Promoter Structure

The expression of the abdominal-A and Abdominal-B genes of the bithorax complex 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. 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).

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. These iab regions are named after the abdominal segments they control. iab-2 through iab-7 regulate abdominal segments 2 through 7, corresponding to parasegments 7 through 12 respectively. It is thought that these mutations define parasegmental cis-regulatory regions that control the expression of abd-A and Abd-B in a fashion similar to the stripe specific enhancer of even-skipped. The expression of Abd-B (and probably also abd-A) exhibit a parasegmental regulation (Busteria, 1989 and Sanchez-Herrero, 1991).

Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. Using markers for the precursors of the somatic cells of the gonad, five discrete steps have been identified in gonadal development:

  1. First, somatic gonadal precursor cells are specified within the mesoderm in parasegments 10 through 12.
  2. After pole cells traverse and exit the midgut they recognize and associate primarily with specific mesodermal cells laterally positioned in the mesoderm of parasegments 11 and 12. These are the migratory gonadal precursors that delaminate from the mesodermal cell sheet.
  3. In a third step, gonadal precursors and pole cells migrate anteriorly, where they contact cells in parasegment 10.
  4. Next, gonadal precursors and pole cells arrest migration at parasegment 10.
  5. Finally, the mesodermal cells partially ensheath the arriving cells, and the cluster coalesces into the gonad.

The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. The proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4, a distal enhancer element, abolish expression of abd A within these cells, blocking the coalescence of the gonad (Boyle, 1995).

Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adult flies. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue (Cumberledge, 1992).

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, 1997).

Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model posits that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12 (Mihaly, 1997).

Using a P-element inserted in the middle of the Fab-7 region (the bluetail transposon), an extensive collection of new Fab-7 mutations have been generated that allow the subdivision of Fab-7 into a boundary element and a Polycomb-response element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the blt transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. Evidence is provided that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing (Mihaly, 1997).

These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the abd-A gene of the BX-C. It is suggested that there are two phases to BX-C regulation. During the first phase, gap and pair-rule genes select the activity state of the iab=6 and iab-7 cis-regulatory domains in PS11 and PS12. Once the activity states have been selected, BX-C regulation switches to the maintenance system. In PS11, where iab-6 is actived while iab-7 is not, the maintenance system must keep iab-7 turned off. This is presumably accomplished by the assembly of a Polycomb group protein silencing complex on iab-7. The Fab-7 boundary must prevent this iab-7 silencing complex from nucleating the assembly of a Polycomb-group protein in iab-6. This is presumably accomplished by blocking interactions between the two domains. In PS-12, where iab-7 is activated, the on state must be maintained (perhaps throught the action of proteins encoded by members of the trithorax group) (Mihaly, 1997).

By generating abd-A muations on rearranged chromosomes that break in the iab-7 region, iab elements switch their target promoter from Abd-B to abd-A. Thus iab elements in breakpoint chromosomes can prevent iab elements from acting on Abd-B and allow them to act on abd-A. These breaks disrupt a mechanism that targets the iab regions to the Abd-B promoter (Hendrickson, 1995).

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein have been investigated. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that both Hb and Kruppel (Kr) are required to set the Abd-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that Hb and Kr are direct regulators of this iab-2 enhancer. The single Kr site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that Kr repression occurs through a local quenching mechanism. The gap repressor Giant (Gt) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. This iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (Pho). These iab-2 Pho sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the Pho sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer (Shimell, 2000).

The iab-2 pattern initiates as a wedge-shaped band in the central region of the embryo at the precellular blastoderm stage. There is an absence of staining in the most ventral cells corresponding to the presumptive mesoderm. Examination of this band at high magnification reveals that the staining is 10-12 cells in width and is graded in both directions from the central region. Only dots of staining, possibly corresponding to the transcribing chromosomes, are observed in weakly expressing cells at the edges of the stripe. Since a parasegment is 4 cells wide at the blastoderm stage, these results indicate that this early expression stripe is approximately three parasegments in width when first activated. As cellularization proceeds, the wide band resolves into two stripes: during gastrulation, weaker, more posterior stripes are added in a pair-rule fashion. During germ-band retraction, epidermal staining fades, and as dorsal closure finishes, staining becomes evident in a subset of cells in the CNS. This CNS staining pattern does not respect the PS7 boundary and is strongest in the anterior of the embryo (Shimell, 2000).

The gap gene product Kr is required to set the iab-2(1.7) anterior expression border. However, since Kr is not expressed anterior to PS5, some other factor must also be required to repress the iab-2(1.7) enhancer in anterior regions. A likely candidate is the Hb protein, which has been shown to be important for repressing the bx and pbx enhancers anterior to PS6. To examine whether Hb plays a role in setting the iab-2(1.7) anterior expression boundary, this construct was crossed into both hb and osk mutant backgrounds. Loss of zygotic hb caused a slight broadening of the initial expression band, indicating an anterior shift in the expression pattern of this enhancer. The presence of maternal Hb likely minimizes the anterior shift in these zygotic hb mutant embryos. Consistent with this view, it has been found that, in an osk mutant background, in which the maternal level of Hb is uniform throughout the embryo, expression from the iab-2(1.7) enhancer is completely abolished. These findings suggest that, as with the bx and pbx enhancers, Hb is important for setting the initial anterior limit of iab-2 enhancer function (Shimell, 2000).

