Sex combs reduced

Promoter Structure

Sequences required for establishment of the SCR embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu, found within the ANTP-C (Pattatucci, 1991b).

Four DNA fragments that had previously been shown to contain putative Scr enhancer elements were found to have functional enhancers; similarly, another Scr fragment was found to contain a functional repressor. Regulation of Scr in the labial segment and the CNS requires the apparently synergistic action of multiple, widely spaced enhancer elements. Regulation in the prothorax also appears to be controlled by multiple enhancers: one complete pattern element and one subpattern element. In contrast, Scr regulation in the visceral mesoderm is controlled by an enhancer(s) located in only one DNA fragment (Gorman, 1995).

Breakpoint mutations located in a 75-kb interval, including the Scr transcription unit and 50 kb of upstream DNA, cause Scr misexpression during development, presumably because these mutations remove Scr cis-regulatory sequences from the proximity of the Scr promoter. Several fragments contain Scr regulatory sequences. Scr expression is controlled by multiple regulatory elements that are separated by more than 20 kb of intervening DNA. Regulatory sequences that direct Scr-like pattern in the anterior and posterior midgut are imbedded in the regulatory region of the segmentation gene fushi tarazu, which is normally located between 10 and 20 kb 5' of the Scr transcription start site (Gindhart, 1995b).

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).

The Sex combs reduced gene specifies the identities of the labial and first thoracic segments in Drosophila. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, a model is proposed in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element that is located physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome (Southworth, 2002).

The two putative negative regulatory elements are located distal and proximal to the 60–70 kb region that includes the chromosome rearrangements that cause the appearance of ectopic sex comb teeth. Although the distal and proximal elements may be different, both putative regulatory elements are referred to as maintenance elements for silencing (MES). In this model, when the Scr gene is active, flanking MESs fail to interact. When the Scr gene is silenced, the flanking MESs preferentially interact in cis to stabilize silencing of genes in between. The interaction of MESs may occur through the binding of different proteins to these elements when silencing is specified, or it may occur by the modification of proteins already bound even when the gene is active. Maintenance of silencing, however, affects only genes that lie between two elements; i.e., silencing requires the ability to form a physical loop of DNA between the two elements. Interaction of the elements on the wild-type homolog would preferentially occur in cis, maintaining silencing in most cells. However, because the silencing elements on the broken chromosome are no longer in cis, they could compete for interactions with the silencing elements on the wild-type homolog. If both elements on the aberration chromosome interact with the elements on the homolog, one configuration might be stable enough to prevent interaction of the two elements in cis on the wild-type chromosome. This would disrupt silencing of the Scr promoter between these two elements, allowing derepression of the wild-type Scr gene. It is believed that deletion chromosomes that contain only one MES are not able to effectively compete with the cis interactions on the wild-type homolog. This model can account for all of the data described so far, and it can also explain the behavior of an old mutation with very anomalous properties. This is the Antp^Scx mutation isolated in 1953 (Southworth, 2002).

The Antp^Scx mutant was isolated originally on the basis of a dominant extra sex combs phenotype. It is lethal when heterozygous to Antp mutant alleles, but is viable when heterozygous to Scr mutant alleles. The Antp^Scx mutant chromosome is cytologically normal and the only molecular lesion identified in the ANTC was the insertion of repetitive DNA very close to the Antp P1 promoter. Given the physical location of the insertion, it is not surprising that the Antp^Scx mutant chromosome fails to complement Antp alleles that specifically lack P1 function, such as Antp^B, Antp^73b, Antp^CB, and Antp¹⁷. There is no difference in the average number of sex comb teeth per first leg in Antp^Scx heterozygous males compared to homozygous wild type or in Antp^Scx/Scr⁴ males compared to +/Scr⁴ males. Males heterozygous for Antp^Scx, however, do have a considerable number of ectopic sex comb teeth (an average of 2.7 per second leg). The ectopic sex comb teeth result from misexpression of Scr in cis and in trans. Males with Scr mutations in cis to Antp^Scx (Scr^E2 Antp^Scx/+ and Scr^E3 Antp^Scx/+) have fewer sex comb teeth per second leg (an average of 0.8); males with Scr mutations on the homolog (Antp^Scx/Scr² and Antp^Scx/Scr⁴) also have fewer sex comb teeth per second leg (an average of 1.3–1.6). Scr mutations both in cis and in trans to Antp^Scx [Scr^E2 Antp^Scx/ Scr⁴;Dp(3;Y)77ab] almost completely eliminate the ectopic expression of Scr (an average of only 0.02 sex comb teeth per second leg). Comparison of the effects of Scr mutations in cis and in trans also suggest that Antp^Scx derepresses the Scr promoter in cis about twice as much as the Scr promoter on the homolog. A molecular mechanism through which the insertion of repetitive DNA ~150 kb upstream of the Scr promoter might be responsible for transcriptional derepression of both the cis promoter and the Scr promoter on the homolog has not been previously suggested (Southworth, 2002).

This model is the first attempt to explain the unusual properties of the Antp^Scx mutant chromosome. It is believed that the repetitive DNA inserted near the Antp P1 promoter on the Antp^Scx mutant chromosome mimics the endogenous regulatory elements involved in the maintenance of silencing (the MES elements). By competing for interactions with the endogenous elements either on the same chromosome or on the homolog, the Antp^Scx insertion disrupts silencing of the Scr promoter in cis or in trans, respectively. In this respect, the Antp^Scx insertion appears to be more effective than a wild-type MES, since deletion chromosomes with a single MES do not interfere with silencing on the homolog. Not only does this model explain the existing data, but it also makes a prediction. The Antp P2 promoter is between the repetitive insertion on the Antp^Scx mutant chromosome and the endogenous regulatory elements in the Scr gene. Interactions between the Antp^Scx insertion and the endogenous elements in the Scr gene in cis should not only derepress the Scr promoter, but should also silence the Antp P2 promoter. Interactions between the Antp^Scx insertion and the endogenous elements in the Scr gene in trans should not silence the Antp P2 promoter. Since the Antp^Scx mutation appears to derepress Scr in cis about twice as much as in trans, about two-thirds of the cells are expected to lack Antp P2 function from the Antp^Scx chromosome. Two mutations (Antp¹ and Antp²³) have been characterized that inactivate the Antp P2 promoter but appear to have normal function for the Antp P1 promoter. These two mutations can be used to examine Antp P2 function on the homologous chromosome in heterozygotes. As expected from this model, Antp^Scx interferes significantly with function of the P2 promoter; Antp^Scx fails to complement both Antp¹ and Antp²³ for viability (no surviving adults were found among several hundred expected). In contrast, deletions that remove the Antp P1 promoter and chromosome aberrations that physically separate the P1 and P2 promoters are all viable when heterozygous to either Antp¹ or Antp²³. With these results, four genetic properties are now associated with the Antp^Scx mutant chromosome: (1) loss of Antp P1 function, (2) loss of Antp P2 function, (3) derepression of the Scr promoter on the mutant chromosome, and (4) derepression of the Scr promoter on the homolog (Southworth, 2002).

