QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 23, 2006
doi: 10.1242/10.1242/dev.02323

This Article Summary Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maeda, R. K. Articles by Karch, F. Search for Related Content PubMed PubMed Citation Articles by Maeda, R. K. Articles by Karch, F.

The ABC of the BX-C: the bithorax complex explained

Department of Zoology and Animal Biology and National Research Centre `Frontiers in Genetics', University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland.

^* Author for correspondence (e-mail: francois.karch{at}zoo.unige.ch)

	SUMMARY

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
As one of two Drosophila Hox clusters, the bithorax complex (BX-C) is responsible for determining the posterior thorax and each abdominal segment of the fly. Through the dissection of its large cis-regulatory region, biologists have obtained a wealth of knowledge that has informed our understanding of gene expression, chromatin dynamics and gene evolution. This primer attempts to distill and explain our current knowledge about this classic, complex locus.

	Introduction

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
In Drosophila, the bithorax complex (BX-C) controls the identity of each of the segments that contributes to the posterior two-thirds of the fly (Fig. 1, also see Box 1). It does this by regulating the expression of the three BX-C homeotic genes: Ultrabithorax (Ubx), abdominal A (abd-A) and Abdominal B (Abd-B). For over ninety years, scientists have been trying to unlock the complexities of the >300 kb cis-regulatory region of the bithorax locus. This work has lead to many ground-breaking discoveries, some of which have helped to shape our modern definition of a gene. Yet, throughout its rich history, the BX-C literature has suffered from a reputation of complexity [for a comprehensive review on BX-C molecular genetics, see Duncan (Duncan, 1987)]. In this primer article, we hope to demystify the BX-C by showing that it can be broken down into a modular array of understandable elements. Many of these elements have now been well-characterized molecularly and fall into a few distinct categories, including initiator elements, maintenance elements/Polycomb-response elements, cell-type specific enhancers, chromatin domain boundaries and promoter targeting sequences (see glossary in Box 2). Although our knowledge of the BX-C is still far from complete, we believe a solid understanding of these basic building blocks, when combined with the wealth of genetic data on the BX-C, will lead to a reasonably clear picture of the functioning of this complex locus.

	A crash course in BX-C genetics

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
Much of the mystery surrounding the BX-C comes from the nature of the early data on the complex. These early data were completely genetic in nature, and, therefore, only described the complex phenotypes of BX-C mutations and the interactions between these mutations (Lewis, 1954; Lewis, 1963). However, although these data are a bit abstract, they provided scientists with their first clues to the complexity of the BX-C and of the vast complexities to be discovered through the exploration of eukaryotic genomes. As such, we begin with a basic introduction to the genetics of the BX-C before proceeding to the molecular data.

In 1978, Ed Lewis published a landmark paper in which he summarized nearly 40 years of his work on the BX-C. In this paper, he reviewed a series of Drosophila mutations (called abx/bx, bxd/pbx, and iab-2 through to iab-8; see Fig. 1) that affect the identity of the posterior two-thirds of the fly: the third thoracic segment (T3) and the eight abdominal segments (A1 to A8; see Fig. 2A) (Lewis, 1978). Phenotypic analysis [which was further extended by Karch et al. (Karch et al., 1985)] defined nine classes of mutations and indicated that each mutation class defined an element that was required for the identity of a single segment (see Box 3). Remarkably enough, these classes of mutations mapped to the chromosome in an order that corresponded to the body segments in which they act. This astonishing correspondence between body axis and genomic organization was later found to be evolutionarily conserved in the homeotic clusters of most animals (for a review, see McGinnis and Krumlauf, 1992).

Although embryos deficient for the whole BX-C never hatch, they live long enough to make identifiable markers for each segment. In these embryos, segments posterior to the second thoracic segment (T2) develop as copies of T2 (see Fig. 2). Because of this, Ed Lewis proposed that T2 represents the ground state of development (i.e. the default state) and that each class of mutation represents a segment-specific function that allows a more posterior segment to differentiate away from the ground state. Furthermore, the fact that mutations affecting individual segment-specific functions always cause homeotic transformations towards the last unaffected, more-anterior segment (and not always to T2), meant that everything required for the development of the more-anterior segments had to be present in the more-posterior segments. Therefore, Lewis proposed that segment-specific functions act in an additive fashion. This idea was supported by the fact that some mutations that affected anterior segment-specific functions also caused slight changes in the more-posterior segments. For example, in flies with defective bxd/pbx function, the A1 segment develops as a copy of T3 (see Fig. 2). Thus, the normal role of bxd/pbx must be to assign segmental identity to A1. Likewise, because A1 is transformed into a copy of T3 instead of T2, the normal role of the abd/bx segment-specific function, required for T3 specification, must be present in the developing A1 segment (Fig. 2). Lewis summarized these findings into two rules: "...a [segment-specific function] derepressed in one segment is derepressed in all segments posterior thereto..." and "...the more posterior a segment... the greater the number of BX-C [segment-specific functions] that are in a derepressed state" (Lewis, 1978). Because of the correlation between chromosomal location and anteroposterior function, Lewis visualized this additive effect as a segmentally sequential opening of genes along the chromosome.