Hb, Kr, and Gt have been classified as short-range repressors whose range of action is limited to approximately 50 to 150 bps. Two major mechanisms of short-range repression are: competitive binding to an overlapping activator binding site, and quenching, which entails interference with function of locally bound activators. Since studies on Hb, Kr, and Gt action have focused primarily on their control of pair-rule genes such as eve, it was of interest to address mechanisms used by these repressors in the alternative context of a homeotic gene regulatory region. The in vitro binding analysis identified five discrete Hb sites on the iab-2(1.7) fragment. One of these sites, Hb2, overlaps extensively with one of the Eve binding sites. Since Eve acts as an activator of iab-2(1.7) expression, Hb may repress by competing with Eve for direct binding to this site. Evidence for a direct competition mechanism has been described for Hb repression through the bx and bxd/pbx control regions of the Ubx homeotic gene. In these cases, the anterior boundary is in PS6 rather than PS7, and Hb competes with Ftz rather than Eve. However, mutational analysis shows that Hb sites other than Hb2 also contribute to iab-2 repression. These additional sites could promote Hb competition with Eve by assisting Hb binding at Hb2 through cooperative interactions. Similarly, the single Gt binding site in iab-2(1.7) overlaps another Eve binding site, suggesting that Gt may also repress by direct competition with Eve in posterior parasegments. In contrast, the single Kr binding site (Kr1) does not overlap Eve sites. A distinct Kr mechanism is also supported by the ability of Kr1 to repress even when relocated 100 bp away from its normal position in the iab-2(1.7) fragment. This flexibility, together with failure of Kr repression when Kr1 is further relocated by 300 bp, is consistent with a short-range quenching mechanism. These results argue against Kr repression by direct interference with basal transcription factors, since 300 bp is small compared to the 20-kb distance between the iab-2 enhancer and the abd-A promoter. Previous studies using a synthetic regulatory region have shown that Kr can repress by a quenching mechanism in vivo (Shimell, 2000).

Any proposed mechanism for Kr action through iab-2, however, must account for the variability of Kr repression within its own expression domain. Specifically, Kr represses the iab-2 enhancer in PS3 and PS5 where Kr concentrations are low, but it does not repress in PS7 where Kr concentrations are high. This observation suggests that simple occupancy of the Kr1 site is not sufficient for iab-2 repression and that another factor acts in concert with Kr. The likely partner is Hb since Kr repression of iab-2 is limited to parasegments that accumulate significant levels of both Kr and Hb. In this view, repression just anterior to PS7 requires both Kr and Hb, whereas repression in more anterior parasegments, where Hb levels are highest, is mediated by Hb alone. Kr-Hb synergy could involve direct contact since the two proteins have been shown to interact when bound to DNA. Whether Kr synergizes with Hb by augmenting Hb binding to DNA in a cooperative manner or by recruiting additional corepressors is not clear. Kr, but not Hb, functions together with the corepressor dCtBP (Shimell, 2000 and references therein).

After Hb and Kr decay during early gastrulation, the repressed state is propagated through later stages of development by the PcG proteins. How the transition from early gap repressors to long-term PcG repressors occurs at the molecular level is not known. Two basic models have been proposed: (1) direct recruitment, and (2) chromatin recognition. Model (1): The gap gene products, especially Hb, have been proposed to help recruit PcG proteins directly to specific DNA sites. Based upon its early time of action, a role for the PcG protein Extra sex combs (Esc) as a molecular bridge between the two sets of repressors has been suggested. However, direct interactions between Esc and gap repressors have not been reported. A better candidate for such a molecular link is dMi-2, which binds directly to Hb and behaves genetically as an enhancer of PcG repression. In its simplest form the direct recruitment model is unlikely because the iab-2, bx, and pbx enhancers all contain Hb sites but do not effectively recruit PcG proteins. These elements fail to maintain A-P boundaries of expression and are unable to attract PcG proteins to sites on chromosomes. Furthermore, the continuous requirement for PRE sequences during development shows that DNA site recognition by PcG proteins can occur long after Hb and Kr have decayed. Model (2):The second model proposes that PcG proteins recognize some feature of silenced chromatin, rather than particular gap repressors. This model is supported by patterns of PcG-dependent silencing that reflect patterns of early gene activity rather than the distributions of gap proteins. In this view, PcG proteins sense the transcriptional off state and then assemble locally to imprint this state through later stages. These two models are not mutually exclusive. Both the Hb-interacting protein dMi-2 and the Kr-interacting protein dCtBP have mammalian homologs that interact with histone deacetylases. Perhaps the gap repressors work by targeting these deacetylases, whose action alters the local acetylation state of the histone tails. This could provide a feature of silenced chromatin that is recognized by PcG proteins and that promotes their association at nearby PREs (Shimell, 2000 and references therein).