It is possible that there are multiple molecular lesions on the Antp^Scx mutant chromosome that were not detected in the molecular analyses. However, it should be emphasized that the Antp^Scx mutant chromosome is cytologically normal, is wild type for Scr function, and has the ability to derepress the Scr gene in trans. Only Scr mutations that have chromosome aberration breakpoints within the Scr locus have the ability to derepress Scr in trans. The model explains how the identified molecular lesion could lead to all of the mutant phenotypes observed (Southworth, 2002).

In the model, trans interactions between MESs occur when the cis interactions are disrupted. Although PREs are believed to normally act in cis to maintain silencing, they are also able to act in trans when included within transgenes. These trans interactions of PREs are enhanced when cis interactions are blocked. In addition, while single PREs appear to partially silence transgenes, silencing is often greater when multiple PREs can interact. A pair of major PREs has also been characterized in about the same position in the Ultrabithorax (Ubx) homeotic gene as the MESs in the Scr gene; i.e., one PRE is ~25 kb upstream of the Ubx promoter and a second PRE is within an intron in the middle of the transcription unit. Therefore, an important question is whether MESs are the same as PREs. They are likely to be distinct elements, but are often in close proximity. Many DNA fragments that contain PREs may also contain MES elements, but these activities may be separable. For example, a 2.9-kb DNA fragment from the Mcp region of the bithorax complex appears to contain at least two different types of regulatory elements. An 800-bp DNA fragment from the central region of the larger fragment is not sufficient for silencing, but it is sufficient for mediating pairing-sensitive interactions between transgenes on different chromosomes. It is also sufficient for mediating long-range interactions between enhancers and promoters in transgenes. Two XbaI restriction fragments from Scr (an 8.2-kb fragment from the second intron and a 10.0-kb fragment 35–45 kb upstream of the promoter) that have been tested in transgenes for PRE activity overlap with the putative MESs. Both fragments appear to partially silence the reporter gene in a transgene assay. This silencing is sensitive to some Polycomb group mutations; however, the two tested fragments differ as to which Polycomb group mutations had effects. Interestingly, only the 8.2-kb fragment exhibited pairing-sensitive silencing, while only the 10.0-kb fragment functioned as a PRE in embryos. The apparent independence of MES function and Polycomb group repression also suggests that MESs may be separate elements that are in close proximity to PREs. It is possible that MESs act to maintain interactions between nearby PREs, thus facilitating the maintenance of silencing. In this respect, MESs may be similar to the pairing-sensitive regulatory elements identified upstream of the engrailed promoter (Southworth, 2002).

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 Trl^13c 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 intrinsic enhancer-promoter specificity and chromatin boundary/insulator function are two general mechanisms that govern enhancer trafficking in complex genetic loci. They have been shown to contribute to gene regulation in the homeotic gene complexes from fly to mouse. The regulatory region of the Scr gene in the Drosophila Antennapedia complex is interrupted by the neighboring ftz transcription unit, yet both genes are specifically activated by their respective enhancers from such juxtaposed positions. A novel insulator, SF1, has been identified in the Scr-ftz intergenic region that restricts promoter selection by the ftz-distal enhancer in transgenic embryos. The enhancer-blocking activity of the full-length SF1, observed in both embryo and adult, is orientation- and enhancer-independent. The core region of the insulator, which contains a cluster of GAGA sites essential for its activity, is highly conserved among other Drosophila species. SF1 may be a member of a conserved family of chromatin boundaries/insulators in the HOM/Hox complexes and may facilitate the independent regulation of the neighboring Scr and ftz genes, by insulating the evolutionarily mobile ftz transcription unit (Belozerov, 2003).

Although intrinsic properties of certain ftz enhancers, such as AE1, can account for their exclusive interaction with the cognate promoters, the same mechanism may not apply to all ftz enhancers in the region. Furthermore, the Scr-distal enhancers, separated from the Scr promoter by the entire ftz gene, would have to overcome the interference from a highly competitive ftz promoter. To test if insulator elements play a role in defining enhancer-promoter interactions in the Scr-ftz region, DNA fragments from the Scr-ftz intergenic region were examined for enhancer-blocking activity. Two tissue-specific enhancers were used in the enhancer-blocking assay, the hairy stripe 1 enhancer (H1) and the rhomboid neuroectoderm enhancer (NEE); these are active in a transverse anterior band and two ventral lateral stripes, respectively. When a neutral DNA spacer from the lambda phage is inserted between the two enhancers, both the lacZ and white reporters are expressed in a composite pattern directed by both H1 and NEE, as shown by whole-mount in situ hybridization. Insertion of a 2.3 kb EcoRI fragment from the Scr-ftz intergenic region reduces the H1-directed white expression and NEE-directed lacZ expression but not the H1-directed lacZ or NEE-directed white expression, indicating a selective block of the distal enhancer activities. The enhancer-blocking activity of the element, named SF1 for the Scr-ftz boundary, appears comparable or even stronger than that of the Su(Hw) insulator from the gypsy retrotransposon. In contrast, other DNA fragments of comparable size from the 10 kb region surrounding SF1 exhibit little or no enhancer-blocking activity. Importantly, the 15 kb intergenic region contains many closely spaced enhancers required for the tissue-specific regulation of Scr and ftz genes. The 2.3 kb SF1 region, however, appears to be devoid of any enhancer activities, as assayed in transgenic embryos with several promoters including those from the white, evenskipped (eve) and ftz genes (Belozerov, 2003).

The ability was tested of SF1 to block a different pair of embryonic enhancers, PE (twist proximal element) and E3 (eve stripe 3 enhancer). When the lambda spacer is inserted between the two enhancers, they direct the white and lacZ reporter expression in the ventral region and in the mid-embryo stripe, respectively. Replacing the spacer with SF1 results in the block of E3-mediated expression of the white reporter and PE-mediated expression of the lacZ reporter. Again, SF1 appears to block the distal enhancers more efficiently than the Su(Hw) insulator. The insulator activity of SF1 is also orientation independent. When the 2.3 kb element is inserted in an inverted orientation between the NEE and H1 enhancers, it blocks the distal enhancers to a comparable level as in the forward orientation. In addition to the enhancer-blocking activity, the 2.3 kb SF1 element also contains a potent chromatin barrier activity as shown by its ability to protect the mini-white transgenes against chromosomal position effects (Belozerov, 2003).

