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 identified 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).

Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb

Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, genetic and molecular interactions between corto and dsp1 were addressed. This study shows that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, a genetic interaction analysis was performed. corto mutants were found to suppress dsp1¹ sex comb phenotypes and enhance Antp^Scx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, it was found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells. These results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state (Salvaing, 2006).

It is concluded that the two ETPs corto and dsp1 interact genetically and that the proteins they encode (1) directly interact in vitro, (2) co-immunoprecipitate in embryos and (3) co-localize on 91 sites in salivary gland polytene chromosomes. These results suggest that the proteins are simultaneously involved in the regulation of several target genes. DSP1 can bind Corto through one of the two HMG-boxes that also mediate DNA binding. It has been suggested that during nucleoprotein complex formation, the HMG-box B of HMGB bends DNA whereas the HMG-box A mediates interaction with transcription factors, thus promoting their contact with targets. DSP1 seems to follow that scheme to enhance the binding of transcription factors as Dorsal or Bicoid to DNA. What therefore could be the role of the DSP1-Corto interaction in the regulation of common targets? First, DSP1 could bring Corto to the chromatin, where it could further interact with other partners. These partners could be PcG factors or GAGA factor, which have previously been shown to interact with Corto (Salvaing, 2003). Nevertheless, this hypothesis is unlikely since no modification of Corto binding to polytene chromosomes was observed in the dsp1¹ strain. Second, DSP1 could inhibit the interaction between Corto and PcG factors or GAF, thus preventing the silencing of targets that bind both proteins. Third, Corto could modify the DNA bending ability of DSP1 and thus modulate its interaction with other factors, for example TrxG complexes. Indeed, the dsp1 gene has been shown to interact with the TrxG genes trx and brm . The results do not allow discrimination between these last two, non-exclusive possibilities (Salvaing, 2006).

The Hox gene Scr is a common target of Corto and DSP1. Both proteins bind a 10-kb XbaI fragment located 37-kb upstream of the Scr transcription start. Genetic studies have shown that this fragment is required for Scr function in the embryo and in the imaginal disc. In embryos, it restricts the expression of a Scr-lacZ fusion gene to the labial and prothoracic segments, whereas in larvae it is required for Scr expression in the first leg imaginal disc and for Scr silencing in the second and third leg imaginal discs. Interestingly, the function of the 10-kb XbaI fragment is sensitive to a subset of PcG and TrxG mutations and has been genetically characterized as an upstream maintenance element of Scr . At the end of embryogenesis, the Scr expression domain is restricted to the labial and prothoracic segments. In consequence, the mean state of this ME in the whole embryo would be silenced. It can thus be assumed that the global situation in embryos mimics that of the T2 and T3 leg imaginal discs. Conversely, since Scr is expressed in S2 cells, it is proposed that the situation in S2 cells rather mimics that of T1 leg imaginal disc cells. Hence, Corto, which is present on the Scr ME whether active (S2 cells) or silenced (embryos), could be present on this ME in all three leg imaginal discs. In contrast, DSP1, which is present on the ME in S2 cells but not in embryos, could bind the ME only in cells where this element is active, hence in T1 leg imaginal disc cells. It is thus proposed that both Corto and DSP1 proteins localize on this Scr ME in the first leg imaginal disc (Salvaing, 2006).

Some trxG mutants as well as the dsp1 null mutant exhibit a reduced sex comb and previous work has shown that dsp1 interacts with certain trxG genes and regulates Scr expression in T1 discs. HMGB, the vertebrate homologue of DSP1, has been reported to activate and stabilize the TFIID-TFIIA-promoter complex and some TrxG factors have been shown to interact with the RNA polymerase II complex, thus facilitating transcriptional elongation. This leads to a proposal that in the T1 leg imaginal disc, DSP1 facilitates the interaction between a TrxG complex and the transcription machinery, thus maintaining Scr activation. Moreover, Corto has been shown to interact with PcG complexes. The binding of Corto to DSP1 could then impede the interaction between Corto and PcG complexes, thus limiting their recruitment. Therefore, the interaction between the two proteins on the ME would lead to a level of Scr transcription compatible with T1 identity. Conversely, in the T2 and T3 leg imaginal discs, since DSP1 does not bind the ME, Corto would be able to interact with PcG complexes, thus enhancing the silencing of Scr (Salvaing, 2006).

In summary, this study has shown that the two ETPs Corto and DSP1 directly interact and are simultaneously found on a Scr ME when active, whereas Corto alone is found on the same ME when inactive. These data suggest that different combinations of ETP favor the recruitment of either PcG or TrxG complexes, participating in the maintenance of the silenced or active state of ME (Salvaing, 2006).