In addition to sites for the gap repressors, the role of iab-2 binding sites was characterized for the recently identified PcG protein, Pleiohomeotic. Pho sites on the iab-2(1.7) fragment are required for pairing-sensitive repression (PSR) of a mini-white reporter. Thus, Pho can mediate this type of gene repression in the context of a homeotic regulatory fragment, analogous to its activity with an engrailed regulatory fragment. Similarly, Pho binding sites are required for function of a different PRE located in the bxd region. Are the iab-2(1.7) Pho sites sufficient for full PcG repression? The results suggest that they are not, since lacZ maintenance in the embryo, as opposed to PSR function during late stages, requires more distally located iab-2 sequences in combination with the iab-2(1.7) fragment. Thus, assays for PSR and for lacZ maintenance are not measuring precisely the same activity. In molecular terms, this could reflect association of distinct complexes at PSR sites as opposed to sites that supply full PRE function. Alternatively, a larger critical number of Pho sites might be needed for lacZ maintenance and fewer sites might suffice for PSR. The iab-2(534) fragment, which enables lacZ maintenance, contains two additional Pho consensus core sites. However, three lines of evidence indicate that Pho is not likely the sole factor that recruits PcG proteins either to PREs or to PSR sites. (1) A multimerized Pho site is insufficient to mediate PSR; (2) in vivo crosslinking studies show heterogeneity among PcG proteins assembled onto DNA from different regions of the engrailed locus., and (3) the DNA-binding GAGA protein has also been implicated in PRE function and has been found associated with PRE sequences in chromatin binding assays. These observations strongly suggest that multiple DNA-binding factors form the landing pad for association of distinct types of PcG complexes. What might be the in vivo role of PSR sites, such as the one on the iab-2(1.7) fragment, which by themselves cannot provide full PRE activity? One possibility is that, in their normal context, they act as secondary recruitment sites to extend and/or stabilize chromatin changes that are nucleated at strong PREs. In agreement with this, is has been found that PC protein first assembles onto core PREs at the blastoderm stage and that high levels of PC association with fragments outside of these core regions do not occur until later in embryogenesis. The scattering of PSR sites throughout large regulatory domains, such as those within the BX-C, might assist assembly and propagation of repressive chromatin complexes over large DNA distances (Shimell, 2000 and references therein).

The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. A study of enhancer traps shows that these regulatory regions function to regulate gene expression to parasegmental domains and that these domains are mediated by Polycomb-mediated repression. P elements are mobile gene vectors that appear to hop around the genome inserting themselves randomly into various genes and regulation regions, mutating these regions in the process of insertion. Eight P elements carrying a beta-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure (McCall, 1994).

Polycomb maintains the segmental expression limits of the homeotic genes in the bithorax complex. Polycomb-binding sites within the bithorax complex were mapped by immunostaining of salivary gland polytene chromosomes. Polycomb bound to four DNA fragments, one in each of four successive parasegmental regulatory regions. These fragments correspond exactly to the ones that can maintain segmentally limited expression of a lacZ reporter gene. Thus, Polycomb acts directly on discrete multiple sites in bithorax regulatory DNA. Constructs combining fragments from different regulatory regions demonstrate that Polycomb-dependent maintenance elements can act on multiple pattern initiation elements, and that maintenance elements can work together. The cooperative action of maintenance elements may motivate the linear order of the bithorax complex (A. Chiang, 1995).

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 lateral dots are a segment-specific neural structure; a paired structure present in the first abdominal segment of the larval CNS and absent in all following abdominal segments. The suppression of lateral dots in segments A3 and A4 requires the presence of two active copies of abdominal-A. The adjacent BX-C regions, iab-3 and iab-4, can act in trans on abd-A not only when the two copies of the BX-C are paired but also, at least to some extent, when pairing is disturbed (Jijakli, 1992).

P elements containing a 7 kb DNA fragment from the middle of the Drosophila bithorax complex insert preferentially into the bithorax complex or into the adjacent chromosome regions. The 7 kb fragment does not contain any known promoter, but it acts as a boundary element separating adjacent segmental domains. An enhancer-trap P element was constructed with the homing fragment and the selectable marker flanked by FRT sites. P insertions can be trimmed down by Flp-mediated recombination to just the lacZ reporter, so that the beta-galactosidase pattern is not influenced by sequences inside the P element. Twenty insertions into the bithorax complex express beta-galactosidase in segmentally limited patterns, reflecting the segmental domains of the bithorax complex where the elements reside. The mapping of segmental domains has now been revised, with enlargement of the abx/bx, bxd/pbx, and the iab-3 domains. The FRT sites in the P elements permit recombination between pairs of elements on opposite chromosomes, to generate duplications or deletions of the DNA between the two insertion sites. Using this technique, the length of the Ultrabithorax transcription unit was varied from 37 to 138 kb, but there was surprisingly little effect on Ultrabithorax function (Bender, 2000).