	The BX-C goes molecular

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
The 300 kb of DNA that covers the BX-C was first cloned during the early 1980s (Bender et al., 1983a; Bender et al., 1983b; Karch et al., 1985). All the mutations affecting the segment-specific functions were found to be associated with rearrangement breakpoints (such as translocations, inversions, deficiencies or insertions of transposable elements). The lesions associated with a given class of mutations always clustered in a relatively small part of the BX-C, and different classes never overlapped (see Fig. 1). The observation that all the mutations in each class are associated with rearrangement breakpoints not only helped to map them onto a DNA map (over 100 mutations have been localized), but also suggested that they did not simply inactivate protein-coding regions (in which case, point mutations would also have been recovered during the numerous screens performed). In 1985, Sanchez-Herrero et al. and Tiong et al. independently presented the first true complementation analysis of the BX-C, which suggested that the whole BX-C only contains three homeotic genes: Ultrabithorax (Ubx), abdominal A (abd-A) and Abdominal B (Abd-B) (Sanchez-Herrero et al., 1985; Tiong et al., 1985). These findings were later supported when it was shown that an Ubx abd-A Abd-B triple-mutant embryo harbored the same phenotype as an embryo carrying a complete deletion of the BX-C (Casanova et al., 1987). Publication of the complete sequence of the complex provided the final confirmation; the BX-C contained only three homeotic genes (Martin et al., 1995).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Diagram of the BX-C. The multicolored bar represents the DNA of the BX-C. Map coordinate numbering follows the numbering established by the original Drosophila Genome Project sequencing of the BX-C (Martin et al., 1995). The three BX-C homeotic genes, Ubx, abd-A and Abd-B are indicated below this bar (with exons indicated by the black horizontal bars and introns indicated by the diagonal lines connecting the bars). The individual cis-regulatory domains are indicated by the different colored regions on this bar. The orange and red regions (abx/bx and bxd/pbx) control Ubx expression. The regions shaded in blue (iab-2, 3 and 4) control abd-A expression. And the regions shaded in green (iab-5 through iab-8) control Abd-B expression. The corresponding adult segments affected by mutations in each cis-regulatory region are indicated on the diagram of the adult fly using the same color code.

But, there was then a contradiction: on the one hand, early genetic analysis revealed the existence of nine classes of mutations that affect segment-specific functions, while, on the other hand, other genetic and molecular analysis indicated that the BX-C only encodes three proteins. The description of the expression patterns of Ubx, abd-A and Abd-B answered this apparent paradox (Beachy et al., 1985; Celniker et al., 1990; Karch et al., 1990; Macias et al., 1990; White and Wicox, 1985). Fig. 3B shows the central nerve cord of a wild-type embryo stained with an antibody directed against Abd-B. Like Ubx and abd-A, although in different parasegments, Abd-B is expressed in an intricate pattern that is finely tuned from one parasegment to the next. By staining various mutant embryos, it was finally understood that the segment-specific functions corresponded to cis-regulatory regions that regulate the expression of Ubx, abd-A or Abd-B in a segment-specific fashion. Mutations in any of the segment-specific regulatory regions alter the expression of its relevant target gene. For example, flies homozygous for the iab-7^Sz mutation have their seventh abdominal segment transformed into a copy of the sixth. Consistent with this, in embryos, the Abd-B expression pattern characteristic for parasegment 12 (PS12, which corresponds to A7, see Box 3) is replaced by the pattern normally present in PS11/A6 (Galloni et al., 1993). The strong correlation between the level of homeotic gene expression and segmental identity also suggested that the level of homeotic gene expression was crucial for determining segmental identity, thus providing a mechanism by which three genes pattern nine segments (Castelli-Gair and Akam, 1995).

Fig. 1 schematically details how the cis-regulatory regions of the BX-C are arranged. The red and orange regions show the regulatory regions that interact with Ubx. They include the abd/bx and bxd/pbx regions that regulate Ubx expression in PS5 and PS6, respectively (Beachy et al., 1985; Little et al., 1990; White and Wicox, 1985). Similarly, iab-2, iab-3 and iab-4 specify the appropriate abd-A expression patterns in PS7, PS8 and PS9, respectively (Karch et al., 1990; Macias et al., 1990; Sanchez-Herrero, 1991). Shown in shades of green are the segment-specific functions that regulate the Abd-B transcription unit. The regulation of Abd-B expression is more complex than that of the other two BX-C Hox genes; however, for the purposes of this review, we will focus only on the short Abd-B transcript (A see Fig. 1; also referred as to Abd-B^m), which is required for the identities of PS10-PS13 (Casanova et al., 1986; Sanchez-Herrero and Crosby, 1988; Kuziora and McGinnis, 1988; Celniker et al., 1989; Zavortink and Sakonju, 1989; Delorenzi and Bienz, 1990). The iab-5, iab-6, iab-7 and iab-8 regions regulate this short transcript in PS10 to PS13, respectively (Boulet et al., 1991; Celniker et al., 1990; Estrada et al., 2002; Sanchez-Herrero, 1991).

