fushi tarazu

Promoter Structure

Two cis-acting control elements are required for ftz expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes, and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer (Hiromi, 1987).

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 fushi tarazu (Pattatucci, 1990b).

ftz has a TATA box and a potential loop forming structure at the 5' end of the gene (Laughon, 1984). The ftz gene has been shown to contain three cis-acting regulatory elements: a zebra element, the neurogenic element, and an upstream element (Hiromi, 1985). The zebra element can drive ftz expression in seven evenly spaced stripes. This is comparable with the stripe-specific expression elements of even-skipped. The upstream element contains two independent enhancers: the distal enhancer directs expression of seven mesodermally restricted stripes, and the proximal enhancer directs expression in stripes that span both ectodermal and mesodermal primordia (Pick, 1990).

The seven-stripe pattern of ftz during early embryogenes is largely specified by the zebra element located immediately upstream of the ftz transcriptional start site. The FTZ-F1 protein, one of multiple DNA binding factors that interacts with the zebra element, is implicated in the activation of ftz transcription, especially in stripes 1, 2, 3, and 6. The predicted amino acid sequence of FTZ-F1 reveals that the protein is a member of the nuclear hormone receptor superfamily (Lavorgna,1991).

The proximal enhancer of ftz, located approximately 3.4 kb upstream of the transcription start site has nine protein binding sites. Ten different sequence-specific DNA-binding complexes that interact with eight of these sites were identified. Some interact with multiple sites, while others bind to single sites in the enhancer. Two of the complexes that interact with multiple sites appear to contain the previously described ftz regulators, FTZ-F1 and Tramtrack/FTZ-F2. In vitro studies allow a narrowing down of the proximal enhancer to a 323-bp DNA fragment that contains all of the protein binding sites. Expression directed by this minimal enhancer element in seven FTZ-like stripes in transgenic embryos is identical to that directed by the full-length enhancer (Han, 1993).

The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. Nine binding sites for multiple Drosophila nuclear proteins have been identifed in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. The nuclear receptor Ftz-F1 interacts with this repeated module. Additional proteins interacting with this module were purified from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Tramtrack and the transcription factor Adh transcription factor 1 (Adf-1) were obtained. While Ttk is thought to be a repressor of ftz stripes, both Adf-1 and Ftz-F1 have been shown to activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1/Adf-1/Ttk binding sites are functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis has identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks (Han, 1998).

It was proposed several years ago that Ttk acts as a repressor of ftz stripes, since the protein is present before and after ftz is expressed in stripes but is not detected during the time that ftz is expressed in stripes (Harrison, 1990). The proximal enhancer used in the current studies contains multiple binding sites for Ttk. Therefore, an initial intention was to test the role of Ttk as a repressor of ftz stripes by simultaneously mutating multiple Ttk binding sites. It was expected that fusion gene expression would initiate earlier and/or persist later in the absence of repression by Ttk. Fusion genes 12 and 13 carry mutations in four Ttk sites, while all five sites are mutated in fusion gene 14. However, three of the five Ttk binding sites overlap with binding sites for activator proteins that are necessary to activate expression of fusion genes (fusion gene 11). Therefore, it was not possible to test whether Ttk represses through its proximal enhancer binding sites, since mutations result in loss of activation due to this overlap. Currently, the role of Ttk in regulating ftz is unclear. Mutation of Ttk binding sites in the zebra element results in premature activation of ftz gene expression, and ectopic expression of Ttk at later stages causes a decrease in ftz expression levels. However, given the observation that most Ttk binding sites also interact with other nuclear proteins, it is difficult to know whether these observations are a result of direct negative regulation of ftz by Ttk. Preliminary results suggest that Ttk can act as a transcriptional activator, raising the possibility either that Ttk interacts with a corepressor to decrease ftz expression levels or that observed effects of Ttk overexpression in embryos are indirect (Han, 1998).

A complex array of activator and repressor elements located within 669 bp proximal to the fushi tarazu transcriptional start site is sufficient to generate the 'zebra-stripe' expression pattern characteristic of the ftz gene. P-element-mediated transformation and ftz promoter/lacZ fusion genes were used to characterize, in detail, several of these transcriptional control elements. By reconstructing promoters with synthetic oligonucleotides containing cis-regulators of stripe expression, it has been shown that these regulatory sites can function as independent units to direct position-specific transcription in the Drosophila embryo. In particular, multiple copies of a positive regulatory site can mediate expression in both the odd- and even-numbered parasegments throughout most of the germ band. Specifically, the fAE3 site serves as an activator recognition site. A protein that binds to this motif is a transcriptional activator of Ultrabithorax and engrailed.. Negative regulatory sites can also transform a continuous pattern of gene expression into discrete stripes. Deletion of the fAE3 site causes ectopic expression of ftz in interstripe regions. This result suggests that fAE3 has a repression function. Four copies of the adjacent fDE site are able to convert a continuous, graded band of expression into a highly resolved pattern of seven stripes, indicating that multiple copies of a single repressor site can selectively repress transcription in this assay. Hairy is somehow required for repression of expression through the fDE1 element. FTZ-F1 can recognize fDE1 and fDE2 sites, both of which are known to serve dual activating and repressing functions. The reconstructed promoter system presented provides an effective means of studying molecular mechanisms governing spatially restricted transcription in the early embryo (Topol, 1991).

