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).

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).

Transcriptional Regulation

Continued: see Fushi tarazu Transcriptional Regulation part 2/2

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