Activity of the homeotic selector genes such as Scr is required to maintain body segment identity throughout the animal life cycle. If SF1 is involved in regulating Scr and ftz genes, its boundary activity would be expected to persist to later stages of development. To test this, the enhancer-blocking activity of SF1 was examined in adult tissues with a transgenic yellow gene. The wild-type activity of yellow is required for the pigmentation of cuticle structures in larval and adult Drosophila. The yellow expression is activated in the adult bristles by the bristle-specific enhancer located in the first intron of the gene. A transgenic mini-yellow gene including the 400 bp upstream sequences and the first intron can produce the dark pigmentation in the bristles in a yellow null background. Similar dark bristles are observed in flies carrying a transgene with the lambda spacer DNA inserted between the bristle enhancer and the mini-yellow gene promoter. When the full-length SF1 is inserted in place of the spacer DNA, it efficiently blocks the B enhancer, reducing the bristle pigmentation to that of the yellow¹ mutant background. Again, the enhancer-blocking activity of SF1 appears slightly stronger than that of the Su(Hw) insulator in a similar assay. Thus the activity of SF1 is present in post-embryonic tissues, consistent with its potential role in regulating the homeotic gene Scr (Belozerov, 2003).

In the Scr-ftz region, at least three distinct types of cis-acting elements define the promoter specificity for no less than ten different enhancers. One type, enhancers such as AE1 distinguish the available promoters based on the core promoter sequence and selectively interact with the TATA-containing ftz promoter. A second type, the Scr-distal T1 enhancer appears to depend on a newly identified 'promoter tethering element' located near the Scr gene for specific interaction. A third type of regulatory DNA, the SF1 boundary/insulator, may be responsible for target promoter specification by the ftz-distal enhancer. The ftz-distal enhancer does not share the same promoter preferences as AE1 and can equally activate TATA or TATA-less promoters. The intergenic position of the SF1 chromatin boundary at the junction of the ftz transcriptional unit and the neighboring Scr gene, and its ability to block the ftz-distal enhancer from a TATA-less, Scr-like promoter suggest that SF1 may be essential for maintaining independent gene regulation in the region. Consistent with this proposed role in regulating the Scr homeotic gene, the boundary activity of SF1 persists through the later stages of development. Another indication of the functional role of the SF1 insulator in the genomic interval is the conservation of the insulator DNA during evolution. While the flanking region has diverged significantly (76% identity) in D.teissieri, the core insulator sequence remains highly conserved (>97% identity) in this species (Belozerov, 2003).

However, it is unclear how SF1, an insulator positioned within the Scr regulatory region, is circumvented by the Scr-distal enhancers located downstream of ftz. Similar questions exist for the Mcp-1, Fab7 and Fab8 boundaries between the Abd-B promoter and the distal iab enhancers in BX-C. A specialized DNA element named promoter targeting sequence (PTS) near the Abd-B promoter may facilitate the enhancers in overcoming the intervening Fab boundaries. An alternative mechanism is based on the recent finding that the Su(Hw) enhancer-blocking activity is abolished by the tandem arrangement of insulators. SF1 or other specialized DNA elements such as the Scr tethering element may interact with similar elements positioned downstream of ftz, thereby 'looping out' the intervening ftz domain and facilitating the Scr enhancer-promoter interactions (Belozerov, 2003).

Chromatin boundary function has been shown to be important for gene regulation in the Hox clusters from fly to mouse. However, the protein components involved in the Hox boundary activity, as well as the mechanism of the boundary function are unknown. Multiple GAGA binding sites have been identified that are essential for the enhancer-blocking activity of the SF1 core insulator. Drosophila GAGA factor may be involved in the SF1 boundary function. Similar findings that GAGA sites are critical for the function of Mcp1 and Fab7 boundary elements from the BX-C have been reported recently. These observations suggest that the chromatin insulators from the ANT-C and the BX-C may share common components and mechanisms, and belong to a family of conserved boundary elements that regulate enhancer-promoter interactions in the Hox complexes (Belozerov, 2003).

It is interesting that the GAGA factor is implicated in the boundary activity in the Drosophila Hox clusters. The GAGA factor has been known to regulate transcription by recruiting chromatin remodeling and transcription initiation complexes. However, its role in boundary/insulator activity may not be attributed to its ability to activate transcription but rather to the ability of this protein to forge links among distant DNA elements through its BTB domain. This property of the GAGA factor is consistent with the looping models proposed for the insulator/boundary mechanism (Belozerov, 2003).

The existence of an independent ftz transcription domain flanked by boundary elements is also consistent with the observed mobility of ftz during evolution. ftz is an 'accessory' gene unique to the invertebrate homeotic complex. Although it has been found in all major arthropod groups, the protein sequence and function of ftz have diverged from the neighboring homeotic genes. Nonetheless, the internal organization of the ftz transcription unit including regulatory sequences is highly conserved, possibly due to its important role in segmentation and neural development. The shift in ftz function appears to coincide with an increased mobility of the transcription unit as a whole, as the 16 kb genomic region is found inverted in certain Drosophila subgenera or missing entirely from the complex in certain insect species. The presence of the SF1 boundary element at the junction of such an evolutionary mobile unit is consistent with its role in maintaining gene independence during evolution (Belozerov, 2003).

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. Evidence is provided for a third mechanism: promoter-proximal tethering elements. The Scr-ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3' of the ftz gene and approximately 25 kb 5' of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1-ftz and T1-Scr interactions. A 450-bp DNA fragment located approximately 100 bp 5' of the Scr transcription start site is essential for T1-Scr interactions and can mediate long-range activation of a ftz/lacZ reporter gene when placed 5' of the ftz promoter. It is suggested that the Scr450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer-promoter interactions at other complex genetic loci (Calhoun, 2002).

Long-range enhancer-promoter interactions in the Scr-Antp interval of the Drosophila Antennapedia complex.

Long-range enhancer-promoter interactions are commonly seen in complex genetic loci such as Hox genes and globin genes. In the case of the Drosophila Antennapedia complex, the T1 enhancer bypasses the neighboring ftz gene and interacts with the distant Scr promoter to activate expression in posterior head segments. Previous studies identified a 450-bp promoter-proximal sequence, the tethering element, which is essential for T1-Scr interactions. To obtain a more comprehensive view of how individual enhancers selectively interact with appropriate target genes, bioinformatic methods were used to identify new cis-regulatory DNAs in the ~50-kb Scr-Antp interval. Three previously uncharacterized regulatory elements were identified: a distal T1 tethering sequence mapping >40 kb from the proximal tethering sequence, a repressor element that excludes activation of Scr by inappropriate enhancers, and a new ftz enhancer that directs expression within the limits of stripes 1 and 5. Many of the regulatory DNAs in the Scr-Antp interval are transcribed, including the proximal and distal tethering elements. It is suggested that homotypic interactions between the tethering elements stabilize long-range T1-Scr interactions during development (Calhoun, 2003).

