the genomic copy of that DNA
fragment. Such binding would tether the P element donor
plasmid before P transposition. Most DNA binding proteins are
bound to hundreds of places throughout the genome; therefore,
homotypic interactions of such proteins would target a P
element to so many places that insertions might appear random.
P elements containing Polycomb response elements may be
homing to many endogenous Polycomb binding sites in this
manner.
Homing to a single chromosomal location, as is reported here,
would require that the DNA binding proteins mediating the
homing be present at only one or a few sites in the genome.
A DNA element unique to the homeotic complexes is perhaps
the boundary element separating segmental domains. Such
boundaries have been best defined by the Fab and Mcp deletions. The homing fragment appears to contain
a boundary element; it can
block Polycomb repression from either side. From its position
in the BX-C, the normal function of this boundary must be to
separate the bxd and iab-2 domains (Bender, 2000).
Trithorax (Trx) and Ash1 belong to the trithorax group (trxG) of transcriptional activator proteins -- this group of proteins maintains homeotic gene
expression during Drosophila development. Trx and Ash1 are localized on chromosomes and share several homologous domains
with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions
between trx and ash1 and the observation that association of the Trx protein with polytene chromosomes is ash1 dependent,
the possibility of a physical linkage between the two proteins was investigated. Endogenous Trx and Ash1 proteins
coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, Trx and Ash1 bind in
vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of
ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that Ash1 and Trx act on Ubx in
relatively close proximity to each other. Finally, Trx and Ash1 appear to interact directly through their conserved SET domains, based on binding assays in vitro
and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that Trx and Ash1 are components that interact either
within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription (Rozovskaia, 1999).
Since genetic experiments suggest that both trx and ash1 are involved in regulation of homeotic gene expression, it was of particular interest to determine whether
binding of the proteins to polytene chromosomes and/or genetic
responsiveness is conferred by the same DNA sequences. To test this, an analysis was performed to see whether both proteins bind in vivo to a
well-characterized TRE-PRE-containing bxd regulatory module
located 25 kb upstream of the Ubx promoter. Indeed, on salivary gland polytene chromosomes, both
proteins are found at the site of insertion of a transgene containing
this 4-kb bxd subregion. This indicates that Trx
and Ash1 DNA binding elements may be close to each other. In addition,
it has been shown that Ash1 is required for full function of the same
regulatory region in vivo. Since this 4-kb region contains
three trx-responsive TREs, this leaves open the possibility
that Trx and Ash1 may function through the same DNA elements.
Experiments aimed at fine mapping of the ash1 response
element(s) within this region of Ubx are currently in
progress. Nonetheless, these results suggest that Trx and Ash1 may act in
concert on one or more bxd TREs. Two interesting possibilities are that both Trx and Ash1 are components of the same
protein complex or that they are interacting components of two separate
protein complexes that form on closely situated TREs. The physical
association between Trx and Ash1 (probably through interaction of their
SET domains) is apparently required for Trx binding to chromosomes,
since Trx is only weakly associated
with chromosomes in ash1 mutant larvae. These close physical and functional associations
on Ubx regulatory DNA provide a biochemical rationale for
the genetic interactions between trx and ash1 mutants (Rozovskaia, 1999).
By applying yeast two-hybrid assays as well as other methodologies, it has been found that the SET domains of both Trx and Ash1 proteins can self-associate. The self-associating Trx fragment (aa 3540 to 3759) spans the ~130-aa
SET domain and includes an additional ~90 aa of upstream sequence. The self-interacting Ash1 region includes the entire SET domain (residues 1318 to 1448) in
addition to upstream sequence (aa 1160 to 1317). An alternative self-associating region of Ash1 (aa 1245 to 1525) also includes the entire SET domain. Mutations
within the SET domain of both Trx and Ash1 prevent self-association. Whether those TRX and ASH1 regions can also undergo
hetero-oligomerization was examined. Indeed, the two polypeptides interact strongly in yeast, as evidenced by activation of both the HIS and lacZ reporters. To
confirm this result, GST pull-down methodology as well as coimmunoprecipitation analysis was performed. A C-terminal Trx polypeptide (Trx SET) was synthesized
and radiolabeled in a coupled transcription-translation system and tested for binding to the relevant Ash1 polypeptide (Ash1 SET) linked to GST. The ASH1-linked resin binds 10- to 20-fold more Trx SET than does GST resin alone.
