Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, the evolution of the genetic switch controlling dimorphic bab expression has been identified and traced. The HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSXF activate bab expression whereas in males DSXM directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits (Williams, 2008).
bab expression in the abdominal epidermis is regulated by two separate CREs, one of which directs gene expression in the anterior abdomen of both sexes, and a second, dimorphic element that regulates female-specific gene expression in segments A5-A7. The dimorphic element, when bound by ABD-B and sex-specific isoforms of the DSX protein, acts as a genetic switch that allows pigmentation in males and represses pigmentation in females. Changes in the activities of both CREs have evolved in the course of the origin of the trait from a monomorphic ancestor. Furthermore, dimorphic CRE function evolved by multiple fine-scale changes within the CRE. These results bear on understanding of how sexually dimorphic traits develop, how new sex- and segment-restricted traits arise, and how CRE functions evolve (Williams, 2008).
Sex-restricted traits are the product of differences in gene expression between sexes, therefore, understanding how such traits develop requires the identification of those genes with sex-limited expression and elucidation of the genetic and molecular mechanisms governing their regulation. This study showed that dimorphic bab expression is regulated by a discrete CRE whose activity is combinatorally regulated by the direct inputs of both region- (ABD-B) and sex-specific (DSX) transcription factors. In females, ABD-B acts in concert with the DSXF isoform through binding sites in the dimorphic element to activate bab expression in the posterior segments. Whereas in males, ABD-B activity is overridden by the repressive activity of the DSXM isoform which binds to the same sites as DSXF and hence, permits the formation of the male-specific posterior pigmentation (Williams, 2008).
The genetic pathways that regulate sex-determination and sexual differentiation differ greatly across the animal kingdom, so this mode of male-specific trait regulation in Drosophila may not apply in detail to other animals. However, the integration of region- and sex-specific regulatory inputs must be a requirement for the production of dimorphic traits. It is suggested that the integration of such combinatorial inputs by cis-regulatory elements, as demonstrated for bab, is a general feature of genetic switches within the pathways regulating the production of dimorphic traits (Williams, 2008).
The origins of sexually dimorphic traits have long been of central interest in evolutionary biology. One of the key questions that Darwin grappled with, as have many others subsequently, was whether dimorphic traits are limited to one sex at their origin, or whether these traits first appear in both sexes and then become restricted to one sex. This question has been particularly important and challenging in terms of genetics and evolutionary theory, as it has not been resolved previously how the effects of mutations could be restricted to one sex (Williams, 2008).
In the simplest genetic scenarios of sexual dimorphism, male-limited traits are the products of the male-limited expression of specific genes. The main evolutionary question then, as it has been phrased in classical genetic terms, is whether male-limited gene expression evolves via: (1) 'alleles' that are expressed only in males; or (2) alleles expressed in both sexes which are then suppressed in females or promoted in males. The elucidation of the regulation and evolution of male-specific pigmentation provides a unique opportunity to reconstruct the genetic path of the evolution of a dimorphic trait (Williams, 2008).
Although posterior male-specific pigmentation is a relatively simple, two-dimensional morphological trait, it is clear that it did not originate via just one of the alternative genetic paths above. Rather, the evolution of this trait has involved three paths: the evolution of male-limited gene expression, of female-limited gene expression, and of non-sex-restricted gene expression. Specifically, this study shows that in the course of the evolution from a monomorphically pigmented ancestor, the activity of the female-specific bab dimorphic CRE expanded into segments A6 and A5 and that the activity of the monomorphic bab anterior CRE retreated from segments A6 and A5 of both sexes. These two combined changes produced the sex-specific repression of bab expression in male segments A5 and A6. In addition, in previous work it was shown that the yellow pigmentation gene gained high-level expression in segments A5 and A6 via the acquisition of ABD-B binding sites in a specific yellow gene CRE, whose activity was male-limited due to repression by Bab (which is apparently indirect) (Williams, 2008).
