cis-Regulatory Sequences and Functions (part 1/3)

Abd-B contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), which is located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including Abd-B. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).

A mutation which is caused by a deletion that leaves 15 kb of the 3' regulatory sequences immediately adjacent to the gene intact but removes 45 kb of the more distant 3' regulatory elements, produces an unexpected homeotic segmental transformation of the fourth through seventh abdominal segments. There is a uniform and moderate level of the Abd-B class A transcript in the posterior abdomen, rather than the normal graded pattern of expression. The gradient of Abd-B expression normally observed in the posterior abdomen appears to be achieved by varying the number of reiterated elements that are active in each segment (Crosby, 1993).

Segment polarity genes are not the only genes to be expressed in a parasegmental register. The abdominal-A and Abdominal-B genes of the bithorax complex specify the identity of most of the Drosophila abdomen. Six different classes of infraabdominal mutations within the BX-C transform a subset of the parasegments affected by the lack of these two genes. It is thought that these mutations define parasegmental cis-regulatory regions that control the expression of abd-A and Abd-B. The expression of Abd-B (and probably also abd-A) exhibit a parasegmental regulation (Sanchez-Herrero, 1991).

Expression of the abdominal-A and Abdominal-B genes of the BX-C of Drosophila is controlled by a cis-regulatory promoter and by distal enhancers called infraabdominal regions. The activation of these regions along the anteroposterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. There is spatially restricted transcription of the infraabdominal regions (infraabdominal transcripts) that may reflect this specific activation. These iab regions are named after the abdominal segments they control, iab-2 through iab-7 regulating abdominal segments 2 through 7 corresponding to parasegments 7 through 12 respectively. The Abd-B gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). The gap genes hunchback, Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in development. The gradients of the Hunchback and Krüppel products seem to be key elements in this restricted activation (Casares, 1995).

Krüppel and Knirps act through the infraabdominal 5 fragment of ABD-B to limit anterior Abd-B expression and regulate the graded Abd-B domain respectively. Both hunchback and Polycomb are required for Abd-B silencing (Busturia, 1993).

Two novel cis-regulatory elements, Mcp and Fab-7, are found in the bithorax complex of Drosophila. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B expression in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. Two new Mcp mutant deletions (McpH27 and McpB116) allow the localization of sequences essential for boundary function to an approximately 0.4 kb DNA segment. These essential sequences closely coincide with an approximately 0.3 kb nuclease hypersensitive region in chromatin. Sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure (Karch, 1994).

A second study reports very similar results. Mutations in Fab-7 result in the misregulation of Abd-B in parasegment 11, and a corresponding homeotic transformation of PS 11 tissues (sixth abdominal segment: A6) into PS 12 (A7). Removal of the Fab-7 element results in a fusion of iab-6 and iab-7 cis-regulatory domains into a single regulatory domain that inappropriately regulates Abd-B in PS11. It is thought that Fab-7 separates iab-6 and iab-7 into distinct chromatin domains or that Fab-7 functions as a silencer element, which attenuates Abd-B expression in anterior regions, including PS 11. A 1.2-kb Fab-7 DNA fragment was placed between divergently transcribed test promoters and challenged with several defined enhancers expressed in early embryos. These include the 300-bp rhomboid neuroectodermal enhancer NEE and the 500-bp even-skipped stripe 3 enhancer. In both cases the Fab-7 interferes with the interaction of these enhancers with the Abd-B proximal promoter (Zhou, 1996).

A minimal fragment of Fab-7 was defined, sufficient for enhancer blocking. It is completely distinct from an adjacent Polycomb-dependent silencer. Identification of the extent of the Fab-7 region is the first step in discovering the basis of its function (Hagstrom, 1996).

Fab-7 functions as an attenuator, which weakens gene expression by reducing enhancer-promoter interactions. Fab-7 selectively blocks distal enhancers in an orientation-independent fashion, and can function when located far from either the distal enhancer or target promoter. Fab-7 is located greater than 35 kb downstream of the Abd-B transcriptional start site, where it functions adequately. Likewise, Fab-7 functions when positioned distant from artificial promoters, when placed between such promoters and distal enhancers. Fab-7 may be related to insulator DNAs that flank genetic loci and functionally isolate neighboring genes (discussed in the Suppressor of Hairy wing). Fab-7 require neither the Su(HW) nor the Mod-Mdg4 proteins involved in the SU(HW) insulator. It is thought that specialized DNA elements, such as the Fab-7 attenuator, might play a general role in controlling the levels of gene expression by modulating enhancer-promoter interactions (Zhou, 1996 and Hagstrom, 1996).

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7') dominantly transforms parasegment 11 into parasegment 12. This chromosomal region contains both a boundary element and a silencer. Previous studies have suggested that removal of a domain boundary element on the proximal side of Fab-7' is responsible for dominantly transforming gain-of-function phenotype. The Fab-7 boundary element maps to two nuclease hypersensitive sites, HS1 and HS2. This article demonstrates that the Fab-7' deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment (the HS3 site) and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a bxd, Ubx/lacZ reporter transgene. Both mini-white and Ubx/lacZ silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste' allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans the HS3 nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. It is suggested that PRE-PRE cooperation, either in trans or in cis, may be an important feature of the silencing process within the BX-C and that boundaries may limit PRE-PRE cooperation (Hagstrom, 1997).

iab-5, iab-6 and iab-7 are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. A 'cis' configuration of gene and regulatory region occurs when the two are on the same chromosome. When gene and regulatory regions are on different chromosomes, the configuration is called 'trans.' The iab-5,6,7 region is able to interact with ABD-B in trans. This interaction is proximity dependent, that is, the gene and the regulatory region, although they are on different chromosomes, must be in close proximity to interact. Transvection is a coined word for activation in trans. Interactions of this type are presumably facilitated by the synapsis of homologs that occurs in somatic cells of Dipterans.