A motif in the zebra element, the FTZ-F1 recognition element (F1RE), has been shown to bind transcription factor FTZ-F1 alpha, a member of the nuclear receptor family. A second, related member of this family, FTZ-F1 beta, also binds to this motif. FTZ-F1 alpha and FTZ-F1 beta both bind as monomers to the 9-bp F1RE in the zebra element, as well as to an imperfect inverted F1RE repeat present in the Drosophila alcohol dehydrogenase gene. These data suggest that FTZ-F1 alpha and FTZ-F1 beta likely coregulate common target genes by competition for binding to a 9-bp recognition element (Ohno, 1994).

The segmentation genes runt and hairy are required for the proper transcriptional regulation of the pair-rule gene fushi tarazu during the blastoderm stage of Drosophila embryogenesis. runt and hairy act on ftz through a common 32 base pair element, designated as fDE1. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by runt and repression by hairy. Examination of reporter genes with mutated fDE1 elements provided further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicate that the opposing effects of runt and hairy are not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to Runt and Hairy (Tsai. 1995).

Binding the the TFIID complex to a target promoter depends on at least three different core promoter elements located within a 50- to 60-base pair sequence flanking the transcription start site, the TATA box, the initiator element (Inr), and the downstream promoter element (Dpe). In general, promoters that lack a TATA sequence must possess conserved copies of the Inr and/or Dpe. Conversely, promoters containing optimal TATA sequences do not require Inr and Dpe elements for the binding of TFIID. The presences of these three elements define two common types of promoters: type I promoters contain a TATA box, whereas type II promoters contain Inr and Dpe sequences. There are numerous examples of shared enhancers interacting with just a subset of target promoters. These "shared enhancer" type of interactions are contrasted with a "competitive interaction" type. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. A transgenic embryo assay was used to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 (located between Sex combs reduced and fushi tarazu) enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer (which selectively activates Abdominal-B, not abdominal-A) from the Bithorax complex (BX-C) preferentially activate the type I, TATA-containing, promoters when challenged with linked TATA-less promoters. The AE1 autoregulatory element in the ANT-C specifically interacts with the ftz promoter, but does not activate the equidistant Sex combs reduced gene. AE1 and IAB5 exhibit a competitive type of interaction. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between type I and type II classes of promoters and exhibit a shared enhancer type of interaction. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters (Ohtsuki, 1998).

Related artificially constructed core promoter sequences were initially used for the analysis of AE1. ftz and eve contain optimal TATA sequences, but lack Inr (INIT) and Dpe (DPE) elements. AE1 also activates white and Tp promoters. white and Tp each contain conserved copies of the INIT and DPE sequences, but lack a TATA sequence (white) or contains a suboptimal TATA (Tp). AE1 can simultaneously activate linked TATA-containing promoters or linked INIT/DPE-containing promoters. In spite of AE1's ability to activate type I and type II promoters, promoter competition can be demonstrated. There is a substantial reduction in white expression when the Tp promoter is replaced with the core eve promoter sequence. This AE1-eve interaction appears to block the expression of the linked white gene. In the absence of eve, white is fully active. These observations are compatible with a promoter-competition mechanism whereby AE1-eve interactions inhibit white (Ohtsuki, 1998).

Similarly, IAB5 prefers the eve promoter. The 1-kb IAB5 enhancer exhibits a preference for TATA-containing promoters. IAB5 was placed downstream of an eve/lacZ fusion gene; the linked CAT reporter gene was placed under the control of the mini-white promoter. There is strong expression of the lacZ reporter gene in the presumptive abdomen, whereas CAT is not expressed above background levels. This result suggests that IAB5 prefers the eve promoter over white. An eve-white chimeric promoter was analyzed in an effort to assess the importance of the core elements, particularly the TATA sequence. An ~20-bp region of the eve sequence (the TATA region) was replaced with the corresponding region of white. This modified eve promoter (evewhite) is attenuated and mediates only weak expression of lacZ in the presumptive abdomen. In contrast, the linked white promoter directs strong expression of CAT. These results suggest that the removal of the eve TATA releases the IAB5 enhancer so that it can now interact with the white promoter (Ohtsuki, 1998).

The 300-bp rhomboid NEE is equally effective in activating the two classes of promoters. Additional experiments were done to determine whether the targeting of IAB5 to eve influences the activities of the nonspecific rho NEE. The latter enhancer is activated by the maternal gradient of Dorsal transcription factor in lateral stripes within the neurogenic ectoderm. A synthetic gene complex was prepared that contains both the NEE and IAB5 enhancers. white and CAT reporter genes were attached to the mini-white promoter, whereas lacZ is driven by eve. The rho NEE activates all three reporter genes, so that white, CAT, and lacZ are all expressed in lateral stripes. In contrast, IAB5 primarily activates the eve promoter, so that only lacZ exhibits strong expression within the presumptive abdomen. These results suggest that IAB5-eve interactions do not influence the nonspecific activities of the rho NEE (Ohtsuki, 1998).