It is important to underscore that none of the genes in this newly-evolved regulatory circuit are globally restricted in their expression to one sex. Rather, the sex-specific features of their expression are controlled by modular CREs that are physically separate from those controlling gene expression in other developing body regions. The properties of these CREs resolve the question of how the effects of mutations can be restricted to one sex. Namely, mutations in a CRE that is under the direct (the female-specific bab dimorphic element) or indirect (the male-specific yellow CRE) control of an effector of sex determination will have sex-limited effects on gene expression. The findings here are a further demonstration of the general principle of how the modular CREs of pleiotropic genes enable the modification of gene expression in and morphology of one body part independent of other body parts, or in this case, the same body part in the opposite sex (Williams, 2008).
It is also notable that none of the CREs analyzed are new to the dimorphically pigmented melanogaster species group. It is clear, then, that the ancestral dimorphic CRE was active in segment A7 and modified to govern sexually dimorphic pigmentation in segments A6 and A5. Thus, in this example, one path is seen to evolving a new dimorphic trait is via the co-option of genetic components that regulate other pre-existing dimorphic traits (Williams, 2008).
One of the major questions concerning the evolution of gene expression is how new gene expression patterns arise. The two most obvious mechanisms would appear to be the gain of new regulatory elements or the gain of new transcription factor-CRE linkages. While the deep ancestry of the dimorphic element ruled out the former, it was expected that the novel sex- and segment-specific regulation of this CRE by DSX and ABD-B in the D. mel. lineage would require the gain of binding sites for these two transcription factors. However, it was found that the both DSX binding sites and most ABD-B sites were present in D. wil. and other monomorphic species and therefore were present in the last common ancestor of both monomorphic and dimorphic species. Thus, the expansion of the dimorphic CRE activity was not due to the wholesale gain of new DSX and ABD-B binding sites (Williams, 2008).
Rather, it was discovered that the expanded, high level activity of the D. mel. dimorphic CRE in segments A6 and A5, relative to the A7-restricted activity of the D. wil. element, was due to an amalgam of changes involving the number, polarity, and topology of transcription factor binding sites. The evolution of dimorphic CRE activity demonstrates how changes beyond the simple gain or loss of binding sites shape CRE evolution. Similarly, changes in the topology and helical phasing of transcription factor binding sites have shaped the evolution of a genetic switch controlling galactose utilization in yeast (Hittinger, 2007). These studies strongly support the view that the relationship between function and sequence variation in CREs is complex. A vast body of work on eukaryotic and prokaryotic transcriptional regulation has shown that binding site polarity and spacing influences the output of regulatory elements. Therefore, it is suggested that one important, but generally unappreciated, class of functionally relevant mutations in CRE and trait evolution involves sequences outside of transcription factor binding sites. CREs thus present a very large target area for potential functionally relevant mutations that quantitatively modulate gene expression and trait development (Williams, 2008).
Finally, these observations concerning the mechanisms underlying the expansion of dimorphic CRE activity help to shed light on another general aspect of the evolution of animal body plans -- the evolution of segmental traits. A large number of studies have demonstrated that some of the major differences among arthropod and vertebrate body plans have involved evolutionary shifts in the spatial boundaries of gene expression along the main body axis. However, the path by which such gene expression patterns are shifted has not been elucidated in any molecular detail. It is submitted here that the expansion of the activity of the dimorphic element from the A7 segment into A6 and A5 is a model of this process. The remodeling of the dimorphic CRE in the course of evolution illustrates that one way such shifts can be accomplished is through numerous small, quantitative incremental changes in the activity of Hox-regulated CREs (Williams, 2008).
The transient early expression of spineless in the leg suggests that spineless plays a role in the establishment of the tarsal region. Support for such a role is provided by the finding that bric a brac (bab) lies downstream of spineless. In wild type, bab expression is initiated in the tarsal region in the mid-third instar; at disc eversion, bab expression can be seen to extend from the middle of the first tarsal segment through the fifth segment. In spineless null mutants, bab expression is abolished in the leg (Duncan, 1998).