Although transvection has been detected in a number of Drosophila genes, transvection of the iab-5,6,7 region is exceptional in two ways. First, interaction in trans with ABD-B does not require that homologs share homologous sequences either within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis (the pairing of homologous chromosomes) (Hopmann, 1995). A second unusual feature of iab-5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements.

The lack of requirement for local synapsis and the tenacity of trans-interaction argue that the iab-5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of iab-5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected (Hopmann, 1995).

The infra-abdominal (iab) elements in the bithorax complex of Drosophila melanogaster regulate the transcription of the homeotic genes abdominal-A and Abdominal-B in cis. iab-6 and iab-7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. The iab regions can regulate their target promoter located at a distant site in the genome in a manner that is much less dependent on homolog pairing than other transvection effects. Breaks in the iab-7 region induce the iab elements to switch their target promoter from Abd-B to abd-A. It is proposed that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter (Hendrickson, 1995).

The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. The observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. The amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B (Sipos, 1998).

It is suggested that the 5' flanking region of the Abd-B promoter contains "tethering elements" that normally mediate cis-interactions between the Abd-B gene and the iab-7 regulatory domain or the other iab regulatory domains. When these upstream tethering elements are deleted, cis-interactions are weakened or eliminated and trans-interactions with tethering elements upstream of the Abd-B gene on the other homologue become stronger. A similar tethering mechanism has been proposed to anchor the upstream enhancers of the white gene to the white basal promoter. In white, the tethering region is thought to be no more than ~95 bp in length. By contrast, the tethering region for the Abd-B gene appears to be larger than 7.6 kb. Moreover, because the strength of trans-regulation increases roughly in proportion to the size of the upstream deletion, it would appear that there may be multiple "tethering elements" distributed throughout this 5' flanking region (Sipos, 1998).

Why does the Abd-B gene have such a large tethering region? Several factors may be important. (1) The regulatory domains that generate the parasegment specific patterns of Abd-B expression are located at a considerable distance from the promoter. (2) The regulation of Abd-B is exceedingly complex, and in each parasegment elements from a different regulatory domain must interact with and control the activity of the Abd-B promoter. (3) Because of the arrangement and parasegment specificity of the iab regulatory domains, at least three of the iab regulatory domains (iab-5, iab-6 and iab-7) must contact the promoter across an intervening DNA segment that contains boundary elements (like Fab-7) and one or more Pc-G-silenced regulatory domains. Moreover, these boundary elements are of sufficient strength to prevent the regulatory domains from contacting heterologous promoters. (4) An additional function of this extensive tethering region may be to ensure cis-preference/cis-autonomy. These data show that interactions between an iab-7 domain and an Abd-B gene on the same homolog are, under normal circumstances, strongly preferred to trans-interaction. However it is difficult to see a priori how the distant iab regulatory domains are able to distinguish a promoter in cis from the same promoter in trans, particularly when the homologs are that tightly paired. One possibility is that the tethering element array in the 5' flanking region of the Abd-B gene helps ensure cis-preference by making multiple contacts with corresponding elements in the regulatory domains. Once these interactions are established in cis, it would be difficult to rebuild the same set of contacts with the promoter on the other homolog. Even under conditions favorable for trans-regulation, such as when there is only a single active iab-7 regulatory domain, the results would suggest that there may only be a partial conversion of the cis-complex into a trans-complex. A similar mechanism could account for the apparent cis-autonomy of Ubx. However, although the presence of an extensive tethering region in the Abd-B 5' flanking region may help explain how cis-preference is maintained, it does not explain why the cis-complex is established in the first place. It is conceivable that the cis-complex is "inherited" from the early stages of embryogenesis, when the BX-C does not appear to be paired. Alternatively, cis-interactions may be established (or reestablished) at a point in the cell cycle, e.g., early G1, prior to the time when the homologs become tightly paired. Evidence is presented that the tenacity of trans-interactions in the Abd-B gene may vary, depending on the tissue and stage of development (Sipos, 1998).

The distribution of Polycomb protein has been mapped at high resolution on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Sheared chromatin was immunoprecipitated and amplified by linker-modified PCR, before using as a probe on a Southern of the entire PX-C walk. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Among the identified sites are peak F (the bxd PRE) and peak G (the bx PRE), both of which contain GAGA consensus sequences. Three other sites, E, D and C correspond to iab2, iab3 and iab4. No PC binding is seen in the regulatory domains iab6, iab7 or iab8, indicating that these domains positively regulate Abd-B expression. These results suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. The Abd-B sites include the iab7/iab8 regulatory region, and the Fab-7 PRE. The Fab-7 PRE does not bind Polycomb. It is proposed that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs (silencing elements) and as trithorax group response elements. It is suggested that a GAGA site in the Antennapedia promoter is both a PRE (binding PC protein) and a TRE (binding GAGA) factor (Strutt, 1997a).