It has been suggested that TATA-containing promoters are intrinsically stronger than TATA-less promoters, possibly because of higher affinity interactions with the TFIID complex. The divergent activities of the IAB5 and NEE enhancers, however, are most easily interpreted on the basis of qualitative, not quantitative, differences in type I and type II core promoter sequences. For example, the insertion of a TATA sequence in the white promoter allows it to compete with a linked eve promoter, whereas the removal of TATA from eve permits activation of white. These alterations in the white and eve promoters, the insertion and removal of TATA, dramatically alter the activities of IAB5, but have virtually no effect on the NEE enhancer. NEE is equally effective in activating the eve, white, evewhite, and whiteTATA promoters, and thereby serves as an internal control for normal promoter function (Ohtsuki, 1998).

These results suggest that the IAB5 and AE1 activators, particularly Ftz, prefer type I promoters. NEE activators, including Dorsal (dl) and bHLH proteins, appear to be promiscuous and work equally well on both classes of core promoters. The authors propose that the TFIID complex adopts different conformations on type I and type II promoters. Basal targets for the Ftz activator may be displayed in a more accessible conformation when TFIID binds TATA. In contrast, basal targets for the Dorsal and bHLH activators may be equally accessible whether TFIID binds TATA or Inr/Dpe elements (Ohtsuki, 1998).

GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophila embryonic S150 extract. The promoter region of fushi tarazu carries several binding sites for GAGA factor, which triggers chromatin remodeling. Deletion of the GAGA factor-binding sites in the ftz promoter is known to markedly reduced the reporter gene expression. The striped expression of ftz is abolished by a mutation of the Trithorax-like gene, which encodes GAGA factor. Transcriptional activation of the ftz gene is observed when a preassembled chromatin template is incubated with GAGA factor and the S150 extract (Okada, 1998).

GAGA factor does not activate transcription on a naked DNA template. GAGA factor has been reported to activate transcription on naked DNA templates in crude extracts but not in transcription systems reconstituted from purified components. GAGA factor-mediated transcriptional activation on a naked DNA template requires the presence of a nonspecific DNA-binding protein, suggesting that GAGA factor functions as an antirepressor by preventing nonspecific inhibitory proteins such as histone H1 from binding to DNA. It is therefore possible that GAGA factor may activate transcription by an antirepressor mechanism in the transcription assay used in the current study. To test this possibility, experiments were carried out starting from a naked DNA template. Since the amount of S150 extract in the preincubation reactions is one order of magnitude lower than that required for full assembly of the chromatin structure on the template, nucleosomes are barely detectable by supercoiling assay after preincubation. In contrast to the preassembled chromatin, little activation (up to 1.5-fold) of ftz transcription is observed after preincubation of the naked template DNA with GAGA factor and the S150 extract. This indicates that only trace levels of activation may be caused by elimination of nonspecific DNA-binding proteins in the presence of GAGA factor. These results also suggest that the GAGA factor-mediated transcriptional activation occurs specifically on the chromatin template. These observations suggest that GAGA factor-mediated chromatin remodeling is required for the proper expression of ftz in vivo (Okada, 1998).

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

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

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

The S150 extract contains a nucleosome-remodeling factor (NURF) that acts with GAGA factor to disrupt the ordered array of nucleosomes near the GAGA factor-binding sites. The chromatin structure within the ftz promoter is specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Micrococcal nuclease assays show that the nucleosome structure surrounding nucleotide 350 in front of the TATA element (and the TATA element itself) of ftz are disrupted by incubation with GAGA factor and the S150 extract. A restriction enzyme assay demonstrates that the AvaII site at 9, the FspI sites at 90 and 317, and the PstI site at 267 on the ftz chromatin template are more susceptible to digestion after incubation with GAGA factor and the S150 extract. Base substitutions of all four GAGA sequences in the ftz promoter at 360, 348, 158, and 46 are required to completely suppress the GAGA factor-mediated chromatin remodeling. These results indicate that chromatin is remodeled throughout the proximal region of the ftz promoter. Both transcriptional activation and chromatin disruption are blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template. In vitro transcription studies have revealed that activation of ftz by FTZ-F1 requires two coactivators, termed MBF1 and MBF2. MBF1 is a bridging molecule that interconnects FTZ-F1 and TATA-binding protein and recruits positive cofactor MBF2 to a promoter carrying the FTZ-F1-binding site. MBF2 activates transcription through its contact with TFIIA. This allows the selective activation of ftz in a FTZ-F1 binding site-dependent manner. It is most likely that the GAGA factor-mediated chromatin remodeling in the proximal region of the ftz promoter is a prerequisite for the formation of active complexes containing FTZ-F1, MBF1, MBF2, TFIIA, and TBP (Okada, 1998).