Enhancers direct localized stripes, bands, and tissue-specific patterns of gene expression in the early Drosophila embryo. They are typically 300 bp to 1 kb in length and contain clustered binding sites for both transcriptional activators and repressors. Enhancers usually activate nearby target genes, although there are examples where they ignore the most proximal promoters and interact with distantly linked genes. Examples include the 3′ enhancers of the dpp gene and the T1 enhancer of Scr. The dpp enhancers fail to activate the neighboring slh and oaf genes but instead activate the expression of the distal dpp gene in imaginal disks. The selective regulation of dpp expression appears to depend on promoter specificity. The oaf and slh promoters are incompatible for activation by the dpp enhancers, despite the fact that they map much closer than does the preferred dpp promoter. Similarly, the distal T1 enhancer jumps over the intervening ftz gene to activate Scr in posterior head segments (Gindhart, 1995b). The failure of the T1 enhancer to activate ftz might also depend on promoter specificity. The T1 enhancer only weakly activates a minimal ftz-lacZ fusion gene, despite the fact that it contains a strong TATA element. However, the possible incompatibility between T1 and the ftz promoter is not sufficient to account for selective T1-Scr interactions, because T1 also fails to activate a Scr-lacZ fusion gene containing the minimal Scr core promoter. A 450-bp tethering element that maps immediately 5′ of the Scr core promoter has been identified (Calhoun, 2002). This element is essential for T1-Scr interactions and is sufficient to mediate long-range T1-ftz interactions when placed immediately 5′ of the ftz promoter (Calhoun, 2003).

A systematic analysis has been conducted of cis-regulatory DNAs in the 50-kb interval that separates Scr and Antp within the Antennapedia complex (ANT-C). An ftz enhancer has been identified that maps 3′ of the ftz transcription unit (ftzDE. This enhancer initiates gene expression within the limits of ftz stripes 1 and 5. The previously identified Scr tethering element contains eight copies of a simple palindromic sequence, TTCGAA. Four tandem copies of this motif are sufficient to mediate T1-ftz interactions in transgenic embryos. A whole-genome survey of high-density clusters of the TTCGAA motif identifies a 389-bp sequence located just 3′ of the Antp transcription unit. This cluster can function as a tethering element when attached to the minimal ftz promoter. It also diminishes the position effects observed for T1-Scr interactions in transgenic strains. A model is proposed whereby proteins that bind the TTCGAA motif in the proximal tethering element and distal cluster mediate the formation of a transcription loop, which stabilizes T1-Scr interactions. The putative loop might depend on the transcription of the cis-regulatory DNAs within the ANT-C, including the tethering element and distal cluster themselves (Calhoun, 2003).

Previous studies have identified three cis-regulatory DNAs in the 50-kb interval that separate the Scr and Antp genes: the T1 and AE1 enhancers and a 450-bp tethering element located immediately 5′ of the Scr core promoter. The tethering element is required for long-range T1-Scr interactions and localized expression in the posterior head segment. AE1 maintains the seven stripes of ftz expression in the germband of elongating embryos. To identify new cis-regulatory DNAs, different genomic DNA fragments from the Scr-Antp interval were assayed in transgenic embryos by using a variety of P element expression vectors (Calhoun, 2003).

Using the Cis-Analyst search algorithm, a new ftz enhancer was identified by scanning the Antp-Scr interval for clusters of cis-regulatory elements that are recognized by transcription factors encoded by maternal (bicoid and caudal), gap (hb, Kr, kni), and pair-rule (ftz) genes. A total of three clusters were identified. Two of the clusters correspond to previously identified cis-regulatory DNAs, the AE1 enhancer, and the ftz zebra element, which initiates ftz expression in early embryos. A third cluster (cluster 3) was also identified that maps just downstream of the ftz transcription unit. A 1.25-kb genomic DNA fragment that encompasses this cluster was inserted into a P element expression vector containing divergently transcribed CAT and lacZ reporter genes. CAT is under the control of the Scr promoter region, whereas lacZ contains the ftz promoter region. Transgenic embryos that contain this reporter gene were collected and hybridized with CAT and lacZ antisense RNA probes. Cluster 3 selectively activates the lacZ reporter gene but fails to induce CAT expression. ftz-lacZ expression is detected in two stripes in cellularizing embryos. Double-staining experiments using a probe that visualizes the endogenous ftz stripes indicates that the newly identified enhancer directs expression in stripes 1 and 5. ftz stripes 1 and 5 flank the expression domain of the gap repressor Krüppel (Kr), suggesting Kr might repress expression in the center of the embryo. In mutant embryos homozygous for a null mutation in the Kr gene, these stripes are expanded into a broad band (Calhoun, 2003).

The newly identified enhancer (cluster 3) is adjacent to the T1 enhancer, which regulates Scr expression in the labial head segment and anterior compartment of the first thoracic segment. Despite its proximity to T1, the new enhancer appears to regulate ftz expression, not Scr. First, the enhancer selectively activates the ftz-lacZ gene and fails to stimulate expression from the Scr promoter, even though the leftward CAT reporter gene contains both the Scr core promoter and the adjacent tethering sequence. In contrast, the T1 enhancer exhibits the opposite regulatory specificity; it selectively activates Scr-CAT and not ftz-lacZ. Another argument that the new enhancer is a component of the ftz locus is the observation that other Drosophila species, such as Drosophila littoralis, contain an inversion that inverts the ftz transcription unit. This inversion includes the 5′ zebra element and AE1 enhancer. It also includes the newly identified enhancer. The 'rightward' chromosomal breakpoint maps between the new enhancer and T1. The new enhancer is referred to as the ftz distal enhancer (ftzDE) and it is suggested that this enhancer is a remnant of the homeotic function seen for Ftz in other insects, such as the flour beetle (Calhoun, 2003).

A promoter-proximal regulatory element located immediately 5′ of the Scr core promoter has been identified. This tethering element is required for specific T1-Scr interactions. When positioned upstream of a ftz-lacZ fusion gene, the T1 enhancer now activates transcription from the heterologous ftz promoter. The 450-bp tethering element contains an overrepresented hexamer motif, TTCGAA. A survey of the entire Drosophila genome using the Flyenhancer search engine identified a relatively small number of short DNA segments (<400 bp) that contain at least five perfect copies of this motif. One of the clusters maps within the Antp-Scr interval, just downstream of the Antp gene. This newly identified distal cluster is also able to function as a tethering element and recruit the T1 enhancer when placed 5′ of the ftz core promoter (Calhoun, 2003).

The newly identified distal cluster maps >40 kb from the Scr promoter. To determine whether it might play a role in the normal regulation of Scr expression, CAT/lacZ fusion genes were created that contain an authentic arrangement of cis-regulatory elements. The tethering element was placed 5′ of the leftward Scr-CAT reporter gene, whereas the T1 enhancer was placed 3′ of the ftz-lacZ reporter gene. The distal cluster was inserted just downstream of the T1 enhancer. Thus, as seen for the normal organization of Scr regulatory elements, the tethering element and distal cluster bracket the remote T1 enhancer (Calhoun, 2003).

As expected, only the Scr-CAT reporter gene is activated by the T1 enhancer. CAT staining is restricted to a groove of cells located between the labial head and first thoracic segments. The ftz-lacZ gene is silent and does not exhibit expression. In the absence of the distal cluster, variable background staining is produced by the Scr-CAT reporter gene. However, extraneous staining is lost in each of the individual lines that contain the distal cluster in the 3′ position. The addition of the distal cluster does not augment T1-Scr interactions. The same levels of CAT staining are observed in the labial-T1 region with or without the distal cluster. The addition of the distal cluster serves to eliminate background staining and to produce a more precise pattern of expression in the labial-T1 region. One interpretation of these results is that proteins bind to the TTCGAA motif in the proximal tethering element and distal cluster and mediate a long-range chromatin loop, which stabilizes T1-Scr. (Calhoun, 2003).

The TTCGAA motif is the most obvious component of the proximal tethering element and distal cluster. To determine whether it is sufficient to recruit the T1 enhancer, different multiples of the motif were placed immediately 5′ of the ftz-lacZ reporter gene. In the complete absence of the motif, there is no activation of ftz-lacZ expression by the T1 enhancer. There is a similar absence of expression when two copies of the TTCGAA motif were placed 5′ of the ftz promoter. However, four tandem copies of the motif led to weak but consistent activation of the ftz-lacZ reporter gene in the labial-T1 region of transgenic embryos. Similar staining was obtained with a fusion gene that contains six copies of the TTCGAA motif. Stronger ftz-lacZ expression was obtained when either the proximal tethering element or distal cluster was placed 5′ of the ftz promoter. These observations suggest that the TTCGAA motif is an important component of the regulatory activities of the tethering element and distal cluster, but additional sequence elements and DNA-binding proteins are required for long-range T1-Scr interactions (Calhoun, 2003).

Creating a TATA element in the minimal Scr promoter and inserting the tethering element 5′ of the minimal ftz promoter are sufficient to swap the regulatory activities of the T1 and AE1 enhancers. When placed between divergently transcribed Scr-CAT and ftz-lacZ reporter genes, T1 now activates ftz-lacZ expression in the labial head segment, and AE1 activates Scr-CAT in seven stripes along the germ band. A limitation of this earlier experiment, however, is that the arrangement of cis-regulatory DNAs does not reflect the in vivo organization seen in the ANT-C. Moreover, the AE1 enhancer retains the capacity to activate ftz-lacZ expression when the minimal 450-bp tethering element is placed 5′ of the ftz promoter. This residual AE1-ftz activity was diminished by placing AE1 5′ of the 3.8-kb T1 enhancer. The intervening T1 enhancer somehow attenuates AE1, either through weak enhancer blocking activity or by simply increasing the distance separating AE1 from the ftz promoter (Calhoun, 2003).

This analysis identified three new cis-regulatory DNAs in the Scr-Antp interval of the ANT-C: a 3′ ftz enhancer, a distal cluster of TTCGAA elements, and negative elements that inhibit AE1-Scr interactions adjacent to the originally defined Scr tethering sequence. The tethering sequence and newly identified distal cluster are themselves transcribed and exhibit similar patterns of transcription even though they map quite far from one another (40 kb). This transcription might promote the formation of a long-range chromatin loop domain that stabilizes T1-Scr interactions (Calhoun, 2003).

The ftz gene was first cloned 20 years ago, and the AE1 enhancer and zebra element were identified just a few years later. The third enhancer was identified by using a computer program to scan the Drosophila genome for clusters of binding sites recognized by segmentation regulatory factors, particularly the gap repressor Kr. The newly identified ftz enhancer has the properties of a primary pair-rule stripe enhancer in that it directs the expression of just two stripes. The 3′ enhancer, although conserved in Drosophila species containing an inversion at the ftz locus, is dispensable for ftz gene function. Previous studies have shown that a ftz minigene lacking 3′ regulatory sequences is nonetheless able to complement ftz-mutant embryos (Calhoun, 2003).

The ftz gene has acquired distinct activities in different insects. In short germband insects such as Tribolium, ftz appears to function in both segmentation and homeosis. The Tribolium Ftz protein contains two peptide motifs, LRALL and YPWM, that mediate interactions with FtzF1 (segmentation) and Exd (homeosis), respectively. When misexpressed in fly embryos, the Tribolium Ftz protein produces both segmentation and homeotic defects. In contrast, the Drosophila Ftz protein contains only the LRALL motif and thereby functions solely in segmentation. It does not produce homeotic defects when misexpressed in transgenic embryos. Ancestral forms of Ftz functioned in both segmentation and homeosis in primitive insects, but the homeotic function has been lost in more modern insects, such as the Diptera. Perhaps the newly identified ftz enhancer is a remnant of the homeotic functions seen in other insects (Calhoun, 2003).

The 450-bp tethering sequence in the promoter-proximal region of the Scr gene is essential for activation by the remote T1 enhancer. The further analysis of this tethering sequence identified multiple copies of a simple palindromic sequence motif, TTCGAA. There are eight copies of this motif in the 450-bp tethering sequence, and the Fly Enhancer program was used to identify additional high-density clusters. One such cluster is also located in the Scr-Antp interval, just downstream of the Antp transcription unit. This newly identified distal cluster can function as a tethering sequence and augment T1-Scr interactions. It also eliminates position effects when placed downstream of the T1 enhancer. Multiple copies of the TTCGAA motif are sufficient to mediate weak T1-ftz interactions in transgenic embryos. This activation is not as robust as that observed for the native tethering sequence. Thus, TTCGAA may be an essential component of the tethering sequence, but additional regulatory elements are likely to play an important role in mediating T1-Scr interactions (Calhoun, 2003).

