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 trx^B11 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).

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements (PREs). The Drosophila PcG protein Pleiohomeotic has been shown to bind to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers (Fritsch, 1999).

To dissect the 1.6 kb Ubx PRE, a Ubx-lacZ reporter gene was used to monitor silencing capacity of PRE subfragments. PBX is an embryonic enhancer and IDE is an imaginal disc enhancer: Both are located about 30 kb upstream of the Ubx transcription start site. PBX directs expression in early embryos in a pattern similar to Ubx with a sharp anterior boundary in parasegment 6 (ps 6). In contrast, if IDE is linked to a reporter gene it activates transcription not only in haltere discs where endogenous Ubx is expressed but also in wing discs where Ubx is not expressed. A PBX-IDE reporter gene is thus active within Ubx expression boundaries in early embryos but is later expressed also outside of the Ubx domain, i.e. in the wing disc. A test was therefore performed to see whether PRE or subfragments thereof would silence this misexpression if inserted into the PBX-IDE reporter gene. The 1.6 kb PRE was inserted between the PBX and IDE enhancers and this reporter gene (PRE_1.6) was introduced into flies. Whereas PBX-IDE transformants without the PRE fragment show nearly uniform beta-galactosidase (beta-gal) expression in wing and haltere discs, beta-gal expression in PRE_1.6 transformants is confined to the posterior compartment of haltere discs. The boundary between beta-gal-positive and beta-gal-negative cells runs through the middle of the haltere disc and apparently coincides with the ps 6 compartment boundary. Thus, IDE activity is completely suppressed anterior to ps 6 but is unaffected in ps 6 itself. This suggests that PRE_1.6 silences the reporter gene anterior to ps 6 and thereby preserves the anterior expression boundary delimited by PBX in the embryo. The expression pattern directed by PBX in the embryo is not silenced by PRE_1.6 (Fritsch, 1999).

Subfragments of the 1.6 kb PRE have been tested for silencing function. PRE silencer is contained in the central 567 bp PRE_D fragment. It was asked whether the silencing mediated by the PRE fragments depends on PcG gene function. A reduction in Pc gene dosage leads to a partial loss of silencing; the extent of the observed misexpression is comparable to the misexpression of the endogenous Ubx gene in Pc heterozygotes. The patterns of PRE_D lines were examined in larvae homozygous for a pho mutation. In each case pho mutant wing and haltere discs show an extensive loss of silencing. These results demonstrate that silencing by PRE_D requires PcG gene function. It was next examined whether Pho protein binds directly to PRE_D. Pho contains a DNA-binding domain with very high similarity to the DNA-binding domain of YY1, which is known to bind to the sequence G/t C/t/a CATN T/a T/g/c. The PRE_D fragment contains several motifs that match versions of this YY1 protein binding site. Oligos spanning each of these motifs were tested for Pho binding in gel-shift assays. Pho protein forms a specific complex with six of the ten tested oligos. These and additional binding tests with other oligos suggest GCCATTAC as an optimal binding site for Pho. To test whether Pho protein binds to the PRE_D construct in vivo, antibodies were generated against the Pho protein. On polytene chromosomes from salivary glands, Pho antibodies bind to approximately 35 different loci. The strongest signal was found at the location of the Bithorax-Complex (BXC), suggesting that Pho protein is bound to the BXC genes. On polytene chromosomes of a PRE_D transformant line, a strong additional signal was found at the transposon insertion site. These data suggest that Pho protein binds directly to PRE_D in vitro and in vivo (Fritsch, 1999).

Are Pho protein binding sites needed for silencing in imaginal discs? All six Pho binding sites in the PRE_D fragment were mutated by altering two or three nucleotides in each CCAT core motif. The introduced base changes abolish binding of Pho protein in vitro. The mutated PRE_D fragment was inserted into the PBX-IDE reporter gene to obtain PRE_Dphomut. These PRE_Dphomut transformants show uniform beta-gal staining in wing and haltere discs that is comparable to transformants carrying the reporter gene without PRE. Thus, mutations in the Pho binding sites abolish PRE function. Taken together, these experiments provide strong evidence that Pho protein binds directly to PRE and is required for silencing (Fritsch, 1999).

Expression of the endogenous Ubx gene was examined in imaginal discs of pho mutants. Animals that are homozygous for pho null mutations develop into pharate adults with only relatively mild homeotic transformations. Consistent with this, it was found that pho¹ and pho^b homozygotes show only slight misexpression of Ubx in wing and antennal discs. The observed misexpression is comparable to the misexpression of Ubx in Pc heterozygotes. In pho mutants, the PRE_D reporter gene shows substantially more misexpression than the endogenous Ubx gene. Thus, silencing of the reporter gene is more sensitive to the lack of pho product than the native Ubx gene. Animals that are mutant for two different PcG mutations often show more severe misexpression of homeotic genes and consequently enhanced homeotic transformations, when compared to the single mutants by themselves. pho homozygotes that are also heterozygous for Pc show very dramatic misexpression of Ubx in wing and other discs. Thus, in this genetically sensitized background due to only one rather than two copies of Pc, pho is required to repress Ubx in all imaginal disc cells (Fritsch, 1999).

What is the role of maternal Pho? Most pho mutant embryos, which lack maternal wild-type pho product, fail to develop altogether and the rare putatively paternally rescued embryos that do develop die with segmentation defects and homeotic transformations. In contrast, if maternal pho product is present, pho homozygotes survive to pharate adults. This suggests that pho function is particularly important in the very early embryo. Mutation of the Pho binding sites in the PRE_Dphomut reporter gene abolish silencing in all disc cells. Thus, it appears that if Pho protein is prevented from binding to PRE, i.e. in the PRE_Dphomut reporter gene, silencing is probably never established. Conversely, silencing of the PRE_D reporter gene is only partially lost in larvae homozygous for a pho null mutation. Thus, in pho homozygous embryos (which contain maternal Pho protein) silencing of the PRE_D reporter is probably established but is subsequently lost in imaginal discs. In summary, these observations strongly suggest that maternally deposited Pho protein is crucial for the establishment of silencing but that zygotic Pho protein is required for complete silencing (Fritsch, 1999).

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).