Lamin is known to interact directly with highly conserved sequences of DNA. Lamin binds with high affinity to scaffold/matrix-associated regions (M/SARs). These DNA sequences are held responsible for mediating the interaction between the nuclear matrix and chromatin. M/SARs are several hundred base pairs long and contain stretches of AT-rich sequences that are likely to form an open chromatin configuration. Indeed, the binding of M/SARs to lamin polymers involves single-stranded regions. In addition, this binding is saturable and requires the minor groove. Lamin polymers also bind to Drosophila centromeric and telomeric sequences. The polymerized alpha-helical rod domain of Lamin, on its own, provides for specific binding to the fushi tarazu M/SAR (Zhao, 1996). The ftz M/SAR functions as an autonomously replicating sequence (ARS) in the budding yeast S. cerevisiae. This M/SAR is found in a 2.57 kb ftz upstream regulatory element. A 189 base pair minimal fragment has ARS function. However, based on growth rates and mitotic stability, its activity is lower than that of the entire SAR. The addition of flanking sequences, including as little as 100 bp of AT-rich DNA to the left of the minimal sequence, can enhance the replicative ability of the ARS. These results implicate lamins in initiation of DNA replication (Amati, 1990a; Amati, 1990b).

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).

A novel boundary element may facilitate independent gene regulation in the Antennapedia complex

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).

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).

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, 1995). 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).

Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. Searching for clusters of predicted transcription factor binding sites can discover active regulatory sequences; 37 regions of the Drosophila melanogaster genome have been identified with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. This study reports the results of in vivo functional analysis of 27 remaining clusters. Transgenic flies were generated carrying each cluster attached to a basal promoter and reporter gene, and embryos were assayed for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three other clusters drive expression in patterns unrelated to those of neighboring genes; the remaining 18 clusters do not appear to have enhancer activity. The Drosophila pseudoobscura genome was used to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. Conservation of binding-site clustering was incorporated into a new genome-wide enhancer screen, and several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns, have been preducted. It is concluded that measuring conservation of sequence features closely linked to function (such as binding-site clustering) makes better use of comparative sequence data than commonly used methods that examine only sequence identity (Berman, 2004).

Each of the 37 pCRMs were assigned an identifier (of the form PCEXXXX). The first nine overlap previously known enhancers of runt, even-skipped, hairy, knirps and hunchback. To determine whether any of the remaining 28 pCRMs also function as enhancers, P-element constructs were generated containing the pCRM sequence with minimal flanking sequence on both sides fused to the eve basal promoter and a lacZ reporter gene. Since the margins of the tested sequences do not precisely correspond to the margins of the clusters, a unique identifier (of the form CEXXXX) was assigned to each tested fragment (identical CE and PCE numbers correspond to the same pCRM) (Berman, 2004).

Multiple independent transgenic fly lines were sucessfully generated for 27 of the 28 pCRMs. Transgenes containing CE8007 could not be generated. This sequence contains five copies of an approximately 358 base-pair (bp) degenerate repeat. One additional pCRM (CE8002) also contains tandem repeats. While it was possible to generate transgenes for CE8002 and assay its expression, these two tandem repeat-containing pCRMs (CE8007 and CE8002) were excluded from subsequent analyses (Berman, 2004).

The expression of these constructs was examined by in situ RNA hybridization to the lacZ transcript in embryos at different stages in at least three independent transformant lines. Nine of the 27 transgenes showed mRNA expression during embryogenesis, while the remaining 18 assayed transgenes showed no detectable expression at any stage during embryogenesis (Berman, 2004).

To identify the genes regulated by the nine pCRMs with embryonic expression, the expression patterns were examined of genes containing the pCRM in an intron and genes with promoters within 20 kb of the CRM. The embryonic microrarray and whole-mount in situ expression data available in the Berkeley Gene Expression Database were used, supplemented with additional whole-mount in situ experiments where necessary. Six of the active pCRMs drive lacZ expression in patterns that recapitulate portions of the expression of a gene adjacent to or containing the pCRM. Four of these new enhancers act in the blastoderm and two during germ-band elongation (Berman, 2004).

CE8001 is 5' of the gene for the gap transcription factor giant and recapitulates the posterior domain (65%-85% egg length measuring from the anterior end of the embryo) of gt expression in the blastoderm. CE8011 is 5' of the gene for the POU-homeobox transcription factor nubbin (nub). The CRM recapitulates the endogenous blastoderm expression pattern of nub, first detected as a broad band extending from 50% to 75% egg length. Although nub expression continues in later embryonic stages, CE8011 expression is limited to the blastoderm stage. CE8010 is 5' of the pair-rule gene odd-skipped (odd) and drives expression of two of its seven stripes: stripe 3 at 55% and stripe 6 at 75% egg length. This CRM also has the ability to drive later, more complex, patterns of expression. During stages 6 and 7, expression is detected in the procephalic ectoderm anlage and in the primordium of the posterior midgut. By stage 13, expression is also detected in the anterior cells of the midgut which will give rise to the proventriculus, the first midgut constriction, the posterior midgut and microtubule primordial as well as cells in the hindgut, all similar to portions of the pattern of wildtype Odd protein expression. CE8024 is 3' of the pair-rule gene fushi-tarazu and drives expression of two of its stripes: stripe 1 at 35% and stripe 5 at 65% egg length. CE8012 is in the third intron of POU domain protein 2 (pdm2) and appears to completely recapitulate its stage-12 expression pattern, which is limited to a subset of the developing neuroblasts and ganglion mother cells of the developing central nervous system. A similar pattern of expression was previously described for the protein product of pdm2. It is worth noting that expression of CE8012 was not detected in the blastoderm stage, whereas the endogenous gene exhibits a blastoderm expression pattern similar to nub. CE8027 is 3' of the gene for the Zn-finger transcription factor squeeze (sqz) and recapitulates the wild-type expression pattern of sqz RNA in a subset of cells in the neuroectoderm at stage 12 (Berman, 2004).

The remaining three active pCRMs cannot be easily associated with a specific gene. CE8005 drives expression in the ventral region of the embryo. It is 3' of a gene encoding a ubiquitously expressed Zn-finger containing protein (CG9650) that is maternally expressed and deposited in the embryo. This strong maternal expression potentially obscures a zygotic expression pattern. Two additional adjacent genes, CG32725 and CG1958, showed no expression in whole-mount in situ hybridization of embryos. CE8016 drives a seven-stripe expression pattern in the blastoderm. It is in the first intron of CG14502 which shows very low level expression by microarrays in the blastoderm, and has no obvious detectable pattern of expression in whole-mount in situ hybridization of embryos. This pCRM is approximately 2 kb 5' of scribbler (sbb), which is expressed maternally, possibly obscuring an early zygotic expression pattern (a few in situ images show a hint of striping). sbb is also expressed later in development in the ventral nervous system. An additional potential target, Otefin (Ote), is also expressed maternally and relatively ubiquitously through germ-band extension. All other nearby genes showed no embryonic expression in whole-mount in situ hybridization or by microarray. CE8020 drives an atypical four-stripe pattern in the blastoderm -- two stripes at 7% and 26% that are anterior to the first ftz stripe and two stripes at 39% and 87%. It is in the first intron of ome (CG32145), which is not expressed maternally and has no blastoderm expression, but is expressed late in salivary gland, trachea, hindgut and a subset of the epidermis. All other nearby genes showed no embryonic expression in whole-mount in situ hybridization or by microarray (Berman, 2004).

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation.

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The 'primary' pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas 'secondary' pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, a comprehensive systems-level examination was conducted of the regulatory architecture underlying pair-rule stripe formation. runt (run), fushi tarazu (ftz) and odd skipped (odd) were found to establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, long-range cis-regulation was observed across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, this study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo (Schroeder, 2011).

The transition from non-periodic to periodic gene expression patterns is a key step in the establishment of the segmented body plan of the Drosophila embryo. The pair-rule genes that lie at the heart of this process have been the subject of much investigation, but important questions, in particular regarding the organization of cis-regulation, have remained unresolved. We have revisited the issue in a comprehensive fashion by combining computational and experimental cis-dissection with mutant analysis and a detailed characterization of expression dynamics, both of endogenous genes and of cis-regulatory elements. This systems-level analysis under uniform conditions reveals important insights and gives rise to a refined and in some respects substantially revised view of how periodic patterns are generated (Schroeder, 2011).

The most important finding of this study is that ftz and odd, which had been regarded as secondary pair-rule genes, closely resemble eve, h and run in nearly all respects and should thus be co-classified with them as primary pair-rule genes. Expression dynamics clearly subdivide the pair-rule genes into two distinct groups, with eve, h, run, ftz and odd all showing an early and non-synchronous appearance of most stripes, whereas the 7-stripe patterns of prd and slp1 arise late and synchronously. The early expression of stripes is associated with the existence of stripe-specific cis-elements that respond to positional cues provided by the maternal and gap genes. Unlike previously thought, maternal and gap input is used pervasively in the initial patterning not only of eve, h and run, but also of ftz and odd. Aided by computational predictions, eight new stripe-specific cis-elements were identifed for ftz, odd, and run. Although molecular epistasis experiments reveal a stronger role for eve, h and run in pair-rule gene cross-regulation, odd clearly affects the blastoderm patterning of other primary factors as well and ftz affects the patterning of odd (Schroeder, 2011).

The revised grouping brings the classification of pair-rule genes in line with their role in transmitting positional information to the subsequent tiers in the segmentation hierarchy. By the end of cellularization, the expression patterns of h, eve, run and ftz/odd are offset against each other to produce neatly tiled overlaps of four-nuclei-wide stripes. In combination, these patterns define a unique expression code for each nucleus within the two-segment unit that specifies both position and polarity in the segment and is read off by the segment-polarity genes. ftz (as an activator of odd) and odd together represent the fourth essential component of this positional code and are thus functionally equivalent to h, eve and run. Placing all components of this code under the same direct control of the preceding tier of regulators presumably increases both the speed and the robustness of the process (Schroeder, 2011).