Distal-less protein can be detected in a central domain in leg discs throughout most of larval development; in mature discs this domain corresponds to the distal-most regions of the leg: the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. Dll3 is the strongest hypomorph in which all of the tarsus is deleted and the tibia and femur are reduced in size. The expression of two genes required for the patterning of the tarsus, al and bric a brac (bab) was examined in Dll3 leg discs. In wild-type discs, al is expressed in the center of the disc and bab in the rest of the presumptive tarsus. In Dll3 leg discs no al or bab expression can be detected in the center of the discs (Campbell, 1998).
Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).
A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).
Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).
The BTB/POZ domain is an evolutionarily conserved protein-protein interaction domain present in the N-terminal region of numerous transcription factors involved in development, chromatin remodeling, and human cancers. This domain is involved in homomeric and heteromeric associations with other BTB/POZ domains. The Drosophila BTB/POZ proteins Bric à brac 1 (Bab1) and Bric à brac 2 (Bab2) are developmentally regulated transcription factors which are involved in pattern formation along the proximo-distal axis of the leg and antenna, in the morphogenesis of the adult ovaries, and in the control of sexually dimorphic characters. Partners of the Bab1 protein have been identified by using the two-hybrid system. The characterization of one of these proteins, called BIP2 for BAB interacting protein 2, is presented. BIP2 is a novel Drosophila TATA-box protein associated factor (TAFII), also named dTAFII155. The BTB/POZ domains of BAB1 and BAB2 are sufficient to mediate a direct interaction with BIP2/dTAFII155. This provides a direct link between these BTB/POZ transcription factors and the basal transcriptional machinery (Pointud, 2001).
Many of the BTB/POZ transcription factors have been shown to be transcriptional repressors. Some of them, but not all, mediate their repressive activity, via their BTB/POZ domain, by recruiting histone deacetylase complexes to promoters of their respective target genes, thereby inducing a repressive chromatin state. Other BTB/POZ proteins function as transcriptional activators. The BTB/POZ domains of BAB1 and BAB2 are sufficient to interact with a potential transcriptional co-activator, the TAFII factor BIP2/ dTAFII155. As BIP2/dTAFII155 is a component of TFIID (Gangloff, 2001), this interaction provides a direct link between the BTB/POZ transcription factors and the basal transcriptional machinery and suggests a new mechanism for the transcriptional regulation by BTB/POZ proteins. Interestingly, interactions between BTB/POZ domains and TAF proteins may mediate either a transcriptional activation or repression. Classical transcriptional activators act through a direct interaction between their activation domain and the co-activators of the basal transcriptional machinery. A number of TAFs have been identified as direct transcriptional targets of activators in vitro (Pointud, 2001).
One interpretation of the interaction between a given BTB/POZ domain and a TAFII is that this BTB/POZ protein is an activator that directly interacts with TFIID or other TBP-free TAFII-containing complexes to activate the transcription of its target genes. An alternative possibility is that the interaction between a BTB/POZ domain and a TAFII mediates repression or anti-activation by interfering with the basal transcriptional machinery. It has been shown that the co-repressor N-CoR can directly interact with TFIIB, hTAFII32, and hTAFII70 to mediate signals from repressors to the basal machinery. BIP2/dTAFII155 and its homolog hTAFII140 contain a PHD-finger at their respective C-termini. While the function of the PHD-finger is unknown, it has been suggested that it is involved in protein-protein interactions. This domain is present in proteins associated with a repressor activity, like histone deacetylases, and in interactions with heterochromatin. The BIP2/dTAFII155 and hTAFII140 proteins could mediate, through their PHD fingers, interactions between BTB/POZ proteins, and repressor activities. BTB/ POZ domains could mediate repression of transcription by recruiting histone deacetylase complexes to generate a close conformation of the chromatin, and/or by direct contacts with members of the basal transcriptional machinery, locking them into a nonfunctional complex or conformation (Pointud, 2001).