Polycomb response elements (PREs) in several genes contain conserved sequence motifs. One of these motifs is the binding site for the protein coded for by the recently cloned gene polyhomeotic (pho), the Drosophila homolog of mammalian YY1. The conserved sequence extends beyond the YY1 core consensus sequence suggesting that parts of Pho may impose additional DNA sequence requirements. In this respect and unlike YY1, PHO has an additional 45 amino acids following the fourth zinc finger. It is also possible that Pho may bind to PREs together with another protein in order to fully exploit the conserved sequence. The conserved sequence motif CNGCCATNDNND, includes the YY1 core consensus CCATNWY. Eight consensus sites have been identified in 6 PREs of the bithorax complex (BX-C): bxd, iab-2, Mcp, iab-6, iab-7 and iab-8. The bxd PRE harbors all three characteristics used to define PREs (maintenance of expression of a lacZ reporter assay throughout development; pairing-sensitive repression of a mini-white reporter, and creation of an additional chromosomal binding site of the PcG-repressing complex in a salivary gland assay). The iab-2 PRE contains two homology boxes (a and b) and has been identifed in the maintenance and pairing-sensitive assays. The Mcp and iab-6 PREs have been characterized in the pairing-sensitive assay. The iab-7 PRE contains two homology motifs, a and b. This PRE has been characterized in all three assays. The iab-8 PRE has been identified in the maintenance assay. The conserved sequence motif is found in three PREs from Sexcombs reduced regulatory regions, and has been identified in the pairing-sensitive assay. The sequence motif found in two PREs from the engrailed regulatory region has been characterized in the pairing-sensitive assay. The sequence motif is also found in polyhomeotic, and has been identified in the pairing-sensitive and salivary gland assays (Mihaly, 1998)

The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4 hyperacetylation has been found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators (Cavalli, 1999).

Fab-7-dependent chromosomal memory of silent or open chromatin states occurs in transgenic Drosophila lines such as FLW-1 and FLFW-1. These lines carry a heat shock-inducible GAL4 driver (hsp70-GAL4) regulating a GAL4-dependent lacZ reporter (UAS-lacZ) flanked by Fab-7 and the mini-white gene. Silencing imposed by Fab-7 on the flanking reporter genes is dependent on the components of the PcG, since heterozygous mutant PcG genes show a relief of white gene repression. Conversely, white gene activity requires the trxG because heterozygous mutations in the different members tested result in a down-regulation of expression. A GAL4 pulse during embryogenesis can impose a mitotically stable reprogramming of the Fab-7 cellular memory module (CMM) from a silenced to an open chromatin state. The maintenance of the activated Fab-7 state is dependent on trithorax (trx) but not on Polycomb (Pc). In a heterozygous Pc- background, Fab-7 can be switched by a GAL4 pulse and be stably maintained, resulting in strong white expression. In contrast, a trx- mutation completely abolishes the mitotic transmission (Cavalli, 1999).

To assess whether the epigenetically activated Fab-7 state correlates with a permanent loss of PcG proteins from the chromatin template, a strong GAL4 induction pulse was administered during embryogenesis in the FLFW-1 line. Polytene chromosomes of third instar larvae were immunostained with antibodies directed against PcG proteins. Surprisingly, all of the PcG proteins tested, Polycomb (Pc) and Posterior sex combs (Psc), Polyhomeotic (Ph), and Polycomb-like (Pcl), are still strongly bound to the Fab-7 transgene irrespective of the epigenetic state. Thus, an epigenetically activated state can be stably propagated in the presence of the protein components of the PcG. These data support previous observations that have demonstrated binding of Pc at cytological sites containing potentially active genes in polytene chromosomes and binding of Ph and Psc proteins at an actively transcribed gene in Drosophila Schneider cells. It has been reported that certain PcG genes may function as activators in specific tissues and at specific developmental times by genetic analyses. Although a role for Pc protein in the maintenance of the activated state of Fab-7 is not observed, it may be possible that other PcG proteins are involved in this process (Cavalli, 1999).

If it is not the removal of PcG repressors on the template, what is the epigenetic tag that marks the activated Fab-7 state? A single embryonic GAL4 pulse was administered to FLFW-1 embryos, and histone acetylation of the Fab-7 transgene as a possible mark was analyzed by immunostaining polytene chromosomes of third instar larvae with specific antibodies against the tetra-acetylated form of H4 and H3 histones. Hyperacetylation of histone H4 is detected at the Fab-7 transgene location in larvae derived from activated embryos, but not from control embryos raised at 18°C. In contrast to H4, no hyperacetylation of histone H3 could be detected in activated FLFW-1 individuals (Cavalli, 1999).

Patterning transcription factors, like the products of many segmentation genes, act only shortly on their downstream genes during early Drosophila development, whereas the PcG/trxG memory system subsequently maintains the embryonically programmed patterns. For this reason, a test was performed to see whether embryonically activated Fab-7 can maintain expression of the reporter gene lacZ in the absence of the primary transcription factor GAL4. The leakiness of the hsp70 promoter prevents the complete disappearance of GAL4 protein during single fly development. To overcome this problem, use was made of the fact that activated Fab-7 can be efficiently propagated through meiosis in the line FLW-1. This allows the crossing out of the GAL4 driver to test lacZ expression in the complete absence of GAL4 in subsequent generations. Upon crossing out GAL4 in activated flies, 20% to 25% of the GAL4-less embryos show substantial levels of homogeneous beta-galactosidase expression in all embryonic cells in two consecutive generations. This percentage correlates well with the fraction of adults showing meiotically stable white derepression. Unfortunately, it is not possible to also test for the meiotic inheritance of H4 hyperacetylated states because of a staining pattern with endogenous bands at the insertion site of the transgene in the FLW-1 line. However, the functional analysis demonstrates that epigenetic inheritance of an active Fab-7 chromatin state results in transcriptional activity of the UAS-lacZ reporter even in the absence of the GAL4 transactivator (Cavalli, 1999).