It is proposed that a common set of proteins bind to both the tethering sequence and distal cluster and form homotypic complexes, which stabilize long-range T1-Scr interactions. It is possible that a chromatin loop forms between the tethering sequence and distal cluster. Alternatively, according to a scanning model for enhancer-promoter interactions, interactions between the tethering sequence and distal cluster might lock the T1 enhancer onto the Scr promoter, after the two encounter one another. In addition to the proposed homotypic interactions between the distal cluster and tethering element, it is conceivable that heterotypic interactions are important for the recruitment of the T1 enhancer to the Scr promoter. The tethering element is sufficient to recruit T1 to either the Scr or ftz promoters in the absence of the distal cluster. These interactions might depend on different classes of proteins. Given that the two tethering elements interfere with activation by AE1, these elements might also serve to isolate the ftz segmentation enhancers away from neighboring homeotic genes. Improper activation of homeotic promoters by segmentation enhancers would be lethal for the developing embryo (Calhoun, 2003).

Regulatory proteins that bind to promoter-proximal sequences, such as the Scr tethering element, might not interact with the basal transcription complex and function as classical activators. Instead, they might regulate gene expression by recruiting distal enhancers. A number of mammalian promoterproximal regulatory proteins might work through this type of mechanism. For example, Sp1 has been shown to mediate the formation of DNA loops when bound to both proximal and distal recognition sequences (Calhoun, 2003).

Previous studies have documented the occurrence of extensive intergenic transcription in the Drosophila Bithorax complex. Many of these transcripts are associated with a number of defined cis-regulatory DNAs, including the Fab-8 insulator and IAB5 enhancer in the extended 3′ regulatory region of the Abd-B gene. It has been suggested that this transcription serves to maintain these critical regulatory elements in an open chromatin conformation during Drosophila development. For example, the Rox RNAs (dosage compensation) serve as docking sites for histone acetyltransferase complexes that are thought to open the chromatin on the male X chromosome and thereby augment gene expression (Calhoun, 2003).

The present study provides evidence for intergenic transcription in the Scr-Antp interval of the ANT-C. Interestingly, some of this transcription occurs in the tissues of parasegment (PS) 3, between the major sites of Scr and Antp expression in PS2 and PS4, respectively. Both homeotic genes are activated in PS3 in older embryos, and it is conceivable that intergenic transcription is required for this expression by maintaining the genes in an open conformation. The transcription of the tethering sequence and distal cluster might help ensure the maintenance of T1-Scr interactions during development (Calhoun, 2003).

Transcriptional Regulation

Drosophila Mi-2 protein binds to a domain in the gap protein Hunchback which is specifically required for the repression of HOX genes. Using LexA-Hb as bait, cDNAs were isolated representing six different genes. dMi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain. dMi-2 homozygotes survive until the first or second larval instar. Mutant embryos and larvae show no obvious mutant phenotypes. Specifically, expression of BXC genes such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B) is completely normal in these mutant embryos. This normal expression may be due to maternally deposited dMi-2 RNAs or proteins that persist through subsequent development. Consistent with this, all early embryos from a dMi-2 deletion stock (including those lacking the gene) show the same high levels of dMi-2 RNA. An attempt was made to generate embryos from mutant dMi-2 germ cells. However, germ cells that are mutant for any of the seven tested dMi-2 alleles fail to develop. This failure can be rescued by a dMi-2 transgene, demonstrating that dMi-2 is essential for the development of germ cells (Kehle, 1998).

dMi-2 protein was tested to see if it participates in PcG repression. As in the case of dMi-2, maternally deposited PcG product often rescues homozygous mutant PcG embryos to a considerable extent. Extensive derepression of HOX genes can be observed if such homozygous embryos are also mutant for another PcG gene. Imaginal discs were examined for derepression of HOX genes as well as the phenotypes of their adult derivatives. Clonal analysis suggests that dMi-2 is required for the survival of somatic cells. Do dMi-2 mutations exhibit gene-dosage interactions with PcG mutations? While larvae heterozygous for Polycomb (Pc) mutations show slight derepression of Ubx, larvae transheterozygous for both Pc and dMi-2 mutations show more extensive derepression. Furthermore, derepression of the HOX gene Sex combs reduced (Scr) in the second and third leg discs of Pc heterozygotes results in the formation of a first leg structure, the sex comb, on the second and third legs. The extent of this homeotic transformation reflects the number of cells that misexpress Scr protein. This homeotic transformation is far stronger in dMi-2/Pc transheterozygotes than in adults heterozygous for Pc alone, which is consistent with more extensive derepression of Scr in the double mutant. These results are further evidence that dMi-2 acts together with PcG proteins to repress HOX genes (Kehle, 1998).

It has been proposed that Hb directly or indirectly recruits PcG proteins to DNA to establish PcG silencing of homeotic genes. The present data suggest that dMi-2 might function as a link between Hb and PcG repressors. Although dMi-2 contains two motifs with similarity to DNA-binding domains (the myb and HMG domains), dMi-2 does not seem to bind to DNA on its own. Therefore, Hb may recruit dMi-2 to DNA. Xenopus Mi-2 was recently purified as a subunit of a histone deacetylase complex with nucleosome remodeling activity. In yeast and in vertebrates, several transcription factors repress transcription by recruiting histone deacetylases. It is possible that in Drosophila, nucleosome remodeling and deacetylase activities of a dMi-2 complex, recruited to homeotic genes by Hb, may result in local chromatin changes that allow binding of PcG proteins to the nucleosomal template. Alternatively, the proposed Hb-dMi-2 complex might directly bind a PcG protein and recruit it to DNA. Finally, the involvement of dMi-2 in PcG silencing suggests that this process may involve deacetylation of histones (Kehle, 1998 and references).

The analysis of the expression of Scr in Antp mutant embryos reveals a case of tissue-specific regulation of Scr expression by Antp. In the epidermis, Antp has been shown to negatively regulate Scr, but it positively regulates Scr in the visceral mesoderm (Reuter, 1990).

Genes that limit locations for transcription of the homeotic gene Sex combs reduced can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is repressed by the three bithorax complex (BX-C) homeotic genes, thus prevented it from inducing prothoracic structures. However, two of the BX-C homeotic genes, Ultrabithorax and abdominal-A, have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX-C gene, Abdominal-B. Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function (Andrew, 1994).