The segmental domains of the BX-C can now be mapped with precision, allowing for investigation of the roles of parasegment enhancers, Polycomb response elements, and boundary elements in the creation of these domains. The enhancer traps reported here redefine several segmental domains. The most proximal group (HC71-1, HC179, and HC16-1) show that the PS5 domain extends at least 20 kb more proximal than the proximal-most bx mutant lesion, almost to the final exon of the Ubx transcription unit. Likewise, the HC184B line extends the limit of the bxd region, mapping 15 kb distal to the most distal bxd mutation so far reported. The most surprising observation is that both the HCJ192B and HCJ200 insertions give lacZ patterns with the anterior edge at PS8, thus extending the iab-3 domain. Two rearrangement breaks proximal to the HCJ200 site had been called iab-4 lesions in a prior analysis. These should now be called iab-3 alleles. There is no contradiction implied by the redefinition, because breaks in the iab-3 domain also lack iab-4 function: they cut the iab-4 domain away from the abd-A gene it is meant to regulate. The iab-4 domain may begin just distal to the HCJ200 site; another P element insertion less than 2 kb from HCJ200 shows lacZ expression with a PS9 anterior boundary (Bender, 2000).

Models for P element homing generally invoke a homotypic interaction between proteins bound to the homing fragment and identical proteins bound to the genomic copy of that DNA fragment. Such binding would tether the P element donor plasmid before P transposition. Most DNA binding proteins are bound to hundreds of places throughout the genome; therefore, homotypic interactions of such proteins would target a P element to so many places that insertions might appear random. P elements containing Polycomb response elements may be homing to many endogenous Polycomb binding sites in this manner. Homing to a single chromosomal location, as is reported here, would require that the DNA binding proteins mediating the homing be present at only one or a few sites in the genome. A DNA element unique to the homeotic complexes is perhaps the boundary element separating segmental domains. Such boundaries have been best defined by the Fab and Mcp deletions. The homing fragment appears to contain a boundary element; it can block Polycomb repression from either side. From its position in the BX-C, the normal function of this boundary must be to separate the bxd and iab-2 domains (Bender, 2000).

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).

Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins

Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. ChIP-on-chip assay was used to compare the distribution of several PcG/TrxG proteins, as well as histone modifications in active and repressed genes across the two homeotic complexes ANT-C and BX-C. The data indicate the colocalization of the Polycomb repressive complex 1 [PRC1; containing the four PcG proteins Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and dRing/Sex combs extra (Sce)] with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state, Pho covers extended chromatin domains over many kilobases. This feature of Pho, observed on many polytene chromosome puffs, reflects a previously undescribed function. At the hsp70 gene, it was demonstrated in mutants that Pho is required for transcriptional recovery after heat shock. Besides its presumptive function in recruiting PcG complexes to their site of action, these results now uncover that Pho plays an additional role in the repression of already induced genes (Beisel, 2007).

This work used two Drosophila tissue culture lines to map the distribution of chromatin proteins required for the transcriptional maintenance of the HOX genes. Although compromising on the precise developmental identity, the tissue culture cells provided a biochemically tractable homogeneous material, which currently would be difficult to obtain from whole animals. This choice was important to obtain the sharply delineated ChIP profiles, which show a highly significant correlation to mapped genetic elements in the two homeotic complexes. As such, the protein patterns obtained seem to reflect a valid situation as found in material from whole animals. In addition, the ChIP profiles uncovered a new function of Pho, which could be confirmed in whole animals (Beisel, 2007).

The results for SF4 cells are consistent with data that used a Schneider cell derivative for ChIP studies. PRC1 binds to discrete sequence elements, whereas H3K27me3 covers large genomic domains, including genic and intergenic regions. These observations indicate that H3K27me3 cannot be solely responsible for PRC1 targeting. How these H3K27 methylated domains influence HOX gene expression and whether the broad methylation pattern is the cause or consequence of gene silencing remains unclear. H3K27me3 may prevent the binding of activating protein factors as e.g., chromatin remodeling complexes and/or prevent the establishment of activating histone modifications. To this regard, a complementary pattern of H3K27me3 and H4ac, which is present in active gene regions, was detected (Beisel, 2007).

Several lines of evidence suggest that PcG proteins propagate their silencing effect by the direct interaction with the promoter region, which results in the inhibition of transcription initiation. In agreement with that, all promoter regions of the silent ANT-C HOX genes are occupied by PRC1. However, the Ubx promoter, which is silent in both cell lines, as well as the silent AbdB transcription units in Kc cells, are devoid of PRC1. Here, probably the numerous PREs, which are occupied by PRC1 in the Ubx and AbdB domains, build up a special chromatin structure that maintains the silent transcription state (Beisel, 2007).