For in vitro coimmunoprecipitation analysis, the same Trx polypeptide was radiolabeled and mixed with unlabeled epitope-tagged (T7) Ash1 SET. The labeled
Trx SET coimmunoprecipitates with the T7-ASH1 SET but not with two unrelated T7-tagged proteins. Similar results were obtained in a reciprocal
experiment. Finally, plasmids encoding the T7-tagged Ash1 SET and HA-tagged Trx SET were transiently cotransfected into COS cells. The epitope-tagged
polypeptides produced in vivo were also found to coimmunoprecipitate (Rozovskaia, 1999).
To address the biological significance of this hetero-oligomerization, conserved residues within the SET domain were mutagenized and their effects on interaction
in yeast were tested. Thirteen different mutations at either single amino acids or nearby pairs of amino acids were constructed, 10 at highly conserved residues and 3 controls at
nonconserved residues within Trx SET. Each of the alterations of conserved amino acids resulted in the loss of most or all of the capacity of TRX SET to interact
with Ash1 SET in yeast. In contrast, the three alterations of nonconserved residues, located within the SET domain or immediately upstream of it, did not
affect the interaction. A more limited mutagenesis analysis of
conserved residues within the Ash1 SET domain shows that conversion of GRG (residues 1310 to 1321) to VRV, PN (1391 and 1392) to AY, I (1414) to A, or
DY (1423 and 1424) to AA results in the loss of most or all of the interaction in yeast. These results argue for the functional significance of the TRX
SET-ASH1 SET interactions seen in yeast and in vitro and suggest that the association in embryos between full-length Trx and Ash1 is direct and involves binding
between their SET domains (Rozovskaia, 1999).
Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. UbxVMB, a visceral mesoderm-specific enhancer of the Ubx gene, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf (Pangolin) binding sites in the UbxVMB enhancer. Thus, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on UbxVMB. A possible target of this activity is Pangolin. Since Pangolin has been shown to be capable of repressing Wingless targets, these results suggest that this repressive activity may be regulated by Notch. It is suggested that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of Pangolin and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with Pangolin (Lawrence, 2001).
These effects of loss of Notch function are not mediated by the Dpp responsive sites but require the integrity of at least one of two Pangolin binding sites on the enhancer, as do other activities of UbxVMB. This regulatory activity of Notch is likely to be different from that which mediates lateral inhibition since the activity of the enhancer is sensitive neither to Su(H) nor to forms of Notch, such as Nintra, that provide constitutive Notch signaling during lateral inhibition. Altogether, these results suggest the existence of an activity of Notch that antagonizes Wg signaling via Dsh. Thus, the removal of Notch function would lower the requirements for Dsh, as was observed. Similar situations have been described before in the development of muscle and peripheral nervous-system precursors in Drosophila and raise the possibility that, in addition to the Frizzled-mediated events, effective Wg signaling requires the downregulation of a Notch signaling event that might be independent of Su(H). These experiments suggest that a possible target of this event is the activity of Pangolin (Lawrence, 2001).
Members of the Pangolin family interact with Arm/ß-catenin to form complexes that can promote the transcription of Wnt/Wg targets in vitro and in vivo. However, with the exception of LEF-1, Tcf family members on their own do not promote the expression of Wnt target genes, and in some instances they can even repress the expression of these targets (Lawrence, 2001 and references therein).