Diverse transcription influences can be insulated by the Drosophila SF1 chromatin boundary

Chromatin boundaries regulate gene expression by modulating enhancer-promoter interactions and insulating transcriptional influences from organized chromatin. However, mechanistic distinctions between these two aspects of boundary function are not well understood. This study shows that SF1, a chromatin boundary located in the Drosophila Antennapedia complex (ANT-C), can insulate the transgenic miniwhite reporter from both enhancing and silencing effects of surrounding genome, a phenomenon known as chromosomal position effect (CPE). It was found that the CPE-blocking activity associates with different SF1 sub-regions from a previously characterized insulator that blocks enhancers in transgenic embryos, and is independent of GAGA factor (GAF) binding sites essential for the embryonic insulator activity. Evidence is provided that the CPE-blocking activity cannot be attributed to an enhancer-blocking activity in the developing eye. The results suggest that SF1 contains multiple non-overlapping activities that block diverse transcriptional influences from embryonic or adult enhancers, and from positive and negative chromatin structure. Such diverse insulating capabilities are consistent with the proposed roles of SF1 to functionally separate fushi tarazu (ftz), a non-Hox gene, from the enhancers and the organized chromatin of the neighboring Hox genes (Majumder, 2009).

This study has characterized the CPE-blocking activity associated with the Drosophila SF1 boundary. The results suggest that SF1 contains at least two non-overlapping boundary activities, a strong embryonic enhancer-blocking activity associated with SF1b element, and strong CPE- blocking activities associated with SF1a and SF1c elements. Mutagenesis and dissection studies indicate that the CPE-blocking activity depends on different cis and trans components from the embryonic enhancer-blocking activity. It was further shown that the CPE-blocking activity is unlikely to be attributed to a late stage enhancer-blocking activity in the developing eye (Majumder, 2009).

Drosophila CPE, manifested predominantly by the enhancement or suppression of miniwhite, was thought to result from the active or repressive chromatin around the transgene insertion sites. CPE-blocking activity, therefore, has been compared to the vertebrate barrier activity and long used as a defining feature for chromatin boundaries in Drosophila. However, the ability of Drosophila boundaries to block both positive and negative CPE argues against a shared mechanism between these elements and the vertebrate barriers such as the β-globin barrier, which counter the progression of silent chromatin by establishing centers of active chromatin (Majumder, 2009).

An alternative explanation for the Drosophila CPE invokes the action of enhancers or silencers near the integrated transgenes. This model is consistent with the ability of boundaries to block both positive and negative effects. It also accommodates the fact that for some Drosophila boundaries the CPE-blocking activity depends on the same cis- and trans- components as the enhancer-blocking activity. However, this hypothesis would predict widespread presence of eye-specific enhancers and silencers in the genome to account for the prevalence of the CPE effect (Majumder, 2009).

This analysis of the SF1 boundary provides the first evidence that the CPE-blocking activity can be separated from the enhancer-blocking activity, suggesting that these two insulating functions may be mediated through distinct mechanisms in Drosophila. It is possible that the CPE-blocking activities in Drosophila form structures that are transcriptionally 'neutral', and able to insulate the weak miniwhite promoter from the effect of local chromatin. It is unclear, however, whether such local chromatin effect can compare, in range or strength, to that of constitutive heterochromatin, or whether such effect influences Drosophila gene promoters in general. A previous study showed that human MAR sequence could facilitate CPE blocking either arranged to flank the reporter or placed upstream in tandem copies. This is distinct from the CPE-blocking behavior of Drosophila boundaries such as suHw and scs, further demonstrating the diverse mechanisms that could influence the regulation of the miniwhite reporter (Majumder, 2009).

The SF1 boundary is located in the Scr-ftz genomic interval in the Drosophila ANT-C, which differs from other Hox clusters in that it contains both homeotic and non-homeotic genes. Proper regulation of these genes requires modulation of enhancer traffic as well as insulation of chromatin-mediated effects. The SF1 compound boundary fulfills both requirements: the SF1b element can restrict long-range enhancers from interfering with the ftz and Scr promoter; and the SF1a and SF1c elements may protect the non-Hox ftz gene from chromatin-mediated regulation, such as the PRE/TRE maintenance of the neighboring Hox genes. Separation and selective association of different types of boundary activities could determine the regulatory role of compound boundaries and provide flexibility in their function (Majumder, 2009).