Although the results support a subdivision between primary and secondary pair-rule genes, they also reveal surprising complexities in the regulatory architecture that are not captured by any rigid dichotomy. The five primary pair-rule genes all have different cis-regulatory repertoires: stripe formation in h appears to rely solely on stripe-specific cis-elements, whereas eve employs a handoff from stripe-specific elements to a late-acting 7-stripe element. In run, ftz and odd, by contrast, the emergence of the full 7-stripe pattern is the result of joint action by stripe-specific elements and early-acting 7-stripe elements, with ftz and odd requiring the 7-stripe element to generate a subset of stripes. This difference in the importance of 7-stripe elements in stripe formation is supported by the results of rescue experiments: whereas the 7-stripe elements of both run and ftz provide partial rescue of the blastoderm expression pattern when the stripe-specific elements are missing, this is not the case for eve. Interestingly, run appears to occupy a unique and particularly crucial position among the five genes: it has both a full complement of stripe-specific elements and an early-acting 7-stripe element, and thus serves as an early integration point of maternal/gap input and pair-rule cross-regulation. Its regulatory region is particularly large and complex, with cis-elements acting over long distances across intervening genes and with partial redundancy between elements. Moreover, the removal of run has the most severe effects on pair-rule stripe positioning among the five genes. Another complexity lies in the fact that the early anterior expression of slp1 and prd, which otherwise exhibit all the characteristics of secondary pair-rule genes, has a clear role in patterning the anteriormost stripes of the primary pair-rule genes. In fact, evidence is provided that the most severe defect observed in the primary pair-rule gene mutants, namely the loss of pair-rule gene stripes 1 or 2 under eve loss-of-function conditions, is an indirect effect attributable to the regulation of these stripes by slp1 (Schroeder, 2011).

This investigation provides important new insight regarding the relative roles of maternal/gap input and pair-rule gene cross-regulation in stripe formation. As described, nearly all stripes of the primary pair-rule genes are initially formed through stripe-specific cis-elements. Cross-regulatory interactions between the pair-rule genes then refine these patterns by ensuring the proper spacing, width and intensity of stripes, as revealed by mutant analysis. However, these two aspects or layers of regulation are not as clearly separated as has often been thought. For example, 7-stripe elements have been regarded primarily as conduits of pair-rule cross-regulation; by contrast, this study found that the 7-stripe elements of run, ftz and odd are activefrom phase 1 onwards and contain significant input from the maternal and gap genes, resulting in modulated or partial 7-stripe expression early. In the case of the KR binding sites predicted within the run 7-stripe element, mutational analysis showed that this input is indeed functional. Conversely, stripe-specific cis-elements receive significant regulatory input from pair-rule genes. This is particularly clear in the case of eve and h: expression dynamics indicate that their 7-stripe patterns become properly resolved without the involvement of 7-stripe elements, and the defects in the h and eve expression patterns that were observe in pair-rule gene mutants occur at a time when only stripe-specific elements are active. In a few cases, pair-rule input into stripe-specific elements has been demonstrated directly, but the comparative analysis of expression dynamics underscores that it is indeed a pervasive feature of pair-rule cis-regulation (Schroeder, 2011).

An important question arising from these findings is how the different types of regulatory input are integrated, both at the level of individual cis-elements and at the level of the locus as a whole. Binding sites for maternal and gap factors typically form tight clusters, supporting a modular organization of cis-regulation. Such modularity is crucial for the 'regional' expression of individual stripes, which can be achieved only if repressive gap inputs can be properly separated between cis-elements. By contrast, binding sites for the pair-rule factors appear to be more dispersed across the cis-regulatory regions and not tightly co-clustered with the maternal and gap inputs. Unlike the gap factors, which are thought to act as short-range repressors, with a range of roughly 100 bp, most pair-rule genes act as long-range repressors, with an effective range of at least 2 kb. Pair-rule factor inputs may therefore influence expression outcomes at greater distances along the DNA, which would allow a single cluster of binding sites to affect multiple cis-elements. Remarkably, the 7-stripe elements of ftz, run, eve, prd and odd all combine inputs from promoter-proximal and more distal sequence over distances of at least 5 kb, and, is seen in the case of odd, the combination can involve non-additive effects. Thus, the refinement of expression patterns through pair-rule cross-regulation might rely on interactions over greater distances, consistent with the 'global' role of pair-rule genes as regulators of the entire 7-stripe pattern. How such interactions are realized at the molecular level and how the inputs from stripe-specific and 7-stripe elements are combined to produce a defined transcriptional outcome is currently unknown (Schroeder, 2011).