In agreement with the observed H3K27me3 pattern in Drosophila cells, in mammalian Hox clusters inactive domains are covered by H3K27 and active domains are found entirely covered by H3K4 methylation. In contrast, the distribution of the enzymes setting the histone marks are completely different. In Drosophila E(Z), Trx, and Ash1 are bound at discrete sequence elements, whereas the mammalian homologues EZH2 and MLL1 localize to extended regions coincident with the methylation signals. MLL1 acts as a functional human equivalent of yeast Set1. Both proteins colocalize with RNA Pol II at the transcription start site of highly expressed genes and catalyze the trimethylation of H3K4 at this location. Only at active Hox genes MLL1 reveals a different binding behavior covering entire active chromatin domains. In contrast, the current data shows that Trx also localizes to promoter regions of silent HOX genes and does not show the spreading behavior of MLL1 but appears at additional discrete sites. A complete colocalization of Trx with PRC1 sites was observed at silent genes, i.e., in this expression state no obvious competition is taking place with regard to binding sites (Beisel, 2007).

The comparison of the AbdB gene with the Dfd gene shows that the maintenance of the active state can be performed in alternative ways. The absence of PcG complexes does not seem to be a prerequisite of the active state as observed at the promoter of Dfd in this study and at regulatory regions of Ubx in imaginal discs (Beisel, 2007).

In the active AbdB domain Ph stays bound in a minor but significant amount, and Psc is present in the active Dfd intron. In this regard, Ph and Psc could serve as recruiting platforms for other PRC1 subunits in case of the gene switching to the off state. However, both proteins have been reported to be associated with active genes. Consistent with this, Ph was also observed in the proximal part of both homeotic complexes binding actively transcribed non-HOX genes. The function of this binding behavior remains elusive (Beisel, 2007).

The transcription of noncoding RNAs (ncRNAs) seem to play an important, although diverse, role in the regulation of the BX-C. Noncoding transcription found through the bxd PRE is crucial for Ubx repression and transcription through Mcp overlaps with AbdB transcription in the embryo. NcRNA transcription in the AbdB domain coincides with an active AbdB gene indicates a nonuniversal, gene specific function for ncRNAs in the BX-C (Beisel, 2007).

In the silent state PRC1 is bound to all PREs in the AbdB domain and might be recruited by the action of sequence-specific factors like Pho and the E(Z) histone methyltransferase activity, which may also mark the entire domain as being inactive. In the active AbdB domain, ncRNA transcription may directly influence the binding of of PRC1 and E(Z) or may trigger the enzymatic activity of Trx. Consistent with this scenario, Trx has been shown to bind single-stranded DNA and RNA in vitro. The switch of Trx into an activating mode could lead to the methylation of histones and/or other proteins setting positive transcriptional marks and modulate their activity, respectively. In this case, the displacement of PcG proteins could be directly caused by the Trx action. The binding of Trx to the promoter regions of the active AbdB transcription units could either be caused by (transient) chromatin looping events bridging Trx-bound PREs with the promoters, or Trx could be recruited independently to the active HOX promoters by interaction with RNA Pol II, similar to MLL1, which is recruited to actively transcribed genes in mammalian cells. Trx- and TAC1-interacting histone acetyltransferases may then be responsible for setting epigenetic marks that maintain the active transcription state. Trx has been shown to be required for transcription elongation and it is localized in the gene body of active Ubx, caused by the interaction with elongation factors. In contrast, other studies that investigated the distribution of PcG and TrxG proteins at the active and repressed Ubx gene in imaginal discs found the same restricted Trx profile did the current study, namely Trx binding at discrete sites. These differences may be explained by the different Trx antibodies used. Trx is most probably proteolytically processed like human MLL which results in two fragments that form a heterodimeric complex. This raises the intriguing question whether the complete heterodimeric Trx complex might get recruited to the promoter and upon gene induction the N-terminal fragment tracked along the gene body together with elongation factors, whereas the C-terminal fragment stayed at the promoter (Beisel, 2007).

Pho maps were generated to investigate its role in the recruitment of PRC1. However, the distribution of Pho suggests that the protein also functions in the gene body of actively transcribed genes. The immunostaining of polytene chromosomes revealed that Pho seems not only to be limited to HOX gene control but plays a general role in gene regulation. The colocalization of Pho with strong signals of active Pol II on polytenes together with the effect of a pho-null mutation on the recovery of induced hsp70 indicates that Pho may be directly involved in the rerepression of highly active genes (Beisel, 2007).

It is difficult to imagine that the spreading of Pho is the result of the ability of this protein to bind sequence specifically to DNA. Instead, a model is proposed in which Pho either acts directly at the Pol II elongation complex or it interacts with a remodeling complex, carrying it along the chromatin fiber. In this line, Pho has been shown to interact with BRM and dINO80, two nucleosome remodeling complexes. Interestingly, heat-shock gene transcription is independent of BRM but involves the recruitment of the TAC1 complex, possibly through multiple interactions with the elongating Pol II complex. The simultaneous action of Trx and Pho at heat-shock genes is striking and might resemble their antagonistic functions at HOX genes. Further studies are necessary to unravel the exact molecular mechanism of Pho in this process (Beisel, 2007).