A weak expression of lacZ in the absence of GAL4 may arise from a heritable loss of PcG-mediated repression, thereby neutralizing the silencing ability of Fab-7 and consequently reflecting the ground state of a nonrepressed chromatin template. If this were the case, it might be expected that in flies carrying an UAS-lacZ construct without Fab-7 (pU/l5), a similar weak homogeneous beta-Gal staining pattern would be observed in all embryos in the absence of GAL4. To test this point, beta-Gal staining was examined in two independent lines carrying the pU/l5 construct but no GAL4 driver. In both cases, most of the embryos were not stained or were stained in a weakly variegated fashion in random cells. This strongly suggests that meiotic inheritance of Fab-7 CMM-activated states does not simply reflect lifting of PcG-mediated silencing but rather the inheritance of an active chromatin state, which is competent for transcriptional activation (Cavalli, 1999).

These findings show in a functional manner that trxG protein complexes recruited at a CMM relieve the requirement for the activating factor for transcriptional maintenance. Hyperacetylation of histone H4 has been identified as an epigenetic mark for the activated Fab-7 state. Unlike the short-lived H4 hyperacetylation induced by transient gene activation at late developmental stages, the mark set at embryonic stages is mitotically stable and inheritable. An important maintenance function of the PcG and trxG protein complexes at CMMs might be to protect epigenetic marks from erasure (Cavalli, 1999).

The Drosophila bithorax complex Abdominal-B gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. Transgene assays have been used to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. The Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. The insulating activity of the Fab-8 boundary in the BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8, together with the previously identified Fab-7 boundary, delimit the first genetically defined higher order domain in a multicellular eukaryote (Barges, 2000).

Chromatin domain boundaries, like scs or gypsy insulators in Drosophila, have been identified in transgene assays through their enhancer-blocking activity. Boundary elements in the bithorax complex (BX-C), such as Fab-7 and Fab-8, have been identified genetically and have been shown to have insulator activity in transgene assays. However, it is not clear whether boundary elements identified in transgene assays will function appropriately in chromosomal contexts such as BX-C. Using gene conversion, the scs or gypsy insulators have been substituted for Fab-7. Both scs and gypsy are very potent insulators in the ectoderm, but surprisingly, the insulating activity of gypsy (but not scs) is lost in the CNS. These results reveal that the Fab-7 boundary must have special properties that scs and gypsy lack, which allow it to function appropriately in BX-C regulation (Hogga, 2001).

These results demonstrate that like Fab-7, both gypsy and scsmin can ensure the functional autonomy of the BX-C iab-6 and iab-7 cis-regulatory domains. However, these two elements differ from Fab-7 and other BX-C boundaries, such as Fab-8, in one critical respect -- they also insulate the Abd-B promoter from the more distal iab-5 and iab-6 cis-regulatory domains. Two models could potentially account for this critical difference. The first postulates that there is some special mechanism in BX-C to facilitate long-distance interactions between the cis-regulatory domains and the three homeotic genes. Evidence for such mechanisms already exists. Studies on transvection in the Abd-B domain indicate that there is a large 'tethering' region upstream of the Abd-B promoter that mediates communication with the iab cis-regulatory domains. One function of this tethering region might be to overcome the action of the Fab boundaries, perhaps through contacts with 'promoter targeting sequences' (PTS) in each cis-regulatory domain. In this model, gypsy and scsmin insulators would differ from the Fab boundaries because they disrupt this interaction mechanism, potentially by interfering with the PTS. An alternative hypothesis comes from the recent finding that when two copies of the gypsy insulator, instead of a single one, are inserted between an enhancer and a promoter, the insulator activity is neutralized. The iab-7 cis-regulatory domain is flanked by Fab-7 and Fab-8 boundaries. It is possible that like the gypsy insulator, the Fab-7 and Fab-8 boundaries can pair and neutralize each other, enabling the more distant iab-5 or iab-6 cis-regulatory domains to contact the Abd-B promoter. In this model, Fab-8 would be unable to neutralize the insulator activity of either gypsy or scs (Hogga, 2001).

Homeotic (or Hox) genes are key determinants in specifying the anteroposterior axis of most animals. The temporal and spatial expression of these genes requires the presence of large and complex cis-regulatory regions. The Abdominal-B Hox gene of the bithorax complex of Drosophila is regulated by several infraabdominal domains, which determine Abdominal-B expression in abdominal segments A5 to A9 (parasegments 10 to 14). Some of the infraabdominal domains have been characterized, including an infraabdominal-8 domain, which has been located 3' to the Abdominal-B transcription unit. The expression and mutant phenotype have been analyzed of a P-lacZ element inserted close to the Abdominal-B m origin of transcription and of derivatives of this transposon. Some of these derivatives represent a particular class of mutations in the bithorax complex, because they transform the eighth and ninth abdominal segments without affecting more anterior metameres. The analysis of these mutations and of transformants carrying sequences upstream of the Abdominal-B m transcription unit has allowed a new infraabdominal-8 regulatory region to be defined, located 5' to the Abdominal-B transcription unit, and has helped to characterize better the complex regulation of the Abdominal-B gene (Estrada, 2002).

Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila bithorax complex

The correct spatial expression of two Drosophila bithorax complex (BX-C) genes, abdominal-A and Abdominal-B, is dependent on the 100-kb intergenic infraabdominal (iab) region. The iab region is known to contain a number of different domains (iab2 through iab8) that harbor cis-regulatory elements responsible for directing expression of abdA and AbdB in the second through eighth abdominal segments. In situ hybridization has been used to perform high-resolution mapping of the transcriptional activity in the iab control regions. Transcription of the control regions themselves is abundant and precedes activation of the abdA and AbdB genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the iab control regions demonstrate colinearity with the sequence of the iab regions along the chromosome and the domains in the embryo under the control of the specific iab regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development (Bae, 2002).

Thus, transcription through these cis-regulatory regions is abundant and subject to a highly ordered developmental program. The early expression of sense transcripts (relative to the direction of abdA and AbdB expression) from the different iab regions is organized into sequential domains along the A-P axis of the developing embryo. This organization is reminiscent of the colinearity exhibited by the BX-C homeotic genes themselves, which are expressed in the same order along the A-P axis of the embryo as they are organized along the chromosome. The intergenic transcripts follow the same rule, because there is colinearity between the location of the iab regions on the chromosome and the anterior limit of transcription in the blastoderm-stage embryo. In this way, regions increasingly closer to AbdB are expressed in increasingly more posterior domains in the embryo, with transcripts from each individual iab region showing unique, spatially restricted patterns of expression. The pattern of transcription from each iab region corresponds to the segmental domain of the embryo that is affected by mutations in each particular iab region. Therefore, it is conceivable that the early sense transcripts could define the domains of activity for cis-regulatory elements within each iab region (Bae, 2002).

The timing of expression in the intergenic region is also significant. If the sense transcripts indeed are capable of defining the domains of activity for cis-regulatory elements in the iab regions, then it would be expected that they are transcribed before the time at which the cis-regulation is required. The iab transcripts in fact are detectable by late stage 4/early stage 5 of embryonic development, before the time at which expression is seen from the abdA or AbdB genes. Earlier studies also noted this temporally restricted order of transcription. The spatial and temporal distribution of the sense transcripts represents the earliest known response of the BX-C to the hierarchical positioning information inherited in the embryo from the gap and pair-rule genes. It appears that the early transcripts represent an initial primed state of the BX-C and, therefore, could act to define the domains of activity for the iab regions in the embryo. This activity has some parallels with the intergenic transcription that has been characterized at mammalian genes. For example, in the Ig genes, germ-line transcription through cis-regulatory elements is thought to activate interactions with regulatory proteins that are necessary to direct the switching of the class of Ig gene expressed in individual cells. The iab transcripts may play a similar role in regulating abdA and AbdB gene expression.

Later in development, all of the intergenic sense transcripts are restricted to expression in the two most posterior abdominal segments. Why the transcription persists late in development is unclear. It is possible that once the domains of activity for the iab regions are established by the early transcripts, other factors may maintain the iab-regulated expression of the target homeotic genes. The transcriptional regulatory proteins of the Polycomb group and Trithorax group are good candidates for this role because they are known to be necessary to maintain expression states for the homeotic genes in the BX-C. They act through cis-regulatory elements in the iab regions, which have been identified as Polycomb response elements or cellular memory modules, to promote either a silenced or activated state throughout development by generating stable, higher-order chromatin structures. It is possible that the transcription detected through the Polycomb response elements early in development primes their segment-specific activity, which is heritable through future cell divisions. Once the Polycomb response elements are activated, they are able to maintain the regulation of the homeotic gene-expression patterns, and, consequently, the iab transcripts are no longer required. One prediction of this model is that ectopic transcription through the iab Polycomb response elements later in development may interfere with Polycomb-mediated silencing. The continued expression of the iab transcripts in abdominal segments 8 and 9 late in development may indicate that homeotic gene expression in these segments is not regulated by the Polycomb or Trithorax group proteins and that the functional role for the transcripts consequently persists (Bae, 2002).

The potential role of the antisense transcripts remains enigmatic. The previously characterized iab4as transcript is known to be processed, although it appears to have no protein-coding potential. Identification of an additional antisense transcript in the iab6 domain may suggest a shared function. One possibility is that the antisense transcripts contribute to the inhibition of the spreading of the sense transcripts from one iab region to another, because they prevent transcription from the opposite DNA strand. Their chromosomal locations, relatively close to the insulator elements Mcp and Fab-7, are consistent with this notion. It is possible that the antisense transcripts may be processed and function to inhibit the sense transcripts by an RNA interference mechanism, similar to the silencing characterized in Schizosaccharomyces pombe. However, despite extensive attempts, no antisense transcripts have been identified in the iab5 or iab7 regions. Further molecular characterization of the sense and antisense transcripts and analysis of genetic mutations at the BX-C will be necessary to further facilitate elucidation of their in vivo functional activities (Bae, 2002).

Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex

In Drosophila, the function of the Polycomb group genes (PcGs) and their target sequences (Polycomb response elements [PREs]) is to convey mitotic heritability of transcription programmes -- in particular, gene silencing. As part of the mechanisms involved, PREs are thought to mediate this transcriptional memory function by building up higher-order structures in the nucleus. To address this question, in vivo the three-dimensional structure was studied of the homeotic locus bithorax complex (BX-C) by combining chromosome conformation capture (3C) with fluorescent in situ hybridization (FISH) and FISH immunostaining (FISH-I) analysis. It was found that, in the repressed state, all major elements that have been shown to bind PcG proteins, including PREs and core promoters, interact at a distance, giving rise to a topologically complex structure. This structure is important for epigenetic silencing of the BX-C, since it was found that major changes in higher-order structures must occur to stably maintain alternative transcription states, whereas histone modification and reduced levels of PcG proteins determine an epigenetic switch that is only partially heritable (Lanzuolo, 2007).

Enhancer loops appear stable during development and are associated with paused polymerase

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. This study generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. These results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase (Ghavi-Helm, 2014).

Drosophila embryogenesis proceeds very rapidly, taking 18 h from egg lay to completion. Underlying this dynamic developmental program are marked changes in transcription, which are in turn regulated by characterized changes in enhancer activity. However, the role and extent of dynamic enhancer looping during this process remains unknown. To address this, 4C-seq (chromosome conformation capture sequencing) experiments were performed, anchored on 103 distal or promoter-proximal developmental enhancers (referred to as 'viewpoints'), and absolute and differential interaction maps were constructed for each, varying two important parameters: (1) developmental time, using embryos at two different stages, early in development when cells are multipotent (3-4 h after egg lay; stages 6-7), and mid-embryogenesis during cell-fate specification (6-8 h; stages 10-11); and (2) tissue context, comparing enhancer interactions in mesodermal cells versus whole embryo. To perform cell-type-specific 4C-seq in embryos, a modified version of BiTS-ChIP (batch isolation of tissue-specific chromatin for immunoprecipitation) was established. Nuclei from covalently crosslinked transgenic embryos, expressing a nuclear-tagged protein only in mesodermal cells, were isolated by fluorescence-activated cell sorting (FACS; (>98% purity) and used for 4C-seq on 92 enhancers at 6-8 h and a subset of 14 enhancers at 3-4 h. The same 92 enhancers, and 11 additional regions, were also used as viewpoints in whole embryos at both time points. The enhancers were selected based on dynamic changes in mesodermal transcription factor occupancy between these developmental stages and the expression of the closest gene. This study was thereby primed to detect dynamic three-dimensional (3D) interactions, focusing on developmental stages during which the embryo undergoes marked morphological and transcriptional changes (Ghavi-Helm, 2014).

All 4C-seq experiments had the expected signal distribution, with high concordance between replicates. To assess data quality further, ten known enhancer-promoter pairs (of the ap, Abd-b, E2f, pdm2, Con, eya, stumps, Mef2, sli and slp1 genes) were compared, and in all cases the expected interactions were recovered. For example, using an enhancer of the apterous (ap) gene, the expected interaction was detected with the ap promoter, 17 kilobases (kb) away, illustrating the high quality and resolution of the data (Ghavi-Helm, 2014).

In chromosome conformation capture assays, interaction frequencies decrease with genomic distance between regions. To adjust for this, the 4C signal decay was modelled as a function of distance using a monotonously decreasing smooth function. Subtracting this trend, the residual interaction signal was converted to z-scores and interacting regions defined by merging neighbouring high-scoring fragments within 1 kb. Using this stringent approach, 4,247 high-confidence interactions were identified across all viewpoints and conditions, representing 1,036 unique interacting regions (Ghavi-Helm, 2014).

Each enhancer (viewpoint) interacted with, on average, ten distinct genomic regions, less than half (41%) of which were annotated enhancers or promoters. Distal enhancers had a higher than expected interaction frequency with other enhancers. Similarly, promoter-proximal elements had extensive interactions with distal active promoters, 98% of which are >10 kb away. Enhancer-promoter interactions, although not significantly enriched, involve active promoters, with high enrichment for H3K27ac and H3K4me3, and active enhancers, defined by H3K27ac, RNA Pol II and H3K79me3. These results are similar to recent findings in human cells and the mouse β-globin locus, indicating similarities in 3D regulatory principles from flies to human (Ghavi-Helm, 2014).

The extent of 3D connectivity is surprising given the relative simplicity of the Drosophila genome. On average, each promoter-proximal element interacted with four distal promoters and two annotated enhancers, whereas each distal enhancer interacted with two promoters and three other enhancers. These numbers are probably underestimates, as 60% of interactions involved intragenic or intergenic fragments containing no annotated cis-regulatory elements. Despite this, the level of connectivity is similar to that recently observed in humans, where active promoters contacted on average 4.75 enhancers and 25% of enhancers interacted with two or more promoters. The multi-component contacts that were observed for Drosophila enhancers indicate topologically complex structures and suggest that, despite its non-coding genome being an order of magnitude smaller than humans, Drosophila may require a similar 3D spatial organization to ensure functionality (Ghavi-Helm, 2014).

Insulators, and associated proteins, are thought to have a major role in shaping nuclear architecture by anchoring enhancer-promoter interactions or by acting as boundary elements between topologically associated domains (TADs). Occupancy data from 0 to 12 h Drosophila embryos revealed a 50% overlap of interacting regions with occupancy of one or more insulator protein. Insulator-bound interactions are enriched in enhancer elements, suggesting that insulators may have a role in promoting enhancer-enhancer interactions. In contrast to mammalian cells, this study observed no association between insulator occupancy and the genomic distance spanned by chromatin loops, although there was a modest increase in average interaction strength. Conversely, 50% of interacting regions are not bound by any of the six Drosophila insulator proteins, suggesting that these 3D contacts are formed in an insulator-independent manner, or are being facilitated by neighbouring interacting regions (Ghavi-Helm, 2014).