The UbxVMB enhancer has provided a good model for the analysis of the role of Pangolin in Wg signaling. The optimal activity of this enhancer requires canonical Wg signaling via Dsh, Arm, and Pangolin, and for this reason it was surprising to observe that in the absence of Notch the activity of UbxVMB is independent of the canonical Wg pathway. The ability of the loss of Notch function to reverse the effects of the loss of function of Wingless signaling is most clearly demonstrated in the case of the UbxVMBM2 mutant enhancer. The activity of this enhancer is independent of Dpp but displays an absolute requirement for Wg signaling. However, while UbxVMBM2 is completely inactive in dsh mutants, it directs expression of a reporter in N;dsh double mutants (Lawrence, 2001).
The response of UbxVMB to the loss of Notch function, like that to Wg signaling, requires the integrity of at least one of the Pangolin sites in the context of the full enhancer and thus raises the possibility that these sites, and perhaps the activity of Pangolin itself, are the targets of Notch. It might be that the activity of UbxVMB is repressed by Pangolin in a Notch-dependent manner and that to signal efficiently, Wg must antagonize this repression. In the absence of Notch, this repression would not be implemented, which would lead to enhancer activity that is independent of Wg. This can account for the widespread and premature activity of the enhancer as well as the diminished requirements for Wg signaling that are observed in the absence of Notch (Lawrence, 2001).
Several observations indicate that Pangolin can act as a repressor, and recent results on the regulation of dpp expression in the VM of Drosophila support this possibility. The mutation of Pangolin binding sites in a Wg-dependent enhancer of the dpp gene results in spatially deregulated high levels of activity of the enhancer. This finding suggests that in this case Pangolin acts, primarily, as a repressor and that one function of Wg/Arm signaling might be to promote a nonrepressed state. Pangolin is also likely to act as a repressor at UbxVMB since the mutation of one Pangolin site, although lowering the overall levels of activity, expands the spatial domain of activity of the enhancer. These results provide further evidence for this repressive activity and suggest that Notch might be involved in it. However, in contrast to results with the dpp enhancer, the mutation of both Pangolin sites in UbxVMB abolishes enhancer activity. This finding indicates that at this enhancer Pangolin is also required as an activator, together with Arm (Lawrence, 2001).
In Drosophila, Pangolin can indeed behave as a repressor and an activator through interactions with different molecules. These results raise the possibility that its activity as a repressor, through interactions with transcriptional corepressors such as Groucho or CtBP, is modulated by a signaling event that depends on Notch. Wg signaling can thus lead to gene expression in two ways, by transcriptional activation through a Pangolin/Arm complex or by antagonizing the repressive activities of Pangolin. This dual activity of Wg signaling would explain the observations that in N;dsh double mutant embryos enhancer activity, although higher than in dsh mutants, is lower than that in N mutants. Thus, in N;dsh embryos the activity of the enhancer results from a derepression (inactivation of Pangolin repressor complexes) without the concomitant Arm activation mediated by Dsh (Lawrence, 2001).
The observations on UbxVMB parallel others in which Notch has been shown to antagonize Wg signaling independently of Su(H), and they raise the possibility that effective Wg signaling requires an antagonism of this repression. Interestingly, Wg and Dsh can bind to Notch, and therefore, the antagonism could be mediated by conformational changes in Notch induced at the cell surface through direct interactions between these molecules (Lawrence, 2001).
On the basis of these observations, it is suggest that in Drosophila, Wg signaling operates by regulating two molecular events: (1) repression of the expression of Wg targets implemented by Notch and (2) the Shaggy/GSK3-dependent degradation of Arm promoted by the Axin/APC complex. The regulation of both processes might be linked through the activity of Pangolin: the first event maintains its activity as a repressor, while the second one prevents its becoming an activator. Wg binding to Notch would modulate the repressive activity of Pangolin; then, through the activity of members of the Frizzled family of receptors, it would modulate the activity of Arm. It may be that Arm can only interact with a nonrepressor form of Pangolin and that, therefore, the 'effectiveness' of Wg signaling is determined by the amount of Notch signaling. A combination of antirepression and activation might help explain the observation that dominant-negative Frizzled and dominant-negative Pangolin have no effect on the activity of this enhancer, as they should have if activation was the only way to get expression. In addition, this would explain the observation that lowering Notch signaling increases the effectiveness of Arm signaling. It will be important to understand how Notch modulates Wg signaling (Lawrence, 2001).