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).

A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands

MicroRNAs (miRNAs) are ~22-nucleotide RNAs that are processed from characteristic precursor hairpins and pair to sites in messages of protein-coding genes to direct post-transcriptional repression. The miRNA iab-4 locus in the Drosophila Hox cluster is transcribed convergently from both DNA strands, giving rise to two distinct functional miRNAs. Both sense and antisense miRNA products target neighboring Hox genes via highly conserved sites, leading to homeotic transformations when ectopically expressed. Sense/antisense miRNAs are also present in the mouse and antisense transcripts are found close to many miRNAs in both flies and mammals, suggesting that additional sense/antisense pairs exist (Stark, 2008).

Hox genes are highly conserved homeobox-containing transcription factors crucial for development in animals. Genetic analyses have identified them as determinants of segmental identity that specify morphological diversity along the anteroposterior body axis. A striking conserved feature of Hox complexes is the spatial colinearity between Hox gene transcription in the embryo and the order of the genes along the chromosome. Hox clusters also give rise to a variety of noncoding transcripts, including microRNAs (miRNAs) mir-10 and mir-iab-4/mir-196, which derive from analogous positions in Hox clusters in flies and vertebrates. miRNAs are ~22-nucleotide (nt) RNAs that regulate gene expression post-transcriptionally. They are transcribed as longer precursors and processed from characteristic pre-miRNA hairpins. In particular, Hox miRNAs have been shown to regulate Hox protein-coding genes by mRNA cleavage and inhibition of translation, thereby contributing to the extensive regulatory connections within Hox clusters. Several Hox transcripts overlap on opposite strands, providing evidence of extensive antisense transcription, including antisense transcripts for mir-iab-4 in flies and its mammalian equivalent mir-196. However, the function of these transcripts has been elusive. This study shows that the iab4 locus in Drosophila produces miRNAs from opposite DNA strands that can regulate neighboring Hox genes via highly conserved sites. Evidence is provided that such sense/antisense miRNA pairs are likely employed in other contexts and a wide range of species (Stark, 2008).

Examination of the antisense transcript that overlaps Drosophila mir-iab-4 revealed that the reverse complement of the mir-iab-4 hairpin folds into a hairpin reminiscent of miRNA precursors. Moreover, 17 sequencing reads from small RNA libraries of Drosophila testes and ovaries mapped uniquely to one arm of the iab-4 antisense hairpin. All reads were aligned at their 5' end, suggesting that the mir-iab-4 antisense hairpin is processed into a single mature miRNA in vivo, which is referred to as miR-iab-4AS. For comparison, six reads were found consistent with the known miR-iab-4-5p (or miR-iab-4 for short) and one read was foudn for its star sequence (miR-iab-4-3p). Interestingly, the relative abundance of mature miRNAs and star sequences for mir-iab-4AS (17:0) and mir-iab-4 (6:1) reflects the thermodynamic asymmetry of the predicted miRNA/miRNA* duplexes. Because they derived from complementary near palindromes, miR-iab-4 and miR-iab-4AS had high sequence similarity, only differing in four positions at the 3' region. However, they differed in their 5' ends, which largely determine miRNA target spectra: miR-iab-4AS was shifted by 2 nt, suggesting targeting properties distinct from those of miR-iab-4 and other known Drosophila miRNAs (Stark, 2008).

Robust transcription of mir-iab-4 sense and antisense precursors was confirmed by in situ hybridization to Drosophila embryos. Both transcripts were detected in abdominal segments in the posterior part of the embryo, but intriguingly in nonoverlapping domains. As described previously, mir-iab-4 sense was expressed highly in abdominal segments A5-A7, showing modulation in levels within the segments: abdominal-A (abd-A)-expressing cells appeared to have more mir-iab-4, whereas Ultrabithorax (Ubx)-positive cells appeared to have little or none. In contrast, mir-iab-4AS transcription was detected in the segments A8 and A9, where Abdominal-B (Abd-B) is known to be expressed. Primary transcripts for mir-iab-4 and mir-iab-4AS were also detected by strand-specific RT-PCR in larvae, pupae, and male and female adult flies, suggesting that both miRNAs are expressed throughout fly development (Stark, 2008).

To assess the possible biological roles of the two iab-4 miRNAs, fly genes were examined for potential target sites by searching for conserved matches to the seed region of the miRNAs. Highly conserved target sites were found for miR-iab-4AS in the 3' untranslated regions (UTRs) of several Hox genes that are proximal to the iab-4 locus and are expressed in the neighboring more anterior embryonic segments: abd-A, Ubx, and Antennapedia (Antp) have four, five, and two seed sites, respectively, most of which are conserved across 12 Drosophila species that diverged 40 million years ago. More than two highly conserved sites for one miRNA is exceptional for fly 3' UTRs, placing these messages among the most confidently predicted miRNA targets and suggesting that they might be particularly responsive to the presence of the miRNA. The strong predicted targeting of proximal Hox genes was reminiscent of previously characterized miR-iab-4 targeting of Ubx in flies and miR-196 targeting of HoxB8 in vertebrates (Stark, 2008 and references therein).