Caudal, targeting Antp P2 and Scr promoters, is a DPE-specific transcriptional factor

The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promoter element (DPE). To understand the basis for core promoter diversity, potential biological functions of the DPE were explored. It was found that nearly all of the Drosophila homeotic (Hox) gene promoters, which lack TATA-box elements, contain functionally important DPE motifs that are conserved from Drosophila melanogaster to Drosophila virilis. It was then discovered that Caudal, a sequence-specific transcription factor and key regulator of the Hox gene network, activates transcription with a distinct preference for the DPE relative to the TATA box. The specificity of Caudal activation for the DPE is particularly striking when a BRE^u core promoter motif is associated with the TATA box. These findings show that Caudal is a DPE-specific activator and exemplify how core promoter diversity can be used to establish complex regulatory networks (Juven-Gershon, 2008).

This study found that the DPE is used extensively in the network of genes that are involved in the development of the early Drosophila embryo. Nearly all of the Drosophila Hox gene promoters, which have been known to be TATA-less, contain functionally essential DPE motifs that are conserved from D. melanogaster to D. virilis. The two Hox genes lacking DPE motifs are also the most recent Hox genes from an evolutionary standpoint. Thus, the DPE is a critical yet previously unrecognized component of the Hox genes (Juven-Gershon, 2008).

The DPE is not only in the Hox genes, but is also present in ftz, gt, h, fkh, cad (zygotic promoter), and en, which are involved in early embryonic development. This finding suggests that the DPE is used broadly throughout the network of genes that mediate the development of the embryo. This hypothesis was tested by analyzing the transcriptional properties of Caudal, a ParaHox protein and sequence-specific DNA-binding factor that regulates ftz, gt, h, and fkh. These studies revealed that Caudal is a DPE-specific activator. The preference of Caudal for activating transcription from DPE- versus TATA-dependent core promoters is seen most distinctly either with the natural ftz enhancer-promoter region or with a core promoter containing a BRE^u motif associated with the TATA box. The effect of Caudal on transcription of two Hox genes, Antp and Scr, was examined and it was found that Caudal activates the TATA-less, DPE-dependent Antp P2 and Scr promoters. These findings thus provide a direct link between Caudal, a DPE-specific activator, and the DPE-containing Hox genes (Juven-Gershon, 2008).

The discovery that Caudal is a DPE-specific activator provides new insight into the basic mechanisms of transcriptional regulation. Previous enhancer-trapping experiments have shown that there are enhancers that activate DPE-dependent promoters but not TATA-dependent promoters; however, neither the cis-acting elements nor the trans-acting factors that are responsible for the DPE-specific activation had been identified. Therefore, these studies demonstrate the existence of a DPE-specific enhancer-binding factor. Moreover, it is likely that other core-promoter-specific enhancer-binding factors, such as TATA-specific activators, will be discovered (Juven-Gershon, 2008).

These experiments uncovered a novel activity of the BRE^u core promoter motif. The BRE^u is a 5' extension of the TATA box that is bound by the TFIIB basal/general transcription factor. Depending on the context, the BRE^u has been found to have either a positive or a negative effect on transcription. In this study, it was found that the BRE^u motif has little effect on basal/unactivated transcription, but potently suppresses the ability of Caudal to activate transcription via the TATA box. In contrast, the BRE^u in its normal upstream location has no effect on Caudal-mediated activation via the DPE. These findings indicate that the BRE^u can contribute to core-promoter-element-mediated transcriptional regulation. Hence, there is a positive linkage between Caudal and the DPE as well as a negative interaction between Caudal and the BRE^u-TATA element. The combination of both positive (DPE) and negative (BRE^u-TATA) interactions yields maximal specificity of Caudal function (Juven-Gershon, 2008).

The new findings lead to the model that transcriptional regulation involves the combined action of sequence motifs in both the core promoter and the enhancer. The ability of Caudal to discriminate among DPE, TATA, and BRE^u-TATA motifs regulates the flow of information from the enhancer-bound activator to the core promoter -- the site of transcription initiation. In this manner, core promoter elements can be viewed as a component of transcriptional circuits. In these transcriptional circuits, connections between enhancers and core promoters are established and modulated according to the properties of the activators and the sequence motifs in the core promoter. Thus, the discovery that Caudal is a core-promoter-specific activator reveals a new strategy with which complex transcriptional networks can be established. Hence, in a broader sense, these findings show how diversity in core promoter structure can contribute to organismal diversity (Juven-Gershon, 2008).

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 (see Tip60). 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).

Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).

This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).

Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).

Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).