If enhancer 3D contacts are involved in transcriptional regulation, then genes linked by interactions with a common enhancer should share spatio-temporal expression. For the four loci examined-pdm2, engrailed, unc-5 and charybde-this is indeed the case. For example, the pdm2 CE8012 enhancer interacts with both the pdm2 and nubbin (nub, also known as pdm1) promoters, located 2.5 and 47 kb away, respectively. Both genes, producing structurally related proteins, are co-expressed in the ectoderm, overlapping the activity of the pdm2 enhancer. Although there are examples of long-range interactions in Drosophila, often involving Polycomb response elements (PREs) and insulator elements, the vast majority of characterized enhancers are within 10 kb of their target gene, with few known to act over 50 kb. However, as investigators historically tested regions close to the gene of interest, characterized Drosophila enhancers are generally close to the gene they regulate. In contrast, although 4C cannot assess the full extent of short-range interactions, it provides an unbiased systematic measurement of the distance of enhancer interactions, far beyond 10 kb (Ghavi-Helm, 2014).

The distance distribution of all significant interactions reveals extensive long-range interactions within the ~180 megabase (Mb) Drosophila genome; 73% span >50 kb, with the median interaction-viewpoint distance being 110 kb. Two striking examples of long-range interactions are the unc-5 and charybde loci. The unc-5 promoter interacts with multiple regions, including a weak but significant interaction with the promoter of slit (sli), at a distance of >500 kb. These genes produce structurally unrelated proteins that are co-expressed in the heart, and are essential for heart formation (Ghavi-Helm, 2014).

A promoter-proximal element near the charybde (chrb) promoter has a strong interaction with the promoter of the scylla (scyl) gene, almost 250 kb away. Both genes are closely related in sequence and co-expressed throughout embryogenesis. These long-range interactions were confirmed by reciprocal 4C, using either the promoter of chrb or scyl, or an interacting putative enhancer as viewpoint. This interaction was further verified using DNA fluorescence in situ hybridization (FISH) in embryos. As a control, the distance was assessed between the chrb promoter (probe A) and an overlapping probe A' or a region on another chromosome (probe D), to determine the distances between regions very close or far away, respectively. Comparing the distance between the chrb and scyl promoters (probes A and B) showed a high, statistically significant co-localization, in contrast to the distance between the chrb promoter and a non-interacting region with equal genomic distance (probes A and C) (Ghavi-Helm, 2014).

The reciprocal 4C revealed several intervening interactions that are consistently associated with loops to both the scyl and chrb promoter. The activity was examined of two of these in transgenic embryos. Both interacting regions can function as enhancers in vivo, recapitulating chrb expression in the visceral mesoderm and nervous system (Ghavi-Helm, 2014).

When considering a 1-Mb scale around this region, the 4C interaction signal drops to almost zero just after the promoters of both genes. This 'contained block' of interactions is reminiscent of TADs, although the boundaries don't exactly match TADs defined at late stages of embryogenesis, which may reflect differences in the developmental stages used. However, the boundaries do overlap a block of conserved microsynteny between drosophilids spanning ~50 million years of evolution, suggesting a functional explanation underlying the maintained synteny. Expanding this analysis across all viewpoints, ~60% of interactions are located within the same TAD and the same microsyntenic domain as the viewpoint. In the case of the chrb and scyl genes, this constraint may act to maintain a regulatory association between a large array of enhancers, facilitating their interaction with both genes' promoters (Ghavi-Helm, 2014).

These examples, and the other 555 unique interactions >100 kb, provide strong evidence that long-range interactions are widely used within the Drosophila genome, potentially markedly increasing the regulatory repertoire of each gene. As enhancer-promoter looping can trigger gene expression, it follows that enhancer contacts should reflect the dynamics of transcriptional changes during development and therefore be temporally associated with gene expression. To assess this, looping interactions were directly compared between the two different time points and tissue contexts. Given the non-discrete nature of chromatin contacts, the quantitative 4C-seq signal was used to identify differential interactions based on a Gamma-Poisson model, and they were defined as having >2-fold change and false discovery rate <10% (Ghavi-Helm, 2014).

Despite the marked differences in development and enhancer activity between these conditions, surprisingly few changes were found in chromatin interaction frequencies, with ~6% of interacting fragments showing significant changes between conditions. Of these, 87 interactions were significantly reduced during mid-embryogenesis (6-8 h) compared to the early time point (3-4 h), and 90 interactions significantly increased. Similarly, 105 interactions had a higher frequency in mesodermal cells, compared to the whole embryo, and For example, a promoter-proximal viewpoint in the vicinity of the Antp promoter identified many interactions, two of which are significantly decreased at 6-8 h, although the expression of the Antp gene itself increases. For one region, the reduction in 4C interaction at 6-8 h corresponds to a loss in a H3K4me3 peak from 3-4 h to 6-8 h, suggesting that this 3D contact is associated with the transient expression of an unannotated transcript. The activity of the other interacting peak was examined in transgenic embryos, and it was shown to act as an enhancer, driving specific expression in the nervous system overlapping the Antp gene at 6-8 h. Along with the two enhancers discovered at the chrb locus, this demonstrates the value of 3D interactions to identify new enhancer elements, even for well-characterized loci like Antp (Ghavi-Helm, 2014).