Very few non-BTB/POZ proteins have been identified as partners of the BTB/POZ domains of Drosophila proteins. The interaction of the Tramtrack BTB/POZ domain with the corepressor dCtBP (C-terminal binding protein) and the BTB/POZ domain of GAGA with the heterologous proteins N-CoR and SMRT in in vitro experiments have been briefly reported. Since the BTB/POZ domains of the Drosophila nuclear proteins are strongly conserved in evolution, it is probable that other Drosophila BTB/POZ proteins interact directly, via their BTB/POZ domain, with one of the central components of the preinitiation complex, like BIP2/dTAFII155, to regulate the transcription of their target genes. Since a given TAF can interact with more than one activator and a single activator can also contact multiple TAFs, it is suggested that BIP2/dTAFII155 could interact with other Drosophila BTB/POZ proteins and/or that different BTB/POZ domains can contact different TAFs. The BTB/POZ transcriptional repressor HIC-1 and its avian homolog gammaFBP-B do not seem to mediate their repressive effect by recruiting histone deacetylase complexes, since they fail to interact with the co-repressors SMRT, N-CoR, and mSIN3A and as their transcriptional repression is unaffected by Histone Deacetylase (HDAC) inhibitors. The repressive function of HIC-1 and gammaFBP-B could be mediated by a direct interaction between their BTB/POZ domains and components of the basal transcriptional machinery such as the vertebrate homolog of BIP2/dTAFII155 (Pointud, 2001).
Although the BTB/POZ domain of BAB1 is sufficient for the interaction with BIP2/dTAFII155, in vitro experiments show that the regions of BAB1 adjacent to the BTB/POZ domain facilitate this interaction. This observation could reflect different folding properties of the different BAB1 fusion proteins or could imply a direct involvement of the BTB/POZ adjacent regions to stabilize the interaction with BIP2/dTAFII155. This could indicate that the regions adjacent to the BTB/POZ domain could give another level of specificity to the BTB/POZ proteins for an interaction with a given protein like a TAF (Pointud, 2001).
The crystal structure of the BTB/POZ domain of promyelocytic leukaemia zinc finger (PLZF) revealed a tightly intertwined dimer with a surface-exposed groove. This pocket is composed of some of the most conserved residues of the BTB/POZ sequence and has a high charge density. The minimal domain of the BIP2/dTAFII155 protein required for the interaction with the BTB/POZ domains of the BAB proteins corresponds to a highly charged region comprising multiple lysine, glutamic acid and aspartic acid residues (aa 859-1091). This charged region of BIP2/dTAFII155 is well conserved in its homologs hTAFII140 and mTAFII140 (Gangloff, 2001). The charged-exposed groove of the BTB/POZ dimer may bind this charged region of BIP2/ dTAFII155 or hTAFII140 through electrostatic interactions (Pointud, 2001).
The BAB proteins may regulate the expression of several genes at different steps of Drosophila development. The BAB2 protein binds to several sites on polytene chromosomes. During leg morphogenesis, bab is required for segmentation and for the specification of segment identity in the tarsus. bab is also required for adult ovary morphogenesis. Kopp (2000) has shown that the BAB proteins are involved in the genetic control of the sexually dimorphic abdominal pigmentation and the abdominal A5-A7 segment morphology in Drosophila melanogaster. Since BIP2/dTAFII155 is expressed in all the tissues that express and require BAB, it is possible that BIP2/dTAFII155 mediates all the BAB functions. The BAB proteins could be transcriptional activators as well as transcriptional repressors, depending on the promoter considered and it will be of great interest to identify BAB target genes and cis-regulatory sequences recognized by the BAB proteins. However, the BAB proteins, unlike most of the other BTB/POZ proteins, do not contain a zinc finger DNA binding domain but a new type of DNA binding domain that is currently being characterizing. Clearly, the identification of bab downstream genes is crucial to understand whether the interaction between BIP2/dTAFII155 and the BTB/POZ domain of the BAB proteins is critical for BTB/ POZ-mediated transcriptional activation or repression (Pointud, 2001).
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