To test whether miR-iab4AS is functional and can directly target abd-A and Ubx, Luciferase reporters were constructed carrying the corresponding wild-type 3' UTRs and control 3' UTRs in which each seed site was disrupted by point substitutions. mir-iab-4AS potently repressed reporter activity for abd-A and Ubx. This repression was specific to the miR-iab-4AS seed sites; expression of the control reporters with mutated sites was not affected. Tested were perform to see whether mir-iab-4AS reduced expression of a Luciferase reporter with the Abd-B 3' UTR, which has no seed sites. As expected, mir-iab-4AS expression did not affect reporter activity, consistent with a model where miRNAs do not target genes that are coexpressed at high levels. In addition to demonstrating specific repression dependent on the predicted target sites, these assays confirmed the processing of the mir-iab-4AS hairpin into a functional mature miRNA (Stark, 2008).

If miR-iab-4AS were able to potently down-regulate Ubx in the fly, its misexpression should result in a Ubx loss-of-function phenotype, a line of reasoning that has often been used to study the functions and regulatory relationships of Hox genes. Ubx is expressed throughout the haltere imaginal disc, where it represses wing-specific genes and specifies haltere identity. When mir-iab-4AS was expressed in the haltere imaginal disc under bx-Gal4 control, a clear homeotic transformation of halteres to wings was observed. The halteres developed sense organs characteristic of the wing margin and their size increased severalfold, features typical of transformation to wing. Consistent with the increased number of miR-iab4AS target sites, the transformation was stronger than that reported for expression of iab-4, for which changes in morphology were confirmed wing-like growth was not found (Stark, 2008).

It is concluded that both strands of the iab-4 locus are expressed in nonoverlapping embryonic domains and that each transcript produces a functional miRNA in vivo. In particular, the novel mir-iab-4AS is able to strongly down-regulate neighboring Hox genes. Interestingly, vertebrate mir-196, which lies at an analogous position in the vertebrate Hox clusters, is transcribed in the same direction as mir-iab-4AS and most other Hox genes, and targets homologs of both abd-A and Ubx. With its shared transcriptional orientation and homologous targets, mir-iab-4AS appears to be the functional equivalent of mir-196 (Stark, 2008).

The expression patterns and regulatory connections between Hox genes and the two iab-4 miRNAs show an intriguing pattern in which the miRNAs appear to reinforce Hox gene-mediated transcriptional regulation. In particular, miR-iab-4AS would reinforce the posterior expression boundary of abd-A, Ubx, and Antp, supporting their transcriptional repression by Abd-B. mir-iab-4 appears to support abd-A- and Abd-B-mediated repression of Ubx, reinforcing the abd-A/Ubx expression domains and the posterior boundary of Ubx expression. Furthermore, both iab-4 miRNAs have conserved target sites in Antp, which is also repressed by Abd-B, abd-A, and Ubx. The iab-4 miRNAs thus appear to support the established regulatory hierarchy among Hox transcription factors, which exhibits 'posterior prevalence,' in that more posterior Hox genes repress more anterior ones and are dominant in specifying segment identity. Interestingly, Abd-B and mir-iab-4AS are expressed in the same segments, and the majority of cis-regulatory elements controlling Abd-B expression are located 3' of Abd-B. This places them near the inferred transcription start of mir-iab-4AS, where they potentially direct the coexpression of these genes. Similarly, abd-A and mir-iab-4 may be coregulated as both are transcribed divergently, potentially under the control of shared upstream elements (Stark, 2008).

These data demonstrate the transcription and processing of sense and antisense mir-iab-4 into functional miRNAs with highly conserved functional target sites in neighboring Hox genes. In an accompanying study (Bender 2008), genetic and molecular analyses in mir-iab-4 mutant Drosophila revealed that the proposed regulation of Ubx by both sense and antisense miRNAs occurs under physiological conditions and, in particular, the regulation by miR-iab-4AS is required for normal development. These lines of evidence establish miR-iab-4AS as a novel Hox gene, being expressed from within the Hox cluster and regulating Hox genes during development (Stark, 2008).

The genomic arrangement of two miRNAs that are expressed from the same locus but on different strands might provide a simple and efficient means to create nonoverlapping miRNA expression domains. Such sense/antisense miRNAs could restrict each other's transcription, either by direct transcriptional interference, as shown for overlapping convergently transcribed genes, or post-transcriptionally, possibly via RNA-RNA duplexes formed by the complementary transcripts. Sense/antisense miRNAs would usually differ at their 5' ends and thereby target distinct sets of genes, which might help define and establish sharp boundaries between expression domains. Coupled with feedback loops or coregulation of miRNAs and genes in cis or trans, this arrangement could provide a powerful regulatory switch. The iab-4 miRNAs might be a special case of tight regulatory integration in which miRNAs and proximal genes appear coregulated transcriptionally in cis and repress each other both transcriptionally and post-transcriptionally (Stark, 2008).

It is perhaps surprising that no antisense miRNA had been found previously, even though, for example, the intriguing expression pattern of the iab-4 transcripts had been reported nearly two decades ago, and iab-4 lies in one of the most extensively studied regions of the Drosophila genome. The frequent occurrence of antisense transcripts suggests that more antisense miRNAs might exist. Indeed, up to 13% of known Drosophila , 20% of mouse, and 31% of human miRNAs are located in introns of host genes transcribed on the opposite strand or are within 50 nt of antisense ESTs or cDNAs. These include an antisense transcript overlapping human mir-196. However, because of the contribution of noncanonical base pairs, particularly G:U pairs that become less favorable A:C in the antisense strand, many miRNA antisense transcripts will not fold into hairpin structures suitable for miRNA biogenesis, which explains the propensity of miRNA gene predictions to identify the correct strand. Nonetheless, in a recent prediction effort, 22 sequences reverse-complementary to known Drosophila miRNAs showed scores seemingly compatible with miRNA processing. Deep sequencing of small RNA libraries from Drosophila confirmed the processing of small RNAs from four of these high-scoring antisense candidates, and the ovary/testes libraries used here showed antisense reads for an additional Drosophila miRNA (mir-312). In addition, using high-throughput sequencing of small RNA libraries from mice, sequencing reads were found that uniquely matched the mouse genome in loci antisense to 10 annotated mouse miRNAs. Eight of the inferred antisense miRNAs were supported by multiple independent reads, and two of them had reads from both the mature miRNA and the star sequence. These results suggest that sense/antisense miRNAs could be more generally employed in diverse contexts and in species as divergent as flies and mammals (Stark, 2008).

A novel function for the Hox gene Abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila

In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), it was discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, an enhancer from the iab-6 regulatory domain was discovered that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. It was determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of Sex peptide in female mates of iab-6 mutant males. It was also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. The results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR (Gligorov, 2013).

The AG synthesizes seminal proteins that are essential for male fertility. These >180 accessory gland proteins ('Acps') are transferred to females during mating and cause post-mating changes in the females known collectively as the post-mating response (PMR). The PMR includes increased rates of egg-laying and ovulation, sperm storage, decreased receptivity to courting males, as well as changes in longevity, feeding, and sleep patterns. The PMR is divided into two phases. The short term response (STR) refers to changes in the above behaviors during the first ~24 hours post-mating. It requires Acps, but not the receipt of sperm. Persistence of the PMR after 24 hr (and for up to ~10 days) is known as the long-term response (LTR). The LTR requires Acps and stored sperm. Many of the roles of Acps were initially discovered by experiments in which whole AG extracts or purified Acps were injected into unmated females, or by whole-tissue ablation in males (Gligorov, 2013).

Each lobe of the AG is composed of a monolayer of approximately 1000 secretory cells comprised of two morphologically distinct cell types. Roughly 96% of these cells are flat, polygonally shaped 'main cells'. The remaining 4% of the cells are large, spherical, vacuole filled 'secondary cells'; these are dispersed among the main cells at the distal tip of the gland. Enhancer trapping and other studies have shown that, in addition to their morphological differences, these two secretory cell types are biochemically distinct. Ablation of the main cells only showed that products of these cells are essential for the PMR. These products include ovulin (Acp26Aa), an Acp that acts in the STR to stimulate ovulation, and the sex peptide (SP, Acp70A), which is the ultimate regulator of most other PMR effects. SP binds to sperm within the mated female, and its active portion is gradually released from the sperm. This binding and release allows SP to affect the female for as long as she contains stored sperm. A network of five other Acps is necessary for SP to bind to sperm and enter storage. The predicted protease CG10586 (Seminase) appears to be necessary for both STR and LTR related events, while the predicted protease CG9997, the predicted cysteine-rich secretory protein (CRISP) CG17575, and the predicted lectins CG1656/1652 appear to be LTR specific. The cellular source of each of these proteins is currently unknown (Gligorov, 2013).

In spite of the detailed characterization of the main cells and several specific Acps, the role of the secondary cells has remained mysterious. No PMR-associated Acps were known to be expressed exclusively in the secondary cells, and no tools have been available to specifically target those cells. This study identified the secondary cells of the male AG as a novel location of Abd-B expression in the adult fly. By screening an extensive collection of cis-regulatory deletions, discovered a 2.8 kb enhancer from the iab-6 cis-regulatory domain was discovered, whose removal completely abolishes Abd-B expression in the secondary cells. Loss of Abd-B expression in the secondary cells causes those cells to develop aberrantly. Moreover, these mutant males provide their mates with substances that initiate the PMR, but are insufficient to maintain it. The results indicate that Abd-B expression in the secondary cells is essential for their proper development and for the production of proteins important for long-term changes in female post-mating responses (Gligorov, 2013).

Continued: abdominal-A Transcriptional regulation part 2/2

abdominal-A: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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