The identification of mutations in Tgfbeta-60A as dominant enhancers of thickveins 6 in the imaginal discs raises the possibility that Tgfbeta-60A is required for optimal signaling by the dpp pathway. To determine if there is a general requirement for Tgfbeta-60A in dpp signaling, the effects of Tgfbeta-60A mutations were examined on dpp signaling in the visceral mesoderm where both dpp and Tgfbeta-60A are expressed. dpp is expressed in two discrete domains in the visceral mesoderm. The anterior domain of dpp coincides with the gastric caecae primordia, which are immediately anterior to the expression domain of Sex combs reduced (Scr) in parasegment (ps) 4. The failure to initiate dpp expression in ps3 in dpp shv mutants results in anterior expansion of Scr expression and arrested outgrowth of the gastric caecae, indicating a role for dpp in repressing Scr in ps3. tkv 6 homozygotes are homozygous viable, so it is not surprising that all the midgut gene expression patterns examined are essentially normal. Scr expression in tkv 6 and Tgfbeta-60A mutants is normal. However, in tkv 6 and Tgfbeta-60A double mutants, the Scr expression extends anteriorly into ps3 as it does in dpp shv mutants, suggesting that Tgfbeta-60A activity is required in ps3 for optimal dpp signaling (Chen, 1998).

Regulation by Polycomb and trithorax group proteins

The expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANTP-C genes Antennapedia, Sex combs reduced and Deformed were examined in mutant trithorax embryos. Each of the genes exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression in response to trx. This implies that each gene has different requirements for trx in different spatial contexts to achieve normal expression levels, presumably depending on the promoters involved and the other regulatory factors (Breen, 1993).

Once established, the Polycomb group (Pc-G) and trithorax group (TRX-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. Isolated Scr regulatory sequences linked to an eye marker produce mosaic patterns of pigmentation in the adult eye, indicating expression is repressed in a clonally heritable manner. The size clones in the adult eye suggests that the event determining expression occurs at least as early as the first larval instar. The amount of repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. (Please refer to the Abdominal B site for further discussion of transvection). Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of Scr in a Polycomb-dependent manner (Gindhart, 1995a).

The extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene (Pattatucci, 1991b).

The consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed (LaJeunesse, 1995).

moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes:

1. Derepression of the Antp gene in the eye-antennal disc causes replacement of adult antennal structures with leg structures.
2. Derepression of the Scr gene in the second and third leg discs causes the appearance of first leg structures in the second and third legs of the adults.
3. Derepression of the Ubx gene in the wing discs causes the appearance of haltere tissue in the adult wing.
4. Derepression of the genes in the BXC (abd-A and Abd-B) causes cells of the fourth abdominal segment of the adult to differentiate structures of a more posterior identity.

moira mutations suppress the derepression phenotypes caused by mutations in another Pc group gene, Polycomblike. moira mutant clones in the haltere differentiate large bristles, characteristic of the anterior wing margin, and often lead to absence or duplication of halteres. Homozygous mor mutations in the posterior wing result in a distorted wing shape; the venation is disrupted and large socketed bristles appear along the posterior wing margin. Leg clones result in the femur and tibia being short and twisted and enlargement of the tarsal segment. Clones of the head cause the shape of the head to be abnormal in the dorsal region and sometimes cause the ocellus to be abnormal or absent. Embryos homozygous for moira mutations have defects in head structures, including truncated lateralgraten and defects in the mouth hooks and dorsal bridge. The first and second midgut constrictions are shifted posterior to their wild-type positions (Brizuela, 1997).

The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).

Loss of maternal brahma function blocks oogenesis; individuals homozygous for extreme brm alleles die as late embryos with no obvious pattern defects (Brizuella, 1994). Since it has not been possible to generate embryos lacking both maternal and zygotic brm function, the exact role of brm in embryonic development is not clear. Information concerning the role of brm after embryogenesis has been derived primarily from the analysis of hypomorphic brm alleles. Individuals trans-heterozygous for certain combinations of brm alleles survive to adulthood and exhibit developmental abnormalities similar to those arising from reduced expression of Antp-C and Bx-C genes, including the transformation of first legs to second legs and the fifth abdominal segment to a more anterior identity (Brizuella, 1994). Because the effect of complete loss of brm function had not been examined, it was unclear whether brm is also involved in other processes. To clarify the role of brm in Drosophila development, mosaic analysis has been used to determine the null phenotype of brm mutations. As an alternative approach, site-directed mutagenesis was used to generate dominant-negative brm mutations and investigate the functions of evolutionarily conserved domains within the Brm protein (Elfring, 1998).

A dominant-negative brm mutation (DNbrm) was generated by replacing a conserved lysine in the ATP-binding site of the Brm protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the high molecular weight (2 million Daltons) Brm complex. Expression of the dominant-negative Brm protein causes peripheral nervous system defects, homeotic transformations, and decreased viability. Individuals bearing one or two copies of the dominant-negative Brm are viable, but frequently exhibit partial transformations of haltere to wing, as evidenced by an increase in haltere size and the appearance of ectopic bristles on the capitellum. Approximately one third of dominant-negative Brm adults exhibit this transformation, which is presumably caused by the decreased expression of the Ultrabithorax gene. Increasing the ratio of dominant-negative Brm to wild-type Brm to 2:1 is lethal. Thus, the dominant-negative brm mutation behaves as an antimorphic allele of brm. Expression of the dominant-negative Brm protein in patterns identical to the segmentation genes hairy or engrailed has no effect on embryonic viability or segmentation. The lack of an embryonic phenotype resulting from embryonic expression of the dominant-negative Brm protein may be caused by the high maternal expression of wild-type Brm protein, which is sufficient to allow embryogenesis to proceed to near completion in the absence of zygotic brm function. Expression of the dominant-negative protein in imaginal tissues after embryogenesis leads to greatly reduced viability. Individuals reared at 20° display partial transformation of first leg to second leg, as evidenced by a reduction in the number of sex comb teeth on the first leg. This phenotype is also seen in adults trans-heterozygous for hypomorphic brm alleles and is presumably caused by decreased expression of the Sex combs reduced (Scr) gene. Adults reared at 20° also display twinning of mechanosensory bristles, a phenotype similar to that observed in clones of brm2 tissue. Expression of the dominant negative protein also has dramatic effects on the size and morphology of the wing; mutant wings are reduced in size, and the L5 and the posterior cross-vein (PCV) are usually absent. Defects in the campaniform sensilla, a class of sensory organs important for flight, are also observed with high frequency. These defects fall into four classes: missing sensilla, duplication or triplication of sensilla, transformation of sensilla into bristles, and the appearance of ectopic sensilla. Ectopic sensilla and bristles are observed most frequently on the L3 vein. Three sensilla (L3-1, L3-2, and L3-3) and no bristles are normally found on this vein. By contrast, approximately one-half of mutant wings display one or two additional sensilla on L3. Ectopic bristles are observed on this vein in approximately one-fifth of mutant wings (Elfring, 1998).