Taken together with previous studies, these data support a coherent and conceptually straightforward model of how stripes are made in theDrosophila blastoderm. In the trunk region of the embryo, the maternal activators BCD and CAD form two overlapping but anti-correlated gradients; they coarsely specify the expression domains of region-specific gap genes, which become refined by cross-repressive interactions among the gap factors. The result is a tiled array of overlapping gap factor gradients, centered around the bilaterally symmetric domains of KR and KNI, which are flanked on either side by the bimodal domains of GT and HB. The same basic principles are used again in the next step: following the initial positioning of stripes by maternal and gap factor input, cross-repressive interactions among the primary pair-rule genes serve to refine the pattern and ensure the uniform spacing and width of expression domains. The significant correlations that were observe between the strength of KR/KNI input and the position of the resulting stripes relative to the respective gradients supports the notion that these factors function as repressive morphogens in defining the proximal borders of the nascent pair-rule stripes. Importantly, owing to the symmetry of the KR and KNI gradients, combined with the largely symmetric positioning of the flanking GT and HB gradients, the same regulatory input can be used to specify two distinct positions, one on either slope of the gradient; this is exploited in a systematic fashion by dual-stripe cis-elements, which account for the majority of stripe-generating elements. Thus, the key to stripe formation is the translation of transcription factor gradients into an array of narrower, partially overlapping expression domains that are stabilized by cross-repressive interactions; the iteration of this process, combined with the duplication of position through bilateral gradient symmetry, creates a periodic array of stripes that is sufficient to impart a unique identity to each nucleus in the trunk region of the embryo (Schroeder, 2011).

With the exception of the anteriormost pair-rule gene stripes, which are subject to more complex regulation (as evidenced by the crucial role of slp1), this model accounts for most of the stripe formation process. Particularly striking is the similarity between the central gap factors and the primary pair-rule factors with respect to regulatory interactions and expression domain positions. The offset arrangement of two pairs of mutually exclusive gap domains, KNI/HB and KR/GT, is mirrored at the pair-rule gene level, with the anti-correlated and mutually repressive stripes of HAIRY and RUN phase-shifted against the similarly anti-correlated expression patterns of EVE and ODD. The parallel suggests that this regulatory geometry is particularly suited to robustly specify a multiplicity of positions. However, such a circuitry of cross-repressive relationships is compatible with a range of potentially stable expression states and thus is insufficient by itself to uniquely define position along the anterior-posterior axis. Therefore, the initial priming of position by the preceding tier of the regulatory hierarchy is crucial. At the level of the pair-rule genes, the extensive repertoire of maternal/gap-driven cis-elements that initiate stripe expression for all four components of the array is thus necessary to ensure that the cross-regulatory dynamics will drive the correct overall pattern. Finally, to achieve a smooth transition between the tasks of transmitting spatial information from the preceding tier of the hierarchy and refinement/stabilization of the pattern, the two layers of regulation need to be closely integrated. This is likely to be facilitated by the fact that in each of the mutually repressive pairs, one gene generates stripes purely through stripe-specific elements (h, eve), whereas the other has an early-acting 7-stripe element that mediates pair-rule cross-repression and acts concurrently with stripe-specific elements (run, odd) (Schroeder, 2011).

Stripe positioning is not always symmetric around the central gap factor gradients. In such cases, the conflicting needs for appropriate regulatory input into the relevant cis-elements appear to have driven the separation of ancestral dual-stripe elements into more specialized elements optimized for generating a single stripe. The co-localization of the cis-elements that generate stripes 1 and 5 within all pair-rule gene regulatory regions, be it in the form of dual-stripe elements or of adjacent but separable single-stripe elements, provides strong evidence that this is indeed a key mechanism underlying the emergence of single-stripe cis-elements. Given the evolutionary plasticity of regulatory sequence, it is not difficult to envision such a separation and subfunctionalization of cis-elements. How the crucial transition from a non-periodic to periodic pattern is achieved in other insects is a fascinating question. Intriguingly, most of the molecular players and general features of their expression patterns are well conserved beyond Diptera, and it is tempting to speculate that dual-stripe cis-regulation might have arisen through co-option as the blastoderm fate map shifted to include more posterior positions. It will be interesting to see to what extent the mechanisms and principles of stripe formation that we have outlined here apply in other species (Schroeder, 2011).

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated in this study, led to a hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. This study investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods deviations from the wild-type patterns of the anterior-most pair-rule stripes were detected in different genetic backgrounds, consistent with Hkb-mediated repression. Moreover, an image processing tool was developed that, for the most part, confirmed the assumptions. Using an hkb misexpression system, specific repression on anterior stripes was detected. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes (Andrioli, 2012).

The aim of this study was to understand the mechanisms underlying the regulation of the anterior pair-rule stripes. The model tested was first proposed for eve 2 regulation. Transcriptional activators do not give enough patterning information, and the presence of repressors is instructive for determining the precise positioning of a particular stripe. The hypothesis was that transcription repressors could be working in a combinatorial manner to determine the correct positioning of the anterior stripes and prevent, in a spatial and temporal manner, the expression of stripe CRMs in the more anterior regions of the embryo by counteracting the activity of activators. There is plenty of evidence supporting this hypothesis, which was further confirmed in this study (Andrioli, 2012).