	Initiation and maintenance phases in BX-C regulation

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
The overall determination of the anteroposterior (AP) axis during the initial stages of embryogenesis in Drosophila is under the control of three classes of transcription factors that are deployed in a cascade. These transcription factors, which are encoded by the maternal, gap and pair-rule genes, subdivide the embryo into 14 parasegments (see Box 3) (for reviews, see Ingham, 1988; Hoch and Jackle, 1993; Kornberg and Tabata, 1993; DiNardo et al., 1994). It is now known that these proteins interact with elements in each of the cis-regulatory regions of the BX-C genes to determine their ultimate expression patterns (White and Lehmann, 1986; Irish et al., 1989; Shimell et al., 1994; Casares and Sanchez Herrero, 1995). For example, the combination of the gap and pair-rule gene products that are present in PS12 allow the iab-7 cis-regulatory region, but not the iab-8 cis-regulatory region, to control Abd-B expression in PS12/A7. However, because the gap and pair-rule genes are only transiently expressed in the early embryo, and the activity states of the segment-specific cis-regulatory regions are fixed for the life of the fly, a system to maintain homeotic gene expression is also required in each cis-regulatory domain (Struhl and Akam, 1985). This maintenance system has been shown to require the products of the Polycomb-Group (Pc-G) and trithorax-Group (trx-G) genes (see Box 4). Although the Pc-G products function as negative regulators, maintaining the inactive state of the cis-regulatory regions not in use, the trx-G products function as positive regulators, maintaining the active state of active regulatory regions (Paro, 1990; Kennison, 1993; Simon, 1995; Pirrotta, 1997). Both the Pc-G and trx-G products are thought to maintain the active or inactive state of each parasegment-specific cis-regulatory region by modifying the chromatin structure of each region. Because of this distinction between the initiation of expression and the maintenance of expression, the elements to which the gap and pair-rule proteins bind have been termed initiators, and the elements to which the Pc-G and trx-G proteins bind have been termed maintenance elements [ME; also known as Pc-G or trx-G response elements (PREs/TREs)].

Box 2. Glossary of specialized terms Boundary element: A DNA element that separates adjacent chromatin/DNA domains. Colinearity: The relationship between the position of a Hox gene along a chromosome and the pattern of its expression along the anteroposterior axis. Gap gene: A class of Drosophila genes that, when mutated, cause embryos to develop with groups of consecutive segments missing. Gene conversion: The transfer of DNA sequences between two homologous sequences; can be a mechanism for mutation if the transfer of material contains one or more mutations. Homeobox: A 180-base-long sequence that is highly conserved among genes encoding Hox proteins. It enables a protein to bind to DNA in a sequence-specific fashion. Homeotic: Adjective of the term homeosis, which was introduced in 1894 by Bateson (Bateson, 1894) to describe phenotypic variation in which "something is changed into the likeness of something else". Initiator element: a DNA fragment that initiates a specific expression pattern of a linked gene. Maintenance element: a DNA fragment that can maintain the expression pattern of a linked gene established during an earlier stage of embryogenesis (by an initiator). Pair-rule gene: A class of Drosophila genes that, when mutated, results in the development of embryos with every second parasegment missing.

 

	Initiators, maintenance elements and segment-specific enhancers

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
Confirmation of the segment-specific and biphasic nature of BX-C gene regulation came from studies using reporter gene constructs. In these experiments, DNA fragments from the various regulatory regions were cloned upstream of a lacZ reporter gene (Lis et al., 1983). By making transgenic flies carrying these reporter constructs and studying their resulting patterns of expression, scientists have been able to identify specific DNA fragments that are required for initiating the segment-specific expression of BX-C genes, maintaining the restricted pattern of their expression, and for producing segment-independent, cell-type specific expression (see Fig. 4 for examples of how initiator and maintenance elements control gene expression patterns during development).

BX-C initiator elements can be defined as being specific types of enhancers that confer a parasegmentally restricted pattern of expression to a reporter gene during early embryogenesis (Simon et al., 1990; Qian et al., 1991; Muller and Bienz, 1992; Busturia and Bienz, 1993; Barges et al., 2000; Zhou et al., 1999; Shimell et al., 2000). For example, Fig. 4A shows the expression pattern of a lacZ reporter gene in an early Drosophila embryo when it is driven by an initiator element derived from the iab-6 regulatory region. As the iab-6 region is responsible for Abd-B expression in PS11, we see that this element can faithfully drive lacZ expression from PS11. Based on these types of assays, we know that initiator elements are able to read an early AP positional address and to transmit this information to a promoter. However, this ability is transient. At later stages of embryogenesis, the strict anterior border of expression derived from this construct is lost and lacZ becomes expressed in all of the parasegments along the AP axis (Fig. 4B). This degeneration of the initial pattern is probably due to the loss of positional information that is provided in the early embryo by the gap and pair-rule gene products. In support of this idea, a few initiator elements have been mapped precisely enough to show a direct correlation with the binding sites for gap and pair-rule gene products (Qian et al., 1991; Zhang et al., 1991; Shimell et al., 1994; Zhou et al., 1999).