If mod(mdg4) functions as a trxG gene, it should play a positive role in controlling the expression of homeotic genes, both during embryonic and later stages of development. To determine whether mod(mdg4) mutations affect homeotic gene expression, their effects were analyzed on the expression of homeotic genes during larval development. The effect of mod(mdg4) mutations were examined on the expression of the Antennapedia (Ant) gene. To this end, a combination of two mod(mdg4) alleles were used, mod(mdg4)¹⁶/mod(mdg4)^E(var)3-93D, resulting in lethality at the early pupa stages. Tissues were taken from live individuals during late larval stages of development. At this time, the Antp protein is expressed in the ventral ganglion in three bands of cells that correspond to the three thoracic segments. In the mod(mdg4)¹⁶/mod(mdg4)^E(var)3-93D mutant individuals examined, the brain lobes are small, the ventral ganglion is malformed, and expression of the Antp protein is undetectable. A second homeotic member of the Antp complex, Sex combs reduced (Scr), is expressed in a stripe of cells located in the most anterior region of the ventral ganglion in wild-type third-instar larvae. This band is not observed in mod(mdg4)¹⁶/mod(mdg4)^E(var)3-93D mutants. Mod(mdg4) also regulates homeotic genes involved in the development of posterior body segments. Ubx is expressed in a band of cells in the ventral ganglion located posterior to the domain of Antp expression. This stripe of Ubx expression is not detectable in the ventral ganglion of mod(mdg4)¹⁶/mod(mdg4)^E(var)3-93D larvae, suggesting that the Mod(mdg4) protein positively regulates Ubx expression. Mutations in mod(mdg4) also affect the expression of the Abdominal B (Abd B) gene, which is expressed in the most posterior region of the ventral ganglion during larval development but is lacking in mod(mdg4)¹⁶/mod(mdg4)^E(var)3-93D mutants. A similar effect is observed for the expression of homeotic proteins in the wing and leg imaginal discs; in mod(mdg4) mutants, these structures often appear malformed, and there is no detectable accumulation of Antp, Scr, Ubx, or Abd-B proteins. These results indicate that several homeotic genes of the Antennapedia and bithorax complexes are not properly expressed in mod(mdg4) mutants, suggesting that the Mod(mdg4) product plays a positive role in regulating their expression, in agreement with its putative role as a trxG gene (Gerasimova, 1998).

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Drosophila Rpd3, referred to here by its alternative name HDAC1, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes (Chang, 2001). \

To demonstrate that the effect of Hdac1 mutations is exerted at the level of expression of homeotic genes, the expressions were examined of Sex combs reduced (Scr) and Ultrabithorax (Ubx) proteins in wild-type and Pc mutant imaginal discs. Scr proteins normally are expressed at high levels in the first leg discs, but are not expressed in the second and third leg discs. In Pc⁴ mutant heterozygotes, however, Scr proteins also can be detected at low levels in second and third leg discs. Consistent with the increase in ectopic sex comb teeth, dramatic increases in the levels of Scr proteins are observed in the second and third leg discs from Pc⁴ mutant heterozygotes that were also heterozygous for any of the Hdac1 alleles except Hdac1³²⁶. In addition, Ubx proteins are marginally detectable only in the peripodial membranes of imaginal wing discs of wild-type or Pc⁴ mutant heterozygous larvae. In larvae heterozygous for both Pc⁴ and an Hdac1 mutation, high levels of Ubx proteins are observed in the medial sections of the wing discs proper. In contrast to the lack of ectopic Scr expression in Pc⁴ heterozygotes carrying the Hdac1³²⁶ allele, a much stronger effect on ectopic Ubx expression is observed; Ubx protein levels in both first and second leg discs are increased substantially. It is highly likely that the expanded Ubx expression reduces Scr expression, resulting in suppressed Pc phenotype (i.e., reduced numbers of ectopic sex comb teeth) in Pc⁴/Hdac1³²⁶ trans-heterozygotes. These results strongly suggest that Hdac1 acts cooperatively with Pc to repress homeotic genes during larval and pupal development (Chang, 2001).

The trithorax group genes are required for positive regulation of homeotic gene function. The trithorax group gene brahma encodes a SWI2/SNF2 family ATPase that is a catalytic subunit of the Brm chromatin-remodeling complex. The Drosophila tonalli (tna) gene was identified by genetic interactions with brahma. tna mutations suppress Polycomb phenotypes and tna is required for the proper expressions of the Antennapedia, Ultrabithorax and Sex combs reduced homeotic genes. The tna gene encodes at least two proteins, a large isoform (TnaA) and a short isoform (TnaB). The TnaA protein has an SP-RING Zn finger, conserved in proteins from organisms ranging from yeast to human and thought to be involved in the sumoylation of protein substrates. Besides the SP-RING finger, the TnaA protein also has extended homology with other eukaryotic proteins, including human proteins. tna mutations also interact with mutations in additional subunits of the Brm complex, with mutations in subunits of the Mediator complex, and with mutations of the SWI2/SNF2 family ATPase gene kismet. It is proposed that Tna is involved in postranslational modification of transcription complexes (Gutiérrez, 2003).

The Antp gene has two alternative promoters, P1 and P2. The Antp^Ns allele derepresses the Antp P2 promoter in the eye-antennal disc and expresses wild-type Antp transcripts from the Antp promoter. Derepression of the Scr gene causes the appearance of extra sex combs on the second and third legs of males. This derepression can be caused by gain-of-function alleles of Scr, such as Scr^Msc, or by loss-of-function mutations in Polycomb group genes, such as Pc³ or Pc⁴. Several trithorax group genes (including brm, mor, osa, kis, skd and kto) were first identified as suppressors of the extra sex combs phenotype caused by derepression of Scr or as suppressors of the antenna to leg transformation caused by derepression of Antp in the Nasobemia (Ns) allele of Antp. Since the tna gene was identified on the basis of genetic interactions with brm, tests were performed to see whether tna mutations could also suppress these two homeotic derepression phenotypes. It was found that all tna mutations strongly suppress the extra sex combs phenotype caused by Pc³, Pc⁴ or Scr^Msc, but only weakly suppress the antenna to leg transformation caused by the Antp^Ns mutation (Gutiérrez, 2003).

Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin

Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex. In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).

It is proposed that Reptin acts as a subunit of the TIP60 HAT complex to generate a repressive chromatin state. This is a novel activity of a HAT complex previously shown to promote transcription. This study shows that Reptin copurifes with the Polycomb complex PRC1. This prompted an investigation of whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. It was shown that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although no interactions were detected between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells. This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression. It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV. Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV, indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, reptin is not considered a bona fide PcG gene, and it is found unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce or with four core components whose activity can be further enhanced by the DNA-binding proteins Zeste and GAGA (Qi, 2006).

Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex, the possibility is considered that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV. In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex. That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests that Reptin functions through the fly TIP60 complex (Qi, 2006).

Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast. Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes. Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast. Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV. Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage. However, no change was found in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (Qi, 2006).

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