Regarding activators, computational analysis predicted Bcd, Hb and Btd binding sites are part of significant clusters in the anterior-most stripe CRM. These predictions agree well with previous genetic data and in vivo DNA binding data from ChIP/chip experiments. Thus, Btd, and above all the widely spread maternal factors Bcd and Hb, might activate anterior stripe CRMs early in the anterior blastoderm. Alternatively, the early broad expression patterns of pair-rule genes could be under the control of dedicated CRMs, although no such elements have yet been reported. It is possible that other regulatory elements could contribute to the expression detected early in the anterior blastoderm, for instance, the CRM responsible for the expression of h head patch or the CRMs responsible for eve 3, eve 5 and h 5, which were proposed to be activated by the maternal factor DSTAT (Drosophila Signal Transducer and Activator of Transcription), which is ubiquitously expressed in the embryo (Andrioli, 2012).

The expression of several gap domains covering all of the anterior regions of the embryo ahead of the seven-striped patterns is consistent with the expected subsequent local repression of pair-rule CRMs activated in the head region. Of these gap domains, Slp1 is a common repressor for anterior pair-rule stripes, but other repressors besides Slp1 were predicted to be necessary for correctly determining the borders of the anterior-most stripes. This study investigated hkb, which, in addition to tll, is the other major gap gene target of the Torso signaling regulation in the terminal system. In the anterior region, hkb is required for the proper formation of the foregut and midgut. Its domain at the anterior tip coincides with the region where the diffused early expression patterns of pair-rule genes first fade. These observations are consistent with local repression roles of Hkb. However, it was not possible to detect derepression of pair-rule genes in the anterior pole of hkb- embryos. One possibility is that the progressive non-detection of the expression of pair-rule genes might correspond to a failure in activation. In fact, Bcd activation was shown to be down-regulated by the Torso-signaling cascade at the anterior tip. Nevertheless, other data suggest that the Torso pathway might induce a repression mechanism at the anterior tip that would be parallel and redundant with Torso-induced inhibition of Bcd. Thus, one might predict that another repressor might still able to act on Hkb targets in the absence of Hkb protein (Andrioli, 2012).

Although no pair-rule derepression was detected in the anterior pole, it was possible to detect subtle deviations in the positioning of eve 1 in hkb- embryos, which was confirmed by morphological measurements using the image processing tool. Enhanced derepression effects were also detected for all anterior-most stripes investigated in slp-;hkb- double-mutant embryos compared to the effects observed in slp- embryos; these results were statistically significant. With the hkb misexpression system, repression effects were detected for h 1, eve 1, run 1 and ftz 1. With the exception of gt repression, no other gap domain disruption was detected in these assays. These results strongly suggest direct repression by Hkb on the CRMs of these stripes. In vivo binding data confirms this possibility. Moreover, with the bioinformatics analysis it was verified that Hkb, along with putative activators, increased the already high significance values of predicted clusters for activators that match these stripe CRMs. Therefore, the combined data suggest that Hkb acts as a repressor for a specific group of anterior pair-rule stripes (Andrioli, 2012).

These data also suggest that there is another possible mechanism underlying the repression that involves the activity of repressors further away from their original sources. One example of this mechanism is expression detected for the ectopic hkb domain, demonstrating that target CRMs are sensitive to Hkb-mediated repression even in the presence of low expression levels of Hkb. The prediction is that low concentrations of Hkb that have diffused away from its endogenous domain could still repress these CRMs. For this mechanism, repressors could fulfill additive repression roles at different anterior subdomains or even contribute to the definition of the anterior borders of stripes that are distantly positioned from where gap domains are detected. Thus, the increased derepression observed in slp-;hkb- embryos would be expected if a combinatorial additive mechanism existed in which each repressor had a small contribution to the overall repression. Following the same rationale, one can predict that at least one other repressor is still responsible for setting anterior border stripes in slp-;hkb- embryos (Andrioli, 2012).

The complexity of the regulation of genes involved in early patterning was postulated to be a condition that is necessary for sensing relatively small differences in the concentrations and combinations of many regulatory factors, which is likely the environment found in the syncytial blastoderm. In agreement with that hypothesis, recent studies revealed that the protein gradients of factors such as Bcd and Dorsal alone are not sufficient to determine all of the spatial limits of target gene expression and that these gradients might combine with other factors to pattern the early embryo. In the head region, it has been suggested that Bcd and the terminal system-mediated activities interact at the level of the target CRMs to generate the proper patterning for the head region of the embryo. In contrast to these studies that focused on gap genes, the current data shed light on a mechanism that is involved in the regulation of the anterior stripe CRMs, with the putative participation of hkb (Andrioli, 2012).

The correct positioning of the anterior pair-rule stripes must be a critical issue in the early developmental patterning of the fly. Even a slightly incorrect positioning of the anterior stripes, for instance, results in the non-formation of the mandibular segment in the slp null mutant. Thus, a complex repression mechanism is necessary to shape the stripes and to avoid inappropriate expression of their CRMs. Therefore, Hkb, Slp1 and other repressors are likely involved in a combinatorial repressive activity in the CRMs of the anterior stripes. Other experiments are necessary to test this hypothesis further and to reveal the underlying molecular mechanisms involved in this regulation (Andrioli, 2012).

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).

Transcriptional Regulation

Continued: see Fushi tarazu Transcriptional Regulation part 2/2

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