One difficulty with this model is the activity of the UbxVMB enhancer in the absence of both Notch and Dsh since, under these conditions, the levels of cytoplasmic Arm are low. It may be that the loss of Notch function alters some parameters of the interaction between Pangolin and Arm that allow very efficient functional association of Pangolin with these low levels of Arm. In this regard there is evidence that the phosphorylation of Tcf can regulate the activity of the Tcf/ ß-catenin complex. Further work should address these issues (Lawrence, 2001).
Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila development. lola like (lolal), also known as batman (ban), functions in both activation and repression of homeotic genes. The 127-amino acid Lolal protein consists almost exclusively of a BTB/POZ domain. This domain is involved in the interaction between Lolal and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Lolal codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Lolal, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7 polycomb response element mediated pairing dependent silencing of mini-white transgene. lolal was also identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts geneticially with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl plays a role in maintaining the repressed state of target genes involving Lolal, which may function as a mediator to recruit PcG complexes (Faucheux, 2003; Mishra, 2003).
Several lines of evidence suggest a close association between Batman and Trl. In 0- to 18-h embryos, increasing the dose of Batman through the use of the Gal4/UAS system increases the formation of the Batman- and Trl-containing complexes on the MHS-70 Ubx PRE fragment, which are fully displaced by both anti-Trl and anti-Batman antibodies. This result suggests that Batman may be a Trl cofactor that modulates its binding to MHS-70. Consistent with this, lowering the dose of ban has the same effect as lowering the dose of Trl in at least two regulatory pathways: the repression of Scr, and pairing-sensitive silencing of a white reporter gene next to an AbdB PRE. In addition, ban function is necessary for the activity of Trl in the activation of Ubx. Finally, the increased lethality of Trl13c mutants when the dose of ban is reduced provides additional evidence for the functional significance of the interaction of ban with Trl (Faucheux, 2003).
The Trl13c allele is a dominant enhancer of the weak homeotic transformation of halter toward wing found in Ubx130 heterozygotes. Flies that are doubly heterozygous for banl(2)k02512 and Ubx130 do not show a significant increase in the expressivity of the Ubx phenotype. In contrast, banl(2)k02512/+; Trl13c/Ubx130 flies display a higher frequency (18%) of strong halter-to-wing transformations compared to their +/+; Trl13c/Ubx130 siblings (6.3%). The synergistic interaction between ban and Trl on the Ubx phenotype indicates that ban function is required together with that of Trl for the activation of the homeotic gene Ubx (Faucheux, 2003).
Taken together, these results suggest that ban cooperates with Trl in order to maintain the activation or the repression of Trl target genes that are necessary for viability and/or normal development of the flies (Faucheux, 2003).
Since Batman binds to many sites that are also targets of Ph and Trl on polytene chromosomes, this binding was further characterized in vitro on a defined PRE. The 70-bp MHS-70 fragment from the bxd PRE in Ubx was chosen. MHS-70 is required in vivo for the maintenance of embryonic silencing of a Ubx enhancer and binds in vitro to both Trl and Ph proteins partially purified from Kc cell nuclear extracts (Faucheux, 2003).