View larger version (27K): [in this window] [in a new window]   Fig. 2. The additivity of segment-specific functions. Diagrams of two Drosophila larvae, anterior to the top. (A) A wild-type larva; (B) a larva mutant for the bxd/pbx segment-specific function. The diagram next to each larva represents the presence or absence of the segment-specific functions that are required to determine a particular segment/parasegment (moving across them horizontally). Underneath these are the three BX-C homeotic genes, Ubx, abd-A and Abd-B. In embryos that lack the entire BX-C, all segments posterior to the second thoracic segment (T2) develop as T2; thus T2 represents the ground state in this model. Because mutations in individual segment-specific functions always cause homeotic transformations towards the last unaffected, more-anterior segment, Ed Lewis proposed that segment-specific functions act in an additive fashion (Lewis, 1978). (A) The wild-type larva shows the segment-specific functions required for the proper development of each segment/parasegment. (B) The mutant larva lacks the bxd/pbx function and therefore has its A1 segment transformed into a copy of T3. Note that ventral pits (a characteristic of T3; arrows) are present in all of the more-posterior segments, indicating that the bxd/pbx segment-specific functions are also required in more-posterior segments (asterisks).

Cell-type or tissue-specific enhancers are a third type of regulatory element that has been identified within the segment-specific, cis-regulatory regions of the BX-C (Simon et al., 1990; Busturia and Bienz, 1993; Pirrotta et al., 1995). In most cases, these elements confer a cell/tissue-specific expression pattern to a reporter gene that is reiterated in all of the parasegments along the AP axis of the embryo. It must be noted, however, that within the BX-C, these enhancers confer a cell/tissue-specific pattern of homeotic gene expression that is restricted parasegmentally. This apparent discrepancy between the expression pattern of homeotic genes and that of transgenic reporter genes when under the control of these enhancers can easily be explained if the enhancers are coordinately regulated by the initiator and maintenance elements (see below).

	Cis-regulatory regions are organized into parasegment-specific chromosomal domains

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 
How can the various enhancers in a cis-regulatory region be coordinately regulated? Two complementary observations (coming from enhancer trap studies and from boundary mutations) have provided compelling evidence that the cis-regulatory regions of the BX-C are organized into parasegment-specific chromosomal domains.

In Drosophila, transgenic animals are generally made using P-element transposons. These transposons insert throughout the genome in a fairly random fashion. If these P-elements contain a basal promoter and a reporter gene, they often respond to nearby enhancer elements. The technique of using P-elements with reporter genes to get a read-out of the enhancers in the vicinity of a P-element insertion is called enhancer trapping (O'Kane and Gehring, 1987). Fig. 5 shows the insertion sites of several enhancer trap transposons that have landed within the BX-C (Galloni et al., 1993; McCall et al., 1994; Bender and Hudson, 2000). The colored line in this figure represents the genomic DNA of the BX-C using the same color coding as that shown in Fig. 3 (see legend for details). If we focus on the three transposons inserted within the 75 kb region marked in orange (between map positions 315,379 and 242,806), we find that all three transposons have similar expression patterns. The anterior border of expression of these enhancer traps is PS5. The abx/bx cis-regulatory region that regulates Ubx expression in PS5 lies within this region. Although the promoters of these three P-elements are obviously trapping different enhancer activities in this 75 kb region of DNA, they are all transcribed in PS5 and in the parasegments posterior to PS5, regardless of where exactly they have inserted. Meanwhile, the anterior parasegmental boundary of expression of the three enhancer traps inserted within the region 232,727 to 192,677 is shifted one parasegment posterior to PS6 (marked in red on Fig. 5). This domain corresponds to the region that contains the bxd/pbx cis-regulatory region that drives Ubx expression in PS6. Once again, although the intensity of expression varies between these three enhancer traps, the anterior border of each one's expression begins at PS6.

By examining the large number of enhancer trap lines isolated in the BX-C (Bender and Hudson, 2000) (some of which are shown in Fig. 5), two striking observations could be made. First, enhancer trap transposons that are spread out over considerable distances often produce the same expression pattern, whereas others located just a few kilobases away produce a different pattern. Second, the anterior border of lacZ expression always progresses towards the posterior by increments of one parasegment. Based on these observations and others, it was proposed that the BX-C enhancers reside in chromosomal domains that are coordinately regulated (Peifer et al., 1987). For example, all elements residing in the 75 kb region between map positions 315,379 and 242,806 (Fig. 5) are turned on and off together. This is why enhancer trap lines inserted in this region display similar patterns of expression. Meanwhile, enhancer traps lying very close to this region, but outside of it, display different patterns of expression (for example, compare the transposons at position 232,727 and 242,806, or the transposons at positions 127,367 and 125,489 in Fig. 5). In this model, enhancer trap transposons behave simply as sensors to the state of a domain, the extent of which can be mapped by comparing the various enhancer trap lines.