An organizational hub of developmentally regulated chromatin loops in the Drosophila Antennapedia complex

Sex combs reduced (Scr) is directed by an unusually long regulatory sequence harboring diverse cis elements and an intervening neighbor gene fushi tarazu (ftz). This study reports the presence of a multitude of Chromatin boundary elements (CBEs) in the Scr regulatory region. Selective and dynamic pairing among these CBEs mediates developmentally regulated chromatin loops. In particular, the SF1 boundary plays a central role in organizing two subsets of chromatin loops: one subset encloses ftz, limiting its access by the surrounding Scr enhancers and compartmentalizing distinct histone modifications; and the other subset subdivides the Scr regulatory sequences into independent enhancer access domains. Tandem pairing of SF1 and SF2, two strong CBEs that flank the ftz domain, providing a mechanism for the endogenous Scr enhancer to circumvent the ftz domain. This study demonstrates how an endogenous CBE network, centrally orchestrated by SF1, could remodel the genomic environment to facilitate gene regulation during development (Li, 2015).

The genomes of insects and mammals are widely populated with CBEs that may serve as anchoring sites for chromatin loops. An increasing body of evidence suggests that in addition to the CBEs that reside between genetic loci and insulate genes, some CBEs can also be found in the introns and in regulatory sequences of a single gene. Using the Drosophila Scr locus as a model, this study has attempted to elucidate the roles of this new class of CBEs in gene regulation. Within a 50-kb Scr regulatory region, there are at least four CBE-like elements that interact with SF1. Evidence is provided that SF1 tethers multiple developmentally regulated chromatin loops through selective and dynamic pairing with these STEs during development. One subset of the loops functionally isolates the ftz gene embedded in the Scr regulatory sequences, while others subdivide and possibly facilitate the Scr early and late regulatory elements. In particular, an STE called SF2 loops with SF1 to enclose and insulate ftz from Scr by blocking the Scr long-range enhancers and repressive chromatin structures. Importantly, association of SF1-SF2 facilitates enhancer bypass in transgenic embryos, suggesting a mechanism that could assist the Scr distal enhancers in circumventing the ftz domain in vivo. These findings validate a mechanism that not only allows CBE-like elements to be tolerated within gene regions but also may provide diverse utility in other genomic functions. This study provides a comprehensive analysis of how an endogenous CBE network, centrally orchestrated by SF1, might provide multilayered control of Scr and ftz gene activities by coordinating dynamic and selective formation of chromatin loops in rapidly developing embryos (Li, 2015).

The chromatin loops tethered by SF1 and STEs may address several major challenges to proper gene regulation in the Scr-ftz-Antp gene region.

First, the loops regulate enhancer access: The Scr regulatory region contains a nested pair rule gene, ftz. The Scr and ftz promoters are located close to each other, and their enhancers are scattered on both sides of ftz. How are enhancer-promoter interactions specified for these two genes? This study has shown that SF1 may play a role by blocking an intergenic enhancer from a Scr-like promoter. However, it remains unclear how ftz is insulated from Scr in the downstream direction. This work showed that SF1 and SF2 pairs transiently to enclose the ftz gene domain, including all its enhancers. The timing and extent of the loop coincide with a reduced access of the ftz promoter to the outside Scr enhancers in vivo. In transgenic embryos, SF1 inserted distal to NEE can also augment the block of the enhancer by SF2B, supporting the notion that an SF1-SF2 loop restricts enhancer access (Li, 2015).

This study further indicates that the SF1-STE loops correlate with domains of enhancer access for the Scr distal regulatory elements. By pairing individually with SF1, these loops could facilitate selected access of these elements to the Scr promoter. Such delineation of enhancer domains by CBE-like elements is reminiscent of the Fab boundaries subdividing the iab enhancer domains in the Abd-B regulatory region. Compared to Fab-7, which was shown to restrict enhancer domains in a tissue-specific fashion, the STEs appear to separate the Scr distal regulatory sequences into early and late regulatory domains. The data further show that the R9/10 region contains a constitutive boundary that may separate as well as insulate neighboring Scr and Antp genes. This region was also known to tether to both Scr and Antp promoters, possibly regulating the access or activity of the Scr distal enhancers (Li, 2015).

Second, the loops result in a separation of distinct chromatin structure: The ftz gene is transcribed in many tissues in which Scr is inactive during early development, and the two genes continue to be expressed in distinct tissues in later stages. How does ftz remain active amid the repressive chromatin assembled in the surrounding Scr regions? Among the STEs, both SF2 and R2 are located at the end of the ftz domain. This study shows that the transient SF1-SF2 loop in 4- to 8-h embryos indeed defines the active ftz domain marked by low H3K9me3 and low H3K27me3 at this stage. The stable SF1-R2 loop also correlates with a small but visible border of distinct chromatin structures between the two genes during late development, possibly protecting ftz from the encroachment of PRE-mediated silencing (Li, 2015).