When tested in an EMSA (electrophoretic migration shift assay) in the presence of a nuclear extract from wild-type embryos, the radiolabeled MHS-70 probe gives rise to the formation of several retarded nucleoprotein complexes. The two slower-migrating complexes are specifically competed against by an excess of unlabeled MHS-70 probe. Batman is involved in the formation of these two complexes, since they are specifically supershifted in the presence of the batC11 antibody but not in the presence of a control anti-FLAG antibody. These complexes are formed with a much higher efficiency when MHS-70 is used as a probe in the presence of a nuclear extract from embryos expressing Batman-FLAG in an otherwise ban+ context. They are completely supershifted in the presence of the batC11 antibody, and partially supershifted in the presence of the anti-FLAG antibody, indicating that both endogenous Batman and Batman-FLAG participate in their formation. Together, these results demonstrate that Batman, as well as Batman-FLAG, binds to MHS-70 (Faucheux, 2003).
Since Batman does not contain much more than a single BTB/POZ protein-protein interaction domain, it was reasoned that its binding to DNA most likely requires an interaction with a DNA binding partner. Trl binds to GAGA sequence motifs found in MHS-70. In addition, Trl's own BTB/POZ domain provides a putative interface for dimerization with Batman. Thus, Trl may be the DNA binding partner mediating the binding of Batman to MHS-70. In order to test this hypothesis, it was first determined whether Trl target sequences are required for the binding of Batman to MHS-70. The formation of the Batman-containing complexes in the presence of a nuclear extract from Da:Gal4; UBF (UBF is the UAS:BAN-FLAG construct) transgenic larvae is specifically competed against by a 100-fold molar excess of the double-stranded GAGA oligonucleotide containing eight d(GA)3 motifs. In contrast, no competition is observed in the presence of a 100-fold molar excess of the MHS-70-derived fragment LS-1/9 in which two terminal d(GA)3 sequences have been mutated. These results indicate that the Batman-containing complexes bind to GAGA repeats in MHS-70, suggesting that their formation involves a GAGA binding factor such as Trl. Indeed, the two Batman-containing DNA-binding complexes are specifically supershifted in the presence of anti-Trl antibody, indicating that they contain Trl (Faucheux, 2003).
The preceding experiments suggest that the binding of Batman to a bxd PRE fragment involves its interaction with Trl or a Trl-containing complex. In order to test whether this interaction occurs independently from binding to a DNA target, protein immunoprecipitation experiments were performed, taking advantage of the tagged Batman-FLAG protein. Anti-FLAG antibody specifically precipitates the Batman-FLAG protein from UBF/+; da:Gal4/+ third instar larvae nuclear extracts. Under these conditions, the Trl protein efficiently coprecipitates with Batman-FLAG from larval nuclear extracts, as well as from embryonic extracts. These results provide further evidence that Batman and Trl are found in the same complexes in vivo, and this independently from binding to DNA (Faucheux, 2003).
The BTB/POZ domain has been shown to function as a hetero- or homo-dimerization interface. Therefore a test was performed to see whether an interaction between Batman and GAF is mediated by the BTB/POZ domains present in both proteins. In a yeast two hybrid assay, the Batman protein interacts with itself as well as with both the 519- and the 581-aa Trl isoforms. In contrast, Batman does not interact with the Trl519Delta[1-103] or Trl581Delta[1-103] variant proteins in which the BTB/POZ domain is deleted. In addition, Batman is unable to interact with the Trl BTB/POZ domain alone (Trl[1-121]). Therefore, it is concluded that the BTB/POZ domain of Trl is necessary but not sufficient to mediate the interaction of the two major Trl isoforms with the Batman BTB/POZ domain (Faucheux, 2003).
In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched "on" and "off." When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. This study has directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, it was shown that transcription through the PRE, in this case from the bxd regulatory region of Ubx, is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. These results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity (Erokhin, 2015).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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Ubx regulation: Table of contents
Ubx regulation: Table of contents
Ultrabithorax:
Biological Overview
| Evolutionary Homologs
| Targets of activity
| Protein Interactions
| Posttranscriptional regulation
| Developmental Biology
| Effects of Mutation
| References
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