One prediction made by the domain hypothesis is the existence of boundary elements, which would act to limit the extent of each domain. In Figs 1 and 5, the boundaries are symbolized by the sharp color transition between the adjacent domains symbolized by the colored rectangles. A boundary is postulated to exist between each of the regulatory domains. Thus far, three boundaries, Mcp, Fab-7 and Fab-8, have been identified through mutational analysis (Gyurkovics et al., 1990; Karch et al., 1994; Mihaly et al., 1997; Mihaly et al., 1998; Barges et al., 2000). The best characterized of them is Fab-7, which separates the iab-6 cis-regulatory domain from the iab-7 cis-regulatory domain. When Fab-7 is deleted, iab-6 and iab-7 fuse into a new functional unit. This fusion disrupts Abd-B regulation in PS11, where normally only iab-6 is active. Usually this results in the inappropriate activation of iab-7 enhancers in PS11, which are turned on by the initiator element in the iab-6 domain. As a consequence, Abd-B expression is regulated in a PS12-like pattern, transforming cell identity from PS11 to PS12 (see Fig. 3C).

Box 3. Segments versus parasegments During early embryogenesis, a Drosophila embryo is rapidly metamerized into 14 parasegments by the products of the maternal, gap and pair-rule genes. In adult animals, these 14 parasegments will form the three head, the three thoracic and the eight abdominal segments. However, although there are similar numbers of segments and parasegments, they are, for the most part, slightly shifted relative to one another. In the thorax and the abdomen, this shift is approximately half a segment, meaning that a parasegment comprises the posterior half of one segment and the anterior half of the next. For example, PS6 comprises the posterior of segment T3 and the anterior segment A1. By chance, this shift is less visible in the adult animal because the visible portion of the adult abdominal segments corresponds primarily to the anterior portion of the segment. Ed Lewis described all of the phenotypes he originally studied according to the adult segments affected (Lewis, 1978). However, in 1985, Martinez-Arias and Lawrence observed that BX-C regulation occurs not through segments but instead through parasegments (PS) (Martinez-Arias and Lawrence, 1985). This observation has been confirmed by the expression patterns of the BX-C homeotic genes in embryos and larvae. The accompanying figure shows the correlation between segments and parasegments in the Drosophila embryo (anterior is to the left), and the expression pattern of each BX-C homeotic gene (darker shades of color indicate higher expression levels). Today, most reports on the regulation of the BX-C use both terminologies depending on whether an embryonic or adult phenotype is being described [the drawing of the embryo is reproduced, with permission, from Hartenstein (Hartenstein, 1993).

 

View larger version (34K): [in this window] [in a new window]

View larger version (25K): [in this window] [in a new window]   Fig. 3. Segment-specific functions are cis-regulatory regions. Three ventral nerve cords dissected out of Drosophila embryos immunostained for the Abd-B protein are shown. Parasegment (PS) borders and parasegment labels (PS10-PS13) are indicated according to the wild-type embryo. Below each cord is a diagram representing the state of each regulatory domain in PS10 through PS12, in which a black line represents a closed/silenced chomatin structure; a colored oval represents an open/active chromatin domain. (A) A wild-type embryonic ventral nerve cord has a distinct pattern of Abd-B expression that begins in PS10 and increases in each parasegment posteriorly. This is diagramed below as a parasegmentally regulated, sequential opening of chromatin domains. (B) The CNS from an iab-7Sz mutant in which the entire iab-7 domain is absent. Its PS12 develops as a copy of PS11 (indicated by the similar staining pattern in PS11 and PS12). (C) A deletion that removes the Fab-7 boundary causes a fusion between iab-6 and iab-7, allowing the stronger iab-7 enhancers to become initiated by the iab-6 initiator element, resulting in an Abd-B expression pattern that is characteristic of PS12 being initiated in PS11, and a homeotic transformation of PS11 into PS12.

Box 4. Polycomb- and trithorax-Group genes The Polycomb-Group (Pc-G) genes (a group of 40 genes) (Jürgens, 1985; Duncan, 1982) keep the homeotic genes of the BX-C repressed in those segments where they have not been activated during early embryogenesis. The Pc-G gene products form large complexes that are thought to package chromatin into a compact, transcriptionally inactive conformation (McCall and Bender, 1996; Boivin and Dura, 1998; Fitzgerald and Bender, 2001). These genes have been named after their founding member, Polycomb (Pc), which was discovered by Ed Lewis as a negative regulator of BX-C activity (Lewis, 1978). Most Pc-G genes have been identified through the mild homeotic transformations that appear when a single Pc-G gene copy is mutated (a haplo-insufficient phenotype). The homeotic transformation that most often occurs in Pc-G mutants is the appearance of extra pairs of sex combs on the legs of the second and third thoracic segment in males, a feature normally found only on the legs of the first thoracic segment. It is for this reason that Pc-G genes often have names like Polycomb, Extra sex combs, Sex combs on midleg and Additional sex combs. So far, two functionally distinct classes of Pc-G protein repressor complexes, PRC1 and PRC2, have been identified. The core PRC1 complex (PCC) contains Polycomb, Polyhomeotic, Posterior sex comb and dRING1 (Sex combs extra – FlyBase) (Francis et al., 2001). The PRC2 complex contains the Enhancer of zeste [E(z)], the Supressor of zeste12[Su(z)12] and the Nurf55/CAF1 proteins. In agreement with a role for Pc-G proteins in mediating chromatin conformational changes, the PRC2 complex has a histone H3 Lys 27 (H3-K27) methyltransferase activity that is mediated by E(z). H3-K27 is an epigenetic chromatin modification that is associated with transcriptionally inactive chromatin (Czermin et al., 2002; Muller et al., 2002; Ng et al., 2000; Tie et al., 2001). Meanwhile, the trithorax-Group (trx-G) genes appear to act counter to the Pc-G genes by maintaining the homeotic genes and their large cis-regulatory regions in a transcriptionally permissive state. Many trx-G genes have been identified through genetic screens for mutations that can suppress the dominant phenotype of Pc-G genes (Kennison and Tamkun, 1988; Shearn, 1989). Thus far, four complexes containing trx-G proteins have been identified, which all have chromatin modification activities, although their modes of action are quite varied (for a review, see Simon and Tamkun, 2002).