Third, the loops facilitate the Scr distal enhancers. The Scr regulatory sequences are interrupted by the ftz gene domain and multiple CBEs, among which SF1 and SF2 contain strong and ubiquitous enhancer-blocking activity. These could pose impediments to the Scr distal enhancers. Previous studies have shown that tandem arrangement of CBEs may lead to reduction or cancellation of their enhancer-blocking function due to changes in chromatin loop configurations. Based on this, it was postulated that pairing between SF1 and SF2 would loop out the ftz domain and allow the Scr distal enhancers to 'bypass' the block of both boundaries. This study has shown that tandem arrangement of SF1 and SF2 indeed neutralizes the block of the distal enhancers in a transgenic setting. This provides a potential mechanism for the Scr distal regulatory elements to overcome multiple CBEs to interact with the Scr promoter (Li, 2015).

This study suggests that the unique SF1-SF2 loop may fulfill multiple functional roles as listed above. Interestingly, the SF1-SF2 interval corresponds to an evolutionarily conserved genomic block (Powell conserved region) that contains the entire ftz gene and is found in a 'flipped' orientation in several Drosophila species. These observations suggest that chromatin loops may shield gene regulation from local chromosome rearrangements, resulting in intermingling as well as interdependence of genes and their regulatory environment during evolution (Li, 2015).

Fourth, the loops result in diverse enhancer-blocking behaviors by STEs . Among the CBEs in the Scr-Antp interval, SF1, SF2, and the R9/10 element exhibit strong and ubiquitous enhancer-blocking activities in the transgenic insulator assay. Genome-wide chromatin immunoprecipitation (ChIP) studies showed that these three elements associate with distinct sets of insulator proteins. While SF1 and SF2 are bound by dCTCF, CP190, and SuHw, R9/10 exhibits strong binding to GAF and Mod(mdg4). Although GAF binds only weakly to SF1, it has been shown to be critical for the enhancer-blocking activity of an SF1 subfragment. GAF also footprints weakly with SF2 but in a nonoverlapping pattern with other insulator proteins. Mod(mdg4) is the only insulator factor that binds significantly to all three elements. These observations suggest that although most known insulator proteins are ubiquitously expressed, selective or combinatorial recruitment of these proteins to various genomic sites by developmentally regulated factors may be involved in regulated boundary activity (Li, 2015).

Two other STEs, R2 and R6, did not exhibit ubiquitous enhancer-blocking activity. The data suggest that R2 and R6 may contain enhancer-blocking activity in labial and thoracic segments. These are the tissues in which Scr and Antp are expressed. In the insulator ChIPseq profile, the R6 region appears to be overdepleted for known insulator proteins compared with the surrounding genome, suggesting that another protein factor(s) may bind there and possibly facilitate interactions with SF1 and other STEs. The results further indicate that although SF1-STE interactions appear to modulate the access of endogenous enhancers, they may not be sufficient to block heterologous enhancers in insulator assays. It is possible that the strength of endogenous chromatin loops is adapted to neighboring regulatory interactions, rather than universally strong. A previously reported endogenous boundary, the 1A2 region in the Drosophila yellow locus, interacts with a full-length Gypsy insulator but exhibits relatively weak enhancer-blocking activity. The results also suggest that major chromatin boundaries, such as SF1, may interact with diverse partners to organize local networks of chromatin loops. These loops may vary in strength, duration, or the tissues in which they form, but they are all physiologically relevant for local gene regulation (Li, 2015).

Certain CBEs are known to allow enhancers to 'bypass' when they are arranged in tandem or interacting in trans. This was taken as evidence that CBEs block enhancers by tethering chromatin loops. An enhancer flanked by pairing CBEs is enclosed in a chromatin loop and blocked from promoters outside the loop, whereas an enhancer and a promoter separated by paired CBEs can interact with each other. Enhancer bypass was first demonstrated for the Gypsy insulator in transgenic Drosophila. Recent studies had shown that boundaries from the Bithorax complex, including Fab-7 and Fab-8, also interact with each other and mediate bypass of heterologous enhancers. Interestingly, the previous data showed that pairing of the full-length Fab-7 and Fab-8 elements did not lead to enhancer bypass in transgenic embryos. This might be due to the absence of the pairing partners in the genome vicinity, an indication of the diverse interactions that could occur between CBEs. The enhancer bypass that were observed in SF1-SF2 paring is the first such example mediated by two authentic pairing CBEs from the Drosophila Antennapedia complex. It provides an explanation of why CBEs not only are tolerated within gene regions but also, indeed, could perform essential functions during gene regulation (Li, 2015).

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