A viewpoint in the vicinity of the Abd-B promoter interacted with a number of regions spanning the bithorax locus, three of which correspond to previously characterized Abd-B enhancers; iab-5, iab-7 and iab-8. The iab-7 and iab-8 enhancers are active in early embryogenesis, and have much reduced or no activity at the later time point. Notably, although the loop to those two enhancers is strong at the early time point, it becomes significantly reduced later in development, when both enhancers' activities are reduced. Conversely, the iab-5 enhancer contacts the promoter at a much higher frequency later in development, at the stage when the enhancer is most active. This locus therefore exhibits dynamic 3D promoter-enhancer contacts that reflect the transient activity of three developmental enhancers. It is interesting to note that in all loci examined, the dynamic contacts of specific elements are neighboured by stable contacts, as seen in the Antp and Abd-B loci. Dynamic changes, therefore, appear to operate in the context of larger, more-stable 3D landscapes (Ghavi-Helm, 2014).

Ninety-four per cent of enhancer interactions showed no evidence of dynamic changes across time and tissue context, which is remarkable given the marked developmental transitions during these stages. To investigate this further, enhancer-promoter interactions were examined of genes switching their expression state between time points or tissue contexts. The ap gene, for example, is not expressed at 2-4 h but is highly expressed during mid-embryogenesis (6-8 h). Despite the absence of expression, the interaction between the apME680 enhancer and the ap promoter is already present at 3-4 h, several hours before the gene's activation. To examine this more globally, differentially expressed genes, going either from on-to-off or off-to-on, were selected. Even for these dynamically expressed genes, there was no correlation with changes in their promoter-enhancer contacts. Similar 'stable' interactions were observed between tissue contexts. Genes predominantly expressed in the neuroectoderm at 6-8 h, for example, have interactions at the same locations in whole embryos and purified mesodermal nuclei at 6-8 h, despite the fact that they are not expressed in the mesoderm at this stage (Ghavi-Helm, 2014).

Pre-existing loops were recently observed in human and mouse cells, and suggested to prime a locus for transcriptional activation. However, why they are formed and how transcription is eventually triggered remains unclear. To investigate this, this study focused on the subset of genes that have both off-to-on expression and no evidence for differential interactions (20 genes; differentially expressed with stable loops (DS) genes). Despite changes in their overall expression, DS genes have similar levels of RNA polymerase II (Pol II) promoter occupancy at both time points. The presence of promoter-bound Pol II in the absence of full-length transcription is indicative of Pol II pausing. Using global run-on sequencing (GRO-seq) data to define a stringent set of paused genes, it was observed that most (75%) DS genes are paused (15 of 20 DS genes), and have a significantly higher pausing index. This percentage is significantly higher than expected by chance when sampling over all off-to-on genes, and is robust to using a strict or more relaxed) definition of Pol II pausing. This association is very evident when examining specific loci, showing Pol II occupancy, short abortive transcripts, and loop formation before the gene's expression. Taken together, these results indicate that 'stable' chromatin loops are associated with the presence of paused Pol II at the promoter (Ghavi-Helm, 2014).

To understand how transcription is ultimately activated, changes were examined in DNase I hypersensitivity at the promoter of DS genes. DNase I hypersensitivity is significantly increased at interacting promoters at the stages when the gene is expressed, suggesting that the recruitment of additional transcription factor(s) later in development might act as the trigger for transcriptional activation (Ghavi-Helm, 2014).

In summary, these data reveal extensive long-range interactions in an organism with a relatively compact genome, including pairs of co-regulated genes contacting common enhancers often at distances greater than 200 kb. Comparing enhancer contacts in different contexts revealed that chromatin interactions are very similar across developmental time points and tissue contexts. Enhancers therefore do not appear to undergo long-range looping de novo at the time of gene expression, but are rather already in close proximity to the promoter they will regulate. Within this 3D topology, highly dynamic and transient contacts would not be visible when averaging over millions of nuclei. As transcription factor binding is sufficient to force loop formation, these results suggest a model where through transcription factor-enhancer occupancy, an enhancer loops towards the promoter and polymerase is recruited, but paused in the majority of cases. The subsequent recruitment of transcription factor(s) or additional enhancers at preformed 3D hubs most likely triggers activation by releasing Pol II pausing. Such preformed topologies could thereby promote rapid activation of transcription. At the same time, as paused promoters can exert enhancer-blocking activity, the presence of paused polymerase within these 3D landscapes could safeguard against premature transcriptional activation, but yet keep the system poised for activation (Ghavi-Helm, 2014).

Quantitative analysis of polycomb response elements (PREs) at identical genomic locations distinguishes contributions of PRE sequence and genomic environment

Polycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position. The phiC31 site-specific integration tool was adapted to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw) reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. The Hox PRE Frontabdominal-7 (Fab-7) was compared with a PRE from the vestigial (vg) gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, the phiC31 tool was used to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT) that is essential for silencing. This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of phiC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design (Okulski, 2011).

Promoter Structure

Continued: see abdominal-B Promoter Structure part 2/3 | part 3/3

Abdominal-B: Biological Overview | Evolutionary Homologs | Transcriptional Regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.