 

In 1999, Zhou and Levine asked this very question and looked for specific DNA fragments that could aid distal enhancers to bypass intervening boundaries (Zhou and Levine, 1999). The result of these experiments was the identification of an element that they called the promoter-targeting sequence (PTS). This element, normally located in the iab-7 domain just adjacent to the Fab-8 boundary, allows distal enhancers to bypass the Fab-8 boundary in transgenic assays. Later, it was shown that this PTS element can enable an enhancer to bypass even the gypsy insulator, suggesting that PTS function is independent of the insulator itself (Zhou and Levine, 1999). Recently, a new PTS element has been found in the iab-6 domain (Chen et al., 2005). On the basis of these results, it now seems likely that each boundary element may be flanked by a PTS element to aid in insulator bypass.

The second set of data that provides some hints as to how boundary elements are bypassed came from experiments performed with the gypsy insulator. When inserted between an enhancer and a promoter, the gypsy insulator is able to prevent enhancer-promoter interactions (Geyer and Corces, 1992). However, it was found that if two gypsy insulators were placed between these same enhancers and promoters, the enhancers were able to bypass the intervening insulators. A model was proposed in which insulators would pair with one another to allow bypass (Cai and Shen, 2001; Muravyova et al., 2001). In the BX-C, where there are often many boundary elements between an enhancer and a target promoter, this is a very attractive model. Perhaps boundary elements interact with one another and allow the appropriate enhancers to reach their target promoters. This model is still untested, although it has been shown that the Mcp element can indeed pair with the gypsy insulator and lead to enhancer bypass (Gruzdeva et al., 2005).

Although these two models are quite attractive, there still remains some doubt regarding their validity. This is largely due to experiments in which the Fab-7 boundary was replaced by the gypsy or scs insulators within the BX-C, by a process called gene conversion. When these experiments were performed, it was found that both the gypsy and scs fragments acted as insulators within the BX-C, blocking all distal enhancers from interacting with the Abd-B promoter (Hogga et al., 2001). This happened even though the PTS elements were left intact in all of these experiments. Therefore, although the BX-C boundaries may work as insulators in a transgenic context, their functioning may be more complicated in their endogenous context.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Reporter constructs identify initiator and maintenance elements. Drosophila embryos immunostained for the ß-galactosidase protein. (A,C) Early embryos at germband extension, where the posterior parasegments have curved around towards the dorsal side; (B,D) later stage embryos. (A,B) Embryos in which lacZ expression is driven by an element from the iab-6 region. (A) In early embryos, lacZ expression is restricted to the posterior of the embryo, with its anterior border positioned at PS11. (B) At later stages of development, the repression of lacZ anterior to PS11 is lost, as the iab-6 element becomes active throughout the embryo. (C,D) Embryos in which lacZ expression is driven by a DNA fragment derived from iab-5. (C) In early embryos, the anterior border of lacZ expression is positioned at PS10. (D) Later in development, the anterior border of lacZ expression is maintained, indicating the presence of both an initiator and a maintenance element on this fragment. ant., anterior; post., posterior.

	Conclusion

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

View larger version (58K): [in this window] [in a new window]   Fig. 5. Enhancer trap transposons within the BX-C. The BX-C is represented as a multicolored bar, as shown in Fig. 1. Transposon insertions of enhancer trap constructs are indicated by triangles above or below the bar. Late-stage embryos are stained for expression of the lacZ reporter gene that is present on each enhancer trap construct. Embryos have been cut along the dorsal midline and flattened to make visualization easier. Numbers in brackets show parasegment borders [adapted from Bender and Hudson (Bender and Hudson, 2000), apart from the enhancer trap lines expressing lacZ in PS9 and PS10 (inserted at 125,489 and 113,864, respectively), which were supplied by D. Fitzgerald and W. Bender].

	ACKNOWLEDGMENTS

 
We are indebted to Drs Dan Fitzgerald and Welcome Bender for sharing results and providing unpublished data for Fig. 5. We also thank Dr Henrik Gyurkovics for stimulating discussions. Finally, we acknowledge Dr Annick Mutero and Maria Gambetta for critical reading of the manuscript. R.M. is supported by the Swiss National Foundation and F.K. by the State of Geneva.

	REFERENCES

TOP SUMMARY Introduction A crash course in... The BX-C goes molecular Initiation and maintenance... Initiators, maintenance elements... Cis-regulatory regions are... Conclusion REFERENCES

 

Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Müller, M., Shanower, G., Schedl, P., Gyurkovics, H. and Karch, F. (2000). 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. Development 127,779 -790.[Abstract]

Bateson, W. (1894). Materials for the Study of Variation. London: Macmillan.

Beachy, P. A., Helfand, S. L. and Hogness, D. S. (1985). Segmental distribution of bithorax complex proteins during Drosophila development. Nature 313,545 -551.[CrossRef][Medline]

Bender, W. and Hudson, A. (2000). P element homing to the Drosophila bithorax complex. Development 127,3981 -3992.[Abstract]

Bender, W. and Fitzgerald, D. P. (2002). Transcription activates repressed domains in the Drosophila bithorax complex. Development 129,4923 -4930.[Abstract/Free Full Text]

Bender, W., Spierer, P. and Hogness, D. S. (1983a). Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster. J. Mol. Biol. 168,17 -33.[CrossRef][Medline]

Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S. (1983b). Molecular genetics of the bithorax complex in Drosophila melanogaster. Science 221,23 -29.[Abstract/Free Full Text]

Boivin, A. and Dura, J. M. (1998). In vivo chromatin accessibility correlates with gene silencing in Drosophila. Genetics 150,1539 -1549.[Abstract/Free Full Text]

Boulet, A., Lloyd, A. and Sakonju, S. (1991). Molecular definition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene. Development 111,393 -405.[Abstract]

Brock, H. W. and van Lohuizen, M. (2001). The Polycomb group–no longer an exclusive club? Curr. Opin. Genet. Dev. 11,175 -181.[CrossRef][Medline]

Busturia, A. and Bienz, M. (1993). Silencers in abdominal-B, a homeotic Drosophila gene. EMBO J. 12,1415 -1425.[Medline]

Busturia, A., Lloyd, A., Bejarano, F., Zavortink, M., Xin, H. and Sakonju, S. (2001). The MCP silencer of the Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for the maintenance of repression. Development 128,2163 -2173.[Abstract/Free Full Text]

Cai, H. N. and Shen, P. (2001). Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291,493 -495.[Abstract/Free Full Text]

Casanova, J., Sanchez-Herrero, E. and Morata, G. (1986). Identification and characterization of a parasegment specific regulatory element of the abdominal-B gene of Drosophila. Cell 47,627 -636.[CrossRef][Medline]

Casanova, J., Sanchez-Herrero, E., Busturia, A. and Morata, G. (1987). Double and triple mutant combinations of the bithorax complex of Drosophila. EMBO J. 6,3103 -3109.[Medline]

Casares, F. and Sanchez Herrero, E. (1995). Regulation of the infraabdominal regions of the bithorax complex of Drosophila by gap genes. Development 121,1855 -1866.[Abstract]

Castelli-Gair, J. and Akam, M. (1995). How the Hox gene Ultrabithorax specifies two different segments: the significance of spatial and temporal regulation within metameres. Development 121,2973 -2982.[Abstract]

Celniker, S. E., Keelan, D. J. and Lewis, E. B. (1989). The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain. Genes Dev. 3,1424 -1436.[Abstract/Free Full Text]

Celniker, S. E., Sharma, S., Keelan, D. J. and Lewis, E. B. (1990). The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 9,4277 -4286.[Medline]

Chan, C. S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J. 13,2553 -2564.[Medline]

Chen, Q., Lin, L., Smith, S., Lin, Q. and Zhou, J. (2005). Multiple promoter targeting sequences exist in Abdominal-B to regulate long-range gene activation. Dev. Biol. 286,629 -636.[CrossRef][Medline]

Chiang, A., O'Connor, M. B., Paro, R., Simon, J. and Bender, W. (1995). Discrete Polycomb-binding sites in each parasegmental domain of the bithorax complex. Development 121,1681 -1689.[Abstract]

Cumberledge, S., Zaratzian, A. and Sakonju, S. (1990). Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex. Proc. Natl. Acad. Sci. USA 87,3259 -3263.[Abstract/Free Full Text]

Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A. and Pirrotta, V. (2002). Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111,185 -196.[CrossRef][Medline]

Delorenzi, M. and Bienz, M. (1990). Expression of Abdominal-B homeoproteins in Drosophila embryos. Development 108,323 -329.[Abstract]

DiNardo, S., Heemskerk, J., Dougan, S. and O'Farrell, P. H. (1994). The making of a maggot: patterning the Drosophila embryonic epidermis. Curr. Opin. Genet. Dev. 4, 529-534.[CrossRef][Medline]

Drewell, R. A., Bae, E., Burr, J. and Lewis, E. B. (2002). Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex. Proc. Natl. Acad. Sci. USA 99,16853 -16858.[Abstract/Free Full Text]

Duboule, D. (1992). The vertebrate limb: a model system to study the Hox/HOM gene network during development and evolution. BioEssays 14,375 -384.[CrossRef][Medline]

