Transvection in the Iab-5,6,7 Region of the Bithorax Complex of Drosophila: Homology Independent Interactions in Trans

The Abdominal-B (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). Here we focus on iab-5, iab-6 and iab-7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cis-regulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the iab-5,6,7 region is able to interact with Abd-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. LEWIS has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues 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 homologues share homologous sequences 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. 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. Evidence is presented that trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. Also, we show that 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-5,6,7 and 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.     

Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity

We have examined the developmental consequences for larval and imaginal segmental cuticular structure of a chromosomal translocation involving a breakpoint in the abdominal region of the bithorax complex (BX-C). This complex makes an essential contribution to the development of metameric differences in part of the thorax and in all abdominal segments. The breakpoint is proximal to the most distal (iab-7) homeobox, and results in the translocation to the Y chromosome of the Ultrabithorax (Ubx) and abdominal-A (abd-A) domains. The genotype deficient for the distal part of the complex shows normal function for Ubx and abd-A but has a phenotype typical for severe Abd-B mutations. Conversely, the distal fragment retains a segment identity function which must represent a contribution from Abd-B in parasegments 13 and 14; the latter metamere is wild type, indicating that it does not require the contribution of Ubx or abd-A. We also constructed a genotype comprising the proximal fragment of this translocation together with an overlapping distal fragment of the BX-C derived from Df(3R)Ubx109. It therefore contained all sequences of the BX-C though in the abdominal region the abd-A and Abd-B domains were not adjacent to each other in the chromosome. This genotype was phenotypically normal and demonstrates that DNA sequences in the abd-A and Abd-B regions do not require cis-arrangement for their activity.     

Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.

A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B 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. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that 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.     

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.     

Fragments of the Fab-3 and Fab-4 Boundaries of the Drosophila melanogaster Bithorax Complex That Include CTCF Sites Are not Effective Insulators

Doklady Biochemistry and Biophysics - The segment-specific regulatory domains of the Bithorax complex (BX-C), which consists of three homeotic genes Ubx, abd-A and Abd-B, are separated by...     

The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain.

In Drosophila the Abdominal-B (Abd-B) domain of the bithorax complex (BX-C) spans over 100 kb and is responsible for specifying the identities of adult abdominal segments five (A5) to nine (A9), inclusive, and correspondingly, neuromeres 10-14 of the embryonic central nervous system. The domain consists of a region coding for two proteins, ABD-BI (54 kd) and ABD-BII (36 kd) and cis-regulatory regions extending from infra-abdominal-5 (iab-5) to iab-9, inclusive. We have used a monoclonal anti-ABD-B antibody to infer that mutants in iab-8 eliminate the expression of ABD-BI in neuromeres 10-13, inclusive, and that mutants in iab-9 eliminate expression of ABD-BII in neuromere 14. ABD-B expression is also analyzed in homozygotes for (i) loss-of-function mutants involving the iab-5, iab-6 and iab-7 regions, (ii) gain-of-function mutants Miscadastral pigmentation (Mcp) and Superabdominal (Sab), and (iii) a trans-regulator, Polycomb (Pc). ABD-B expression along the antero-posterior axis is colinear with the chromosomal order of the cis-regulatory regions. The behavior of rearrangement-associated iab-6 and iab-7 mutants suggests that the enhancer-like region, iab-5, and possibly also iab-6, may be shared between the abd-A and Abd-B domains. Such sharing is proposed as a factor that tends to keep gene complexes intact during evolution.     

Interacting insulators from the Drosophila melanogaster bithorax complex can form independent expression domains

Regulatory region of three bithorax complex genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) can be divided into nine iab domains, capable of directing expression of one of the genes in certain abdominal parasegment of Drosophila. In the Abd-B regulatory region, three insulators were identified, including Fab-7 and Fab-8, which flanked the iab-7domain, and Mcp, which separated the Abd-B and abd-A regulatory regions. It was suggested that boundary insulators formed a barrier between active and repressed chromatin. In the present study, using the yellow and white reporter genes and different combinations of known insulators, Mcp, Fab-7, and Fab-8, it was demonstrated that only specific interaction of two insulators was capable of isolation of active and repressed chromatin, i.e., the formation of independent expression domains.     

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.     

Segmental determination in Drosophila central nervous system: analysis of the abdominal-A region of the bithorax complex.

The bithorax complex (BX-C) comprises several genes required for the diversification of posterior segments in Drosophila. The BX-C genes control segment differences not only in the epidermis but in other tissues as well, especially in the central nervous system. We have examined the control of one segment-specific neural structure: the lateral dots, a paired structure present in the first abdominal segment of the larval CNS and absent in all following abdominal segments. Our results show that the suppression of lateral dots in segments A3 and A4 requires the presence of two active copies of one of the BX-C genes, abdominal-A (abd-A). We also show that the adjacent BX-C regions, iab-3 and iab-4, can act in trans on abd-A not only when the two copies of BX-C are paired but also, at least to some extent, when pairing is disturbed.     

Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos.

The abdominal-A (abd-A) and Abdominal-B (Abd-B) genes of the bithorax complex (BX-C) specify the identity of most of the Drosophila abdomen. Six different classes of infraabdominal (iab) 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. By staining embryos mutant for different iab mutations with anti-abd-A and anti-Abd-B antibodies I show here that the expression of Abd-B (and probably also abd-A) exhibit a parasegmental regulation. I have also studied the significance of the chromosomal order of parasegmental iab regulatory sequences, and the possible presence of chromosomal 'boundaries' between them, by looking at the expression of abd-A and Abd-B in embryos carrying the Uab and Mcp mutations. These data are discussed in the light of models of parasegmental-specific regulatory regions within the BX-C.     

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.     

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.     

A Polycomb and Gaga Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex

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(1)) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7(1) is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7(1) deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment 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 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(1) allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor.