The Bithorax Complex (BX-C) of Drosophila melanogaster is a genomic area of 300 Kb, located in the chromosome 3R, that comprises the necessary elements for determining the correct positioning along the anterior-posterior axis of the structures that give rise to the posterior thoracic and abdominal region of the adult fly (Lewis 1978, Karch et al. 1985).
This region of the body is composed of 9 segments (one thoracic and eight abdominal), and it derives from the embryonic parasegments PS5-PS14. At this point it is convenient to clarify that, during the embryonic development of Drosophila, the embryo is divided into a parasegmental organization, where each parasegment corresponds to the posterior compartment of an adult segment and the anterior compartment of the next one (for example, PS13 corresponds to the posterior seventh abdominal segment, A7, and the anterior A8). The expression of Hox genes follows this division (Martinez-Arias and Lawrence 1985), and therefore I will refer to parasegments from now on.
Ed Lewis and the BX-C
The studies of Edward Lewis of the Bithorax Complex showed that the expression of the homeotic phenotypes along the anterior-posterior axis followed the position of the alleles that caused each one of them along the chromosome. He proposed that this was the result of the sequential activation of gene products located in the BX-C (following the principle of colinearity explained in the first section). He stated that, once a gene was activated in a given parasegment, it would remain active until the posterior end of the animal. Finally, he proposed that the activation of each gene was controlled by cis- acting elements which controlled each gene in an independent manner. Interestingly, Lewis proposed that the genes were maintained by default in a inactive state due to the repressive activity exerted by the gene Polycomb (Pc) (Lewis 1978).
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In this initial model, Lewis proposed the existence of at least nine "segment-specific functions". Later studies (Sanchez-Herrero et al. 1985, Tiong et al. 1985, Casanova et al.
1987, Martin et al. 1995) showed that, in fact, the BX-C was only composed of three Hox genes: Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B).
The Bithorax Complex Hox genes
If the BX-C only contains 3 genes, how does it control differential gene expression in 9 different parasegments? The vast amount of mutations studied inside the BX-C that showed homeotic phenotypes allowed, less than 10 years after the predictions of Ed Lewis, to propose a model on the function of the BX-C. The proposed model stated that the BX-C is divided into 9 independent regulatory domains. Each domain is responsible for the activation of a different expression pattern for a single gene from a given parasegment towards the posterior end of the embryo (Figure I). The unique combination of Hox expression patterns generated in this manner specifies the identity of each parasegment. In this way, the domain iab-2 would be responsible for assigning an expression pattern to the gene abd-A from PS7, iab-3 from PS8, etc (Peifer et al. 1987).
The expression pattern of each one of the Hox genes, and the corresponding regulatory domains, are depicted in Figure I.
Figure I. Genetic composition of the BX-C
Schematic representation of the expression pattern of the three Hox genes present in the BX-C in the embryo (A) and its correspondence with the adult body (B). The color scheme represents the regulatory domains of the BX-C that drive expression of each HOX gene, represented in B. Figure modified from (Maeda and Karch 2006).
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Ubx becomes active in PS5 of the embryo, and remains present all along the axis until PS12 (Akam et al. 1985, Hogness et al. 1985). This expression pattern is regulated by the domains abx/bx and bxd/pbx, and is essential for the determination of the identity of the third thoracic segments (T3) and the first abdominal segment (A1) of the adult fly (Casanova et al. 1985, White and Wilcox 1985, Muller and Bienz 1991).
abd-A begins to be expressed in PS7 of the embryo, remaining active until the anterior compartment of PS13, and determines the identity of the abdominal segments A2, A3 and A4 of the adult fly. The cis-regulatory sequences that confer this expression pattern are located in the domains of the BX-C iab-2, iab-3 and iab-4 (Karch et al. 1990, Macias et al. 1994).
Abd-B is expressed from the embryonic PS10, determining the identity of the last abdominal segments (A5, A6, A7, A8 and A9) of the adult. Its expression is regulated by the domains iab-5, iab-6, iab-7 and iab-8,9 (Casanova et al. 1986, Celniker et al. 1989, Celniker et al. 1990, Sanchez-Herrero 1991). Interestingly, this gene can start its transcription from different promoters, giving rise to two different protein isoforms (ABD-Bm and ABD-Br) (Casanova et al. 1986, Zavortink and Sakonju 1989).
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Regulation in the BX-C: The "open for business" model
Once established the fact that 9 regulatory domains control the differential expression of each of the 3 Hox genes of the BX-C in each of the embryonic parasegments between PS5 and PS14, how does this regulation take place? Adapting the existence of cis- regulatory domains to the model proposed by Ed Lewis that required sequential activation of each gene in a colinear manner, the prediction made in (Peifer et al. 1987) was that the domains are either "inactive" or "active" in each parasegment, depending on their chromatin state. This would mean that the regulatory regions present on each of the domains would be able to drive transcription of their target Hox gene only if they were on a parasegment where the domain was "open". Accordingly, the chromatin of the BX-C, repressed in anterior parasegments of the embryo, would be progressively "opened"
(turned to a permissive state) as we travel along the anterior-posterior axis. This is known as the "open for business" model. For a comprehensive review on the subject refer to (Maeda and Karch 2015).
This model was definitely confirmed years later, by the insertion in various areas of the BX-C of P-elements containing a lacZ gene, able to do enhancer trapping. Each one of the lines containing a P-element expressed lacZ from a different parasegment. This pattern was highly reproducible and depended on the region of the BX-C in which the P-element had landed (Figure II). For example, a line containing a P-element that landed on the region of iab-2 shows a lacZ expression pattern that begins in PS7 and remains active until the posterior end of the embryo. This is explained by the promoter of the lacZ reporter gene "trapping" the enhancers from the iab-2 region in the parasegments where this region is in an active state (Bender and Hudson 2000).
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Figure II. P element homing in the BX-C leads to restricted lacZ expression patterns Dissected embryos showing restricted lacZ expression patterns coming from P elements inserted in various locations of the BX-C. Reproduced from (Maeda and Karch 2006), the figure is an adaptation from (Bender and Hudson 2000).
One year later, a modification of this study, but based on the insertion of a lacZ gene whose expression was controlled by UAS sites instead of by enhancer trapping, showed that the GAL4 transcription factor was unable to access its target UAS sites inserted in a given regulatory domain in those parasegments where the domain was predicted to be inactive. Based on this observation, the authors proposed that the inactivation of the regulatory domains actually corresponded with a reduction in chromatin accessibility (Fitzgerald and Bender 2001).
The demonstration that the activation of the regulatory domains corresponded to the switch of a repressive chromatin mark to an active mark requires the possibility of purifying nuclei specific to single parasegments, a prerequisite that became only recently
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available through the elegant work of the Bender's laboratory (Bowman et al. 2014). In this work, Bowman et al. created a series of Gal-4/Gal80 enhancer trap lines, that when combined appropriately, generated embryos in which Gal-4 is active in specific single parasegments (Figure III,A).
This technique showed that, in different parasegments, the profile of H3K27 trimethylation (a common mark characteristic of Polycomb-repressed chromatin) in the chromatin of the BX-C was different (Figure III,B). The H3K27me3 pattern was high in the domains predicted to be inactive in each parasegment. Active domains, on the other hand, presented low H3K27me3 and high H3K27 acetylation, a typical chromatin mark of active enhancers.
This established for the first time a direct correlation between the Lewis model and the increasing/decreasing of chromatin accessibility in the domains of the BX-C along the anterior-posterior axis.
Figure III. A parasegment-specific Gal-4 system for cell sorting
A. Different lines expressing combinations of Gal4+Gal80 drive expression of a reporter gene in a single parasegment, during embryonic development.
B. H3K27me3 profile of the BX-C in nuclei extracted from whole embryos (mixed) and from single parasegments between PS4 and PS7. CTCF binding (red lines) marks the location of the boundaries between domains.
Modified from (Bowman et al. 2014).
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