Duncan, I. M. (1982). Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia gene complexes of Drosophila melanogaster. Genetics 102,49 -70.[Abstract/Free Full Text]

Duncan, I. (1987). The bithorax complex. Annu. Rev. Genet. 21,285 -319.[CrossRef][Medline]

Estrada, B., Casares, F., Busturia, A. and Sanchez-Herrero, E. (2002). Genetic and molecular characterization of a novel iab-8 regulatory domain in the Abdominal-B gene of Drosophila melanogaster. Development 129,5195 -5204.[Medline]

Fitzgerald, D. P. and Bender, W. (2001). Polycomb group repression reduces DNA accessibility. Mol. Cell. Biol. 21,6585 -6597.[Abstract/Free Full Text]

Francis, N. J., Saurin, A. J., Shao, Z. and Kingston, R. E. (2001). Reconstitution of a functional core polycomb repressive complex. Mol. Cell 8,545 -556.[CrossRef][Medline]

Fritsch, C., Brown, J. L., Kassis, J. A. and Muller, J. (1999). The DNA-binding Polycomb group protein Pleiohomeotic mediates silencing of a Drosophila homeotic gene. Development 126,3905 -3913.[Abstract]

Galloni, M., Gyurkovics, H., Schedl, P. and Karch, F. (1993). The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 12,1087 -1097.[Medline]

Garber, R. L., Kuroiwa, A. and Gehring, W. J. (1983). Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila. EMBO J. 2,2027 -2036.[Medline]

Geyer, P. K. and Corces, V. G. (1992). DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6,1865 -1873.[Abstract/Free Full Text]

Gruzdeva, N., Kyrchanova, O., Parshikov, A., Kullyev, A. and Georgiev, P. (2005). The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication. Mol. Cell. Biol. 25,3682 -3689.[Abstract/Free Full Text]

Gyurkovics, H., Gausz, J., Kummer, J. and Karch, F. (1990). A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation. EMBO J. 9,2579 -2585.[Medline]

Hagstrom, K., Muller, M. and Schedl, P. (1996). Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev. 10,3202 -3215.[Abstract/Free Full Text]

Hartenstein, V. (1993). Altas of Drosophila Development. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Hoch, M. and Jackle, H. (1993). Transcriptional regulation and spatial patterning in Drosophila. Curr. Opin. Genet. Dev. 3,566 -573.[CrossRef][Medline]

Hogga, I. and Karch, F. (2002). Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing. Development 129,4915 -4922.[Abstract/Free Full Text]

Hogga, I., Mihaly, J., Barges, S. and Karch, F. (2001). Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex. Mol. Cell 8,1145 -1151.[CrossRef][Medline]

Ingham, P. W. (1988). The molecular genetics of embryonic pattern formation in Drosophila. Nature 335, 25-34.[CrossRef][Medline]

Irish, V. F., Martinez-Arias, A. and Akam, M. (1989). Spatial regulation of the Antennapedia and Ultrabithorax homeotic genes during Drosophila early development. EMBO J. 8,1527 -1537.[Medline]

Jürgens, G. (1985). A group of genes controlling the spatial expression of the bithorax complex in Drosophila. Nature 316,153 -155.[CrossRef]

Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I., Celniker, S., Crosby, M. and Lewis, E. B. (1985). The abdominal region of the bithorax complex. Cell 43, 81-96.[Medline]

Karch, F., Bender, W. and Weiffenbach, B. (1990). abdA expression in Drosophila embryos. Genes Dev. 4,1573 -1587.[Abstract/Free Full Text]

Karch, F., Galloni, M., Sipos, L., Gausz, J., Gyurkovics, H. and Schedl, P. (1994). Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster. Nucleic Acids Res. 22,3138 -3146.[Abstract/Free Full Text]

Kaufman, T. C., Seeger, M. A. and Olsen, G. (1990). Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster. Adv. Genet. 27,309 -362.[Medline]

Kellum, R. and Schedl, P. (1991). A position-effect assay for boundaries of higher order chromosomal domains. Cell 64,941 -950.[CrossRef][Medline]

Kennison, J. A. (1993). Transcriptional activation of Drosophila homeotic genes from distant regulatory elements. Trends Genet. 9, 75-78.[CrossRef][Medline]

Kennison, J. A. and Tamkun, J. W. (1988). Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila. Proc. Natl. Acad. Sci. USA 85,8136 -8140.[Abstract/Free Full Text]

Kornberg, T. B. and Tabata, T. (1993). Segmentation of the Drosophila embryo. Curr. Opin. Genet. Dev. 3,585 -594.[CrossRef][Medline]

Kuziora, M. A. and McGinnis, W. (1988). Different transcripts of the Drosophila Abd-B gene correlate with distinct genetic sub-functions. EMBO J. 7,3233 -3244.[Medline]

Le Calvez, J. (1948). In (3R) SSAr: Mutation Aristapedia, hétérozygote dominante, homozygote lethal chez Drosophila melanogaster. Bull. Biol. Fr. Belg. 82, 97-113.[Medline]

Lewis, E. B.