Identification and characterization of a parasegment specific regulatory element of the abdominal-B gene of Drosophila

We have characterized mutations of the Abdominal-B gene of the bithorax complex of Drosophila. We conclude that the gene contains two distinct genetic elements: one has a morphogenetic role and acts in parasegments 10, 11, 12, and 13, while the other acts on parasegment 14 and has primarily or exclusively a regulatory function. Evidence indicates that the latter suppresses the activity of the morphogenetic element of Abd-B and of other genes responsible for the development of sclerotic plates. The regulatory element also suppresses those BX-C genes and other homeotics that, in the absence of Polycomb or extra sex combs function, can become active in parasegment 14.     

Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis

The Drosophila bithorax complex (BX-C) controls segmental development by selectively deploying three protein products, Ubx, abd-A and Abd-B, within specific segments along the body axis. Expression of these products within any one segment (or, more accurately, parasegment) is affected by mutations clustered in a particular region of the BX-C. The regulatory regions defined by this genetic analysis span 20-50 kb and there is one region for each segmental unit. Here we describe regulatory elements from several of these regions, identified by fusion to a Ubx-lacZ gene and analysis in germline transformants. A small DNA fragment from the abx region programs expression with an anterior boundary in the second thoracic segment (parasegment 5). This anterior limit is appropriate, since the abx region normally controls Ubx in parasegment 5. Other regulatory regions of the BX-C that control development of parasegments 6, 7 or 8 contain similar regulatory elements that program expression with anterior limits in parasegments 6, 7 or 8, respectively. These experiments define a class of BX-C regulatory elements that control expression along the anterior-posterior axis. The early appearance of the lacZ patterns in embryos suggests a role for these elements in the initial activation of expression from the BX-C.     

Double and triple mutant combinations of bithorax complex of Drosophila

We have constructed double and triple mutant combinations for the Ubx, abd-A and Abd-B genes of the bithorax complex and have examined the homeotic transformations they produce in the larval and adult patterns. Embryos hemizygous for the triple combination exhibit a metameric pattern consisting of parasegments 5-12 being transformed into parasegment 4. In addition, parasegment 13 develops like a mixture of parasegment 3 and 4, and parasegment 14 is abnormal. The same phenotype is displayed by embryos homozygous for DfP9, lacking all the BX-C DNA, >300 kb. This result strongly supports the notion that the BX-C contains only three genes which account for all the developmental functions of the complex. The phenotypes of the different double combinations also support the same view; the Ubx abd-a comthoracic and several abdominal functions. The abd-A Abd-B combination exhibits the same phenotype of DpP10 DfP9, lacking all the abdominal functions except those specific for A1. Our results also indicate that each BX-C gene becomes active autonomously regardless of the presence or functional state of the other BX-C genes.     

Trans-regulatory functions in the Abdominal-B gene of the bithorax complex

We have investigated the functional organization of the Abdominal-B gene in the bithorax complex using the expression of the Ultrabithorax gene as an assay for Abdominal-B trans-regulatory functions. Using Polycomb mutants to relax the normal spatial control of Ultrabithorax expression, we have examined the effects of Abdominal-B mutations on the expression of Ultrabithorax protein products in parasegment 14. The results support the hypothesis that the Abdominal-B gene contains two trans-regulatory functions: the m element active in parasegments 10-13 and the r element acting exclusively in parasegment 14.     

Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex

The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.     

The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain

The Drosophila bithorax complex Abdominal-B (Abd-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. We use transgene assays 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. We also show that 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. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in 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.     

Boundary swapping in the Drosophila Bithorax complex

Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, we tested whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, we exchanged the Fab-7 and Fab-8 boundaries within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, we find that the Fab-7 boundary can almost completely replace the Fab-8 boundary. Our results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries.     

The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.     

How do single homeotic genes control multiple segment identities?

Each of the homeotic genes of the bithorax complex of Drosophila defines the identities of more than one body segment. The mechanisms by which this occurs have been elusive. In a recent report, Castelli-Gair and Akam⁽¹⁾ analyze in detail the control of parasegment 5 and parasegment 6 identities by the bithorax complex gene Ubx. Their results indicate that differences in the spatial and temporal expression patterns of Ubx are critical in determining differences between these parasegments. However, dose effects observed by others indicate that parasegment-specific differences in the level of Ubx expression are also important. For the other BX-C genes, parasegment-specific expression of protein isoforms, or combinatorial control dependent on the expression patterns of other spatially restricted regulators, may also play a role.     

Homoeosis in Drosophila: anterior and posterior transformations of Polycomb lethal embryos

Lethal embryos homozygous for Polycomb (Pc) mutations show transformations of segment-specific cuticular features to those of more anterior or posterior segments; the frequency and extent of such changes show differences which depend on the genotype and the region. The mesothorax of Polycomb lethal embryos often shows posterior transformations of the anterior- and posterior-most portions of the segment, and anterior transformations of the medial portion. A comparison of Polycomb embryos also bearing various genetic lesions of the bithorax gene complex (BX-C) shows that the penetrance of anterior transformation and the extent of posterior transformation in the appears independent of posterior transformation, even though cells undergoing each of these changes lie in close proximity in the developing embryo. It has been shown previously that in Polycomb lethal embryos posterior transformations require the normal function of the BX-C. We show here that anterior transformations of the mesothorax and other segments require the normal function of the Sex combs reduced (Scr) locus, also necessary for the normal development of the prothorax and some head segments. Similar observations are also presented for a Polycomblike mutation. We suggest that in Polycomb embryos there are errors in the clonal transmission of determined states resulting in expression of the BX-C and Scr+ loci at abnormal locations, and that such events are probabilistic in nature and show marked regional differences in frequency.     

Initiator elements function to determine the activity state of BX-C enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain.