Ultrabithorax

How do the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro? Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax and abdominal-A. There is a selective interaction of the Extradenticle with certain UBX and ABD-A proteins. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms (Johnson, 1995).

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution was determined, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax, located amino-terminally to its homeodomain, which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals (Passner, 1999).

The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide identified in the 5' region of the mouse Hoxb-1 (the mammalian homolog of Drosophila labial) gene that is sufficient to direct a labial-like autoregulated expression pattern in Drosophila embryos. Labial and Extradenticle, their binding sites separated by only 4 bp, bind DNA as a heterodimer in a head-to-tail orientation. Mutations in base pairs predicted to contact the HOX N-terminal arm results in a change in HOX preference in the heterodimer, from Labial to Ultrabithorax. These results demonstrate that Extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site (Chan, 1996).

The homeobox gene extradenticle (exd) acts as a cofactor of Hox function in both Drosophila and vertebrates. It has been shown that the distribution of the Exd protein is developmentally regulated at the post-translational level; in the regions where exd is not functional, Exd is present only in the cell cytoplasm, whereas it accumulates in the nuclei of cells requiring exd function. Maternal EXD mRNA lasts for a few hours and is undetectable by stage 9 of embryogenesis. Protein produced by maternal RNA is stable. Both maternally and zygotically derived protein translocates into nuclei, suggesting that the proteins translocated from both mRNAs are functional and also suggesting that regulation of protein distribution is not dependent on transcriptional control (Azpiazu, 1998).

The subcellular localization of Exd is regulated by the BX-C genes; to varying degrees, BX-C genes can prevent or reduce nuclear translocation of Exd. Embryos homozygous for the deficiency Df(3R)P9, lacking the entire Bithorax complex, contain Exd at high levels in the nuclei of epidermal cells of both thoracic and abdominal segments. Only in the Keilin's organs of each segment does Exd remain cytoplasmic. This rise in the level of nuclear Exd in the abdominal segments (in comparison with the wild-type distribution) already indicates an involvement of the BX-C genes with the subcellular distribution of the product. In contrast, the thorax-determining genes Sex combs reduced (Scr) and Antp do not appear to affect Exd localization, as Scr Antp homozygous embryos exhibit normal distribution of Exd. To discriminate the roles of individual BX-C genes in the nuclear translocation of Exd, the distribution of Exd protein was examined in two more BX-C mutant combinations: Ubx- abd-A+ Abd-B+ and Ubx- abd-A- Abd-B+. Embryos homozygous for the first combination, defective only for Ubx function, show an increased level of nuclear Exd in the first abdominal segment as compared to wild type. In more posterior abdominal segments the levels and distributions of Exd are normal. In the second combination, lacking Ubx and abd-A functions, Exd is detected at high levels in the nuclei of the abdominal segments A1 to A4. In Df(3R)P9 (Ubx abd-A Abd-B) embryos, Exd nuclear localization extends to A8: all these results indicate that each BX-C gene is capable of preventing or reducing the nuclear translocation of Exd (Azpiazu, 1998).

The inhibition of Exd nuclear transport by overexpression of BX-C genes causes exd-like phenotypes. This was shown by inducing ectopic Ubx expression with several Gal4 lines during embryonic development and examining the segmental transformations produced. In the presence of normal exd function, Ubx specifies the pattern of the first abdominal (Al) segment. In contrast, in embryos lacking exd function, Ubx specifies a pattern resembling a more posterior segment, of A3-A5 type. Under conditions in which levels of Gal4 activity are high, larvae develop all segments anterior to A2 with an A3-A5 pattern. This segment pattern closely resembles that found in the A1 segment of zygotic exd larvae and is the same overall pattern observed after heat shock-inducing Ubx expression in zygotic exd embryos. These observations indicate that high levels of Ubx protein are able to produce an exd-like phenotype, in good agreement with the observed negative effect of BX-C genes on the nuclear translocation of Exd (Azpiazu, 1998).

The Ultrabithorax and Antennapedia genes of Drosophila encode homeodomain proteins that have very similar DNA binding specificities in vitro but specify the development of different segmental patterns in vivo. Cooperative interactions occur between UBX protein and Extradenticle, that selectively increase the affinity of UBX for a particular DNA target. UBX and EXD bind to neighboring sites on this DNA and interact directly to stabilize the DNA-bound form of UBX (Chan, S. K., 1994).

Specific amino acid residues at the amino end of the Ultrabithorax homeodomain are required to specifically regulate Antennapedia transcription; and in the context of a Deformed protein, these amino-end residues are sufficient to switch from Deformed- to Ultrabithorax-like targeting specificity. Although residues in the amino end of the homeodomain are also important in determining a Deformed-like targeting specificity, other regions of the Deformed homeodomain are also required for full activity (Lin, 1992).

DFD and UBX bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. However, there are striking differences between the two homeodomains in their specific interactions with DNA. Sequence differences within the selected binding sites have differential effects on protein binding, depending on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an AT base-pair from TA in the minor groove (Draganescu, 1995).

A comparison was made among the DNA sequence preferences of homeodomains encoded by four Drosophila HOM proteins. One of the four, ABD-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the UBX and ANTP homeodomains display indistinguishable preferences outside the core while DFD differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity (Ekker, 1994).

The Ultrabithorax, abdominal-A, and Antennapedia homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress P1, whereas ANTP activates it. Homeoproteins can use the same regulatory element but in very different ways. Chimeric UBX-ANTP proteins and UBX deletion derivatives demonstrate that functional specificity in P1 regulation is dictated mainly by sequences outside the homeodomain, with important determinants in the N-terminal region of the proteins (Saffman, 1994).

Naturally occurring binding sites for UBX contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more UBX molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation. Cooperative binding is dependent on sequences outside the homeodomain (Beachy, 1993).

Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of the capitellum, a structure on the third thoracic segment, into the wing, the analogous structure on the second thoracic segment. This mutant phenotype is also caused by genetically reducing the cellular concentration of UBX. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of UBX protein is reduced by mutations in Ubx (Mortin, 1992).

Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. The Ubx-HD binding to a single optimal target site displays significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA is unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains show that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH is greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo (Li, 1996).

The presence of the LIM domain of mammalian Isl-1 (Drosophila homolog: Islet) inhibits binding of the homeodomain to its DNA target. This in vitro inhibition can be released either by denaturation/renaturation of the protein or by truncation of the LIM domains. A similar inhibition is observed in vivo using reporter constructs. LIM domains in a chimeric protein can inhibit binding of the Ultrabithorax homeodomain to its target. The ability of LIM domains to inhibit DNA binding by the homeodomain provides a possible basis for negative regulation of LIM-homeodomain proteins in vivo (Sanchez-Garcia, 1993).

Hox transcription factor Ultrabithorax Ib physically and genetically interacts with disconnected interacting protein 1, a double-stranded RNA-binding protein

Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx). The Ubx.DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated (Bondos, 2004).

A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax

Hox transcription factors are essential for shaping body morphology in development and evolution. The control of Hox protein activity in part arises from interaction with the PBC class of partners, pre-B cell transcription factor (Pbx) proteins in vertebrates and Extradenticle (Exd) in Drosophila. Characterized interactions occur through a single mode, involving a short hexapeptide motif in the Hox protein. This apparent uniqueness in Hox-PBC interaction provides little mechanistic insight in how the same cofactors endow Hox proteins with specific and diverse activities. This study identified in the Drosophila Ultrabithorax (Ubx) protein a short motif responsible for an alternative mode of Exd recruitment. Together with previous reports, this finding highlights that the Hox protein Ubx has multiple ways to interact with the Exd cofactor and suggests that flexibility in Hox-PBC contacts contributes to specify and diversify Hox protein function (Merabet, 2007).

The current view of Hox-PBC interactions is that they all occur through a single mode, involving a short hexapeptide (HX) motif. The importance of the Hox HX motif in mediating interaction with PBC proteins is extensively supported by its requirement in in vitro interaction assays and by crystallographic studies that showed that the HX provides most if not all major contacts. In contrast, in vivo functional support for a role of the HX in mediating interaction with PBC proteins is still limited, mainly because effects of HX mutations during development have only been assessed for two vertebrate Hox proteins, Hoxa-1 and Hoxb-8, and for three Drosophila proteins, Labial (Lab), Ubx, and Abdominal-A (AbdA) (Merabet, 2007 and references therein).

Mutation of the HX in Hoxb-8 results in dominant phenotypes, which are at present difficult to interpret with regard to Pbx recruitment. Hoxa-1 HX mutation mimics Hoxa-1 loss of function, including defects in the hindbrain that could relate to loss of Pbx recruitment because inactivation of Pbx2 and Pbx4 in the zebrafish affects hindbrain patterning. However, addressing in vertebrates whether phenotypes resulting from HX mutations are consequences of defects in Pbx interaction will require examination of combinations of Hox-1 paralogous and Pbx gene mutations. In Drosophila, mutation of the HX in Lab, the only representative of Hox-1 class genes, results in an hyperactive protein when assayed for its potential to activate transcription through an evolutionarily conserved Hoxb-1 autoregulatory element. This hyperactivity results from the loss of an inhibitory action of the HX on Lab DNA binding. In this context, it was proposed that HX-mediated recruitment of Extradenticle (Exd) acts to mask the DNA-binding inhibitory activity of the HX motif (Merabet, 2007 and references therein).

Although the Hoxa-1 and Lab studies support, yet not exclusively, a role of the HX in mediating recruitment of PBC class proteins during development, work on Drosophila Ubx and AbdA has provided evidences for HX-independent mode of Exd recruitment. Regarding Ubx, a truncated protein lacking N-terminal sequences (including the HX) was shown in vitro to retain Exd recruitment potential and to interact weakly with Exd in yeast two-hybrid assays. More specifically, mutation of the HX does not affect the capacity to recruit Exd on a Hox/Exd consensus target sequence in vitro and to repress in an Exd-dependent manner the limb-promoting gene Distalless (Dll), which has served as a paradigm to study Hox-Exd interactions. Concerning AbdA, the HX-deficient protein was shown to recruit Exd on the Dll regulatory element that mediates Dll repression, consistent with its retained ability to repress Dll (Merabet, 2007).

Thus, Hox-PBC interactions are not limited to HX-mediated interactions, highlighting that another Hox protein motif, yet to be identified, may also assume this function. The Ubx C terminus [sequences downstream of the homeodomain (HD), UC], important for Ubx segment identity functions and shown to increase the ability of the Ubx HD to associate with Exd in yeast two-hybrid assays, harbors an 8-aa peptide previously termed UbdA as well as a QA repression domain responsible for changes in Ubx activity. Although evolutionarily conserved, the precise function of the UbdA motif, only present in Ubx and AbdA proteins but absent from any other Drosophila Hox protein, is not known. This work reports on the function of the UbdA motif in the context of the Ubx protein. Because this motif is specific to Ubx and AbdA, which share the HX-independent mode of Exd recruitment, the analysis focused on the possible implication of this motif in mediating Exd recruitment (Merabet, 2007).

The results strongly support that the UbdA motif mediates Exd recruitment by the Ubx protein. This finding is first established by the requirement of the motif for Exd recruitment in the process of Dll regulation: mutation of the motif impairs the capacity of Ubx to mediate interaction with Exd on Dll regulatory sequences in vitro, which correlates with the reduced ability of UbxUbdA to perform Exd-dependent repression of Dll in vivo. Evidence is provided that mutation of the UbdA motif does not result in a globally defective protein: the UbdA mutated protein still binds DNA with appropriate affinities as a monomer, still represses the wing promoting genes dSRF, sal, and vg and still activates the dpp target gene in the visceral mesoderm. Thus, mutating the UbdA motif selectively affects a subset of Ubx functions. Importantly, the conclusion that the UbdA motif mediates Exd recruitment is also supported by the demonstration that the motif provides de novo Exd recruitment potential to a Hox protein that has been rendered deficient for this function. This finding is shown both in vitro by the potential of the motif to confer Exd recruitment to Antp on a Hox/Exd consensus sequence and on a cis-regulatory sequence of the Antp/Exd target gene tsh, and in vivo by its potential to restore Exd-dependent activation of tsh. Complexity in Hox-Exd Interactions (Merabet, 2007).

The Ubx protein provides a so far unique situation wherein two identified protein motifs within the same Hox protein have the potential to perform the recruitment of the Exd cofactor, which raises the question of whether these two motifs are effectively used for Exd recruitment by Ubx. Previous work has shown that an HX-deficient Ubx protein was altered in its segment identity specification: whereas Ubx specifies A1 segment identity, the mutated form specifies A2-like identity. Interestingly, in a context deficient for zygotic Exd contribution, Ubx also specifies A2-like identity, suggesting that the HX motif is required for Exd-dependent A1 specification. These observations support that within this context, the HX is the motif used to perform Exd recruitment, although definitive support awaits characterization of Ubx-Exd interaction on a Ubx downstream target gene involved in segment identity specification. Considering the finding that the UbdA motif mediates Exd recruitment in the process of Dll regulation, it is proposed that depending on the developmental context, i.e., on the target gene regulated, Ubx uses different protein motifs for Exd recruitment. The contextual (gene-specific) use of the HX and UbdA protein motifs introduce a first level of complexity in Ubx-Exd interactions (Merabet, 2007).

A second level of complexity in the Ubx-Exd relationship is illustrated by the regulation of the dpp target gene. In this case, it was found that neither the HX nor the UbdA motif was required for Exd-dependent activation by Ubx. The possibility that these two motifs were acting in a redundant way was excluded by the observation that a Ubx protein mutant for both motifs still activates dpp. Thus, other protein motifs, yet to be identified, could confer an additional mode of Exd interaction, further increasing the diversity by which Ubx could contact the Exd cofactor. Alternatively, the dispensability of the HX and UbdA motifs for dpp activation may also suggest that Ubx/Exd contacts are not required. The latter hypothesis is supported by the existence in dpp regulatory regions of Exd-binding sites that are not closely associated to Hox-binding sequences and by the previous observation that Exd can improve Ubx monomer binding to dpp regulatory sequences in a manner that does not require the formation of a Ubx-Exd-DNA tripartite complex. In any case, the regulation of dpp suggests further complexity in the Ubx-Exd relationship, which, by extension, highlights that the functional interplay of Hox-PBC proteins is likely to be more diverse than the current view (Merabet, 2007).

Although previous studies showed that HX-deficient Hox proteins retain the capacity to interact with Exd and to mediate Exd-dependent functions, motifs responsible for alternative modes of interaction were not identified. This work identifies a so far unique HX-alternative mode of PBC recruitment, introducing the notion of flexibility in Hox-PBC contacts. This interaction mode was not anticipated from previous crystallographic studies because the truncated Ubx protein used was lacking the UbdA motif. Given the divergence of the primary sequences of the HX and UbdA motifs, their distinct location in the protein, and the absence of functional redundancy, the UbdA- and HX-mediated interaction modes are likely to be structurally distinct (Merabet, 2007).

These findings have also implications with regard to Hox protein diversity and specificity. Flexibility in Hox-PBC interactions allows addressing of the issue of diversity from a mechanistic point of view: depending on the motif involved in the interaction, which likely relies on the target sequence, the Hox-PBC complex may adopt different conformations, which in turn set structural bases for distinct activities. This process therefore provides cues to explain how diversity can be generated through qualitatively distinct interaction modes involving the same protein partners. Furthermore, because the UbdA motif is only found in Ubx and AbdA, it likely endows these two proteins with a specific Exd interaction mode. This mode may serve to distinguish Ubx and AbdA from other Drosophila Hox proteins, therefore providing basis for Hox protein specificity. Finally, this study questions whether additional HX-independent modes of PBC interaction exist. It was reported previously that the HX-deficient Lab protein retains Exd interaction potential and in vivo Exd-dependent activity. As Lab does not bear a UbdA motif, it supports further flexibility in Hox-PBC interaction. Addressing the issue of diversity in Hox-PBC interaction thus appears as a necessary step to understand the mechanisms underlying Hox protein activity in development and evolution (Merabet, 2007).

Non-requirement of a regulatory subunit of Protein Phosphatase 2A, PP2A-B', for activation of Sex comb reduced activity in Drosophila melanogaster

The Drosophila HOX transcription factor, Sex combs reduced (SCR), is required for determining labial and the first thoracic segmental identity. A Protein Phosphatase 2A holoenzyme assembled with the PP2A-B′ regulatory subunit is proposed to specifically interact with, and dephosphorylate, the SCR homeodomain activating SCR protein activity. To test this hypothesis further, a null mutation was created in the PP2A-B′ gene, PP2A-B′^Δ, using Flip-mediated, site-specific recombination. The number of sex comb bristles, salivary gland nuclei and pseudotracheal rows are SCR-dependent and were counted as a measure of SCR activity in vivo. Adults and larvae homozygous for PP2A-B′^Δ showed no decrease in SCR activity. In addition, no evidence of functional redundancy of PP2A-B′ with other regulatory subunits, Twins (TWS) and Widerborst (WDB), for dephosphorylation and activation of SCR activity was observed. In conclusion, a PP2A holoenzyme containing the PP2A-B′ regulatory subunit has no role in the dephosphorylation and activation of SCR, and analysis of functional redundancy of PP2A regulatory subunits uncovered no evidence supporting a role of PP2A activity in dephosphorylation and activation of SCR (Moazzen, 2009).

Although the gene that encodes PP2A-B' is dispensable for viability, PP2A-B' is functional. The analysis of functional redundancy between PP2A-B' and TWS/WDB showed that removal of PP2A-B' in a genetic background deficient for one or both of the tws and wdb loci significantly increased the number of sex comb bristles. This suggests that the PP2A holoenzyme containing either TWS, the B regulatory subunit, or WDB, a B' regulatory subunit, may functionally substitute for the loss of PP2A-B'. Although PP2A-B' is dispensable for development, it may have an essential and specific role in biological processes not assayed in this study like the immune response, mating behaviour or circadian rhythm (Moazzen, 2009).

Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. This study investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. The Med19 subunit was found to directly bind Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, a role was identified for Med19 in Hox-dependent target gene activation. A conserved, animal-specific motif was found that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded 'co-factor' that acts together with Hox proteins through their homeodomains in regulated developmental transcription (Boube, 2014).

The finely regulated gene transcription permitting development of pluricellular organisms involves the action of transcription factors (TFs) that bind DNA targets and convey this information to RNA polymerase II (PolII). Hox TFs, discovered through iconic mutations of the Drosophila melanogaster Bithorax and Antennapedia Complexes, play a central role in the development of a wide spectrum of animal species. Hox proteins orchestrate the differentiation of morphologically distinct segments by regulating PolII-dependent transcription of complex batteries of downstream target genes whose composition and nature are now emerging. The conserved 60 amino acid (a.a.) homeodomain (HD), a motif used for direct binding to DNA target sequences, is central to this activity. Animal orthologs of the Drosophila proteins make use of their homeodomains to play widespread and crucial roles in differentiation programs yielding the very different forms of sea urchins, worms, flies or humans. They do so by binding simple TAAT-based sequences within regulatory DNA of developmental target genes. One crucial aspect of understanding how Hox proteins transform their versatile but low-specificity DNA binding into an exquisite functional specificity involves the identification of functional partners. Known examples include the TALE HD proteins encoded by extradenticle (exd)/Pbx and homothorax (hth)/Meis, which assist Hox proteins to form stable ternary DNA-protein complexes with much-enhanced specificity. This involves contacts with the conserved Hox Hexapeptide (HX) motif near the HD N-terminus, or alternatively, with the paralog-specific UBD-A motif detected in Ubx and Abdominal-A (Abd-A) proteins. Other TFs that can serve as positional Hox partners include the segment-polarity gene products Engrailed (En) and Sloppy paired, that collaborate with Ubx and Abd-A to repress abdominal expression of Distal-less. Finally, specific a.a. residues in the HX motif, the HD and the linker separating them play a distinctive role in DNA target specificity, allowing one Hox HD region to select paralog-specific targets (Boube, 2014).

Contrasting with knowledge of collaborations involving Hox and partner TFs, virtually nothing is known of what transpires at the interface with the RNA Polymerase II (PolII) machinery itself to generate an appropriate transcriptional response. The lone evidence directly linking Hox TFs to the PolII machine comes from the observation that the Drosophila TFIID component BIP2 binds the Antp HX motif (Boube, 2014).

Another key component of the PolII machinery is the Mediator (MED) complex conserved from amoebae to man that serves as an interface between DNA-bound TFs and PolII. MED possesses a conserved, modular architecture characterized by the presence of head, middle, tail and optional CDK8 modules. Some of the 30 subunits composing MED appear to play a general structural role in the complex while others interact with DNA-bound TFs bridging them to PolII. Together, these subunits and the MED modules they form associate with PolII, TFs and chromatin to regulate PolII-dependent transcription (Boube, 2014).

The analysis of a Drosophila skuld/Med13 mutation isolated by dose-sensitive genetic interactions with homeotic proboscipedia (pb) and Sex combs reduced (Scr) genes led to a view that MED is a Hox co-factor. However, how MED might act with Hox TFs in developmental processes has not been explored. This work pursues the hypothesis that Hox TFs modulate PolII activity through direct binding to one or more MED subunits. Starting from molecular assays, Med19 was identified as a subunit that binds to the homeodomain of representative Hox proteins through an animal-specific motif. Loss-of-function (lof) Med19 mutations isolated in this work reveal that Med19 affects Hox developmental activity and target gene regulation. Taken together, these results provide the first molecular link between Hox TFs and the general transcription machinery, showing how Med19 can act as an embedded functional partner, or 'co-factor', that directly links DNA-bound Hox homeoproteins to the PolII machinery (Boube, 2014).

Hox homeodomain proteins are well-known for their roles in the control of transcription during development. Further, much is known about the composition and action of the PolII transcription machine. However, virtually nothing is known of how the information of DNA-bound Hox factors is conveyed to PolII in gene transcription. The Drosophila Ultrabithorax-like mutant affecting the large subunit of RNA PolII provokes phenotypes reminiscent of Ubx mutants, but the molecular basis of this remains unknown. The lone direct evidence linking Hox TFs to the PolII machine is binding of the Antp HX motif to the TFIID component BIP2. This study undertook to identify physical and functional links between Drosophila Hox developmental TFs and the MED transcription complex. The results unveil a novel aspect of the evolutionary Hox gene success story, extending the large repertory of proteins able to interact with the HD to include the Drosophila MED subunit Med19. HD binding to Med19 via the conserved HIM suggests this subunit is an ancient Hox collaborator. Accordingly, loss-of-function mutants reveal that Med19 contributes to normal Hox developmental function and does so at least in part via its HIM element. Thus this analysis reveals a previously unsuspected importance for Med19 in Hox-affiliated developmental functions (Boube, 2014).

A fundamental property of the modular MED complex is its great flexibility that allows it to wrap around PolII and to change form substantially in response to contact with specific TFs. Recent work in the yeast S. cerevisiae places Med19 at the interfaces of the head, middle and CDK8 kinase modules. Med19 is thus well-positioned to play a pivotal regulatory role in governing MED conformation (see Model for the role of Med19 at the interface of Hox and MED). The results raise the intriguing possibility that MED structural regulation and physical contacts with DNA-bound TFs can pass through the same subunit. In agreement with this idea, recent work identified direct binding between mouse Med19 (and Med26) and RE1 Silencing Transcription Factor (REST). This binding involves a 460 a.a. region of REST encompassing its DNA-binding Zn fingers. The present work goes further, in identifying a direct link between the conserved Hox homeodomain and Med19 HIM that is the first instance for a direct, functionally relevant contact of MED with a DNA-binding motif rather than an activation domain (Boube, 2014).

Med19 contributes to developmental processes with Antp (spiracle eversion), Dfd (Mx palp), and Ubx (haltere differentiation). Other phenotypes identified indicate further, non-Hox related roles for Med19. As shown in this study, complete loss of Med19 function leads to cell lethality that can be conditionally alleviated when surrounded by weakened, Minute mutation-bearing cells. These observations, that uncouple HIM-dependent functions from the role of Med19 in cell survival/proliferation, are compatible with reports correlating over-expression of human Med19/Lung Cancer Metastasis-Related Protein 1 (LCMR1) in lung cancer cells with clinical outcome. Further, RNAi-mediated knock-down of Med19 in cultured human tumor cells can reduce proliferation, and tumorigenicity when injected into nude mice. A recent whole-genome, RNAi-based screen identified Med19 as an important element of Androgen Receptor activity in prostate cancer cells where gene expression levels also correlated with clinical outcome. It will be of clear interest to examine how, and with what partners, Med19 carries out its roles in cell proliferation/survival (Boube, 2014).

The role played by mammalian Med19 and Med26 in binding the REST TF, involved in inhibiting neuronal gene expression in non-neuronal cells, provides an instance of repressive Med19 regulatory function. This study found that Med19 activity is required in the Drosophila haltere disc for transcriptional activation of CG13222/edge and bab2, but is dispensable for Ubx-mediated repression of five negatively-regulated target genes. Ubx can choose to activate or it can repress, at least in part through an identified repression domain at the C-terminus just outside its homeodomain. Conversely Med19, which binds the Ubx homeodomain, appears to have much to do with activation (Boube, 2014).

Concerning the mechanisms of Ubx-mediated repression, one illuminating example comes from analyses of regulated embryonic Distal-less expression. Ubx can associate combinatorially with Exd and Hth, plus the spatially restricted co-factors Engrailed or Sloppy-paired in repressing Distal-less . Engrailed in turn is able to recruit Groucho co-repressor, suggesting that localized repression involves DNA-bound Ubx/Exd/Hth/Engrailed, plus Engrailed-bound Groucho. Groucho has been proposed to function as a co-repressor that actively associates with regulatory proteins and organizes chromatin to block transcription. The yeast Groucho homolog Tup1 interacts with DNA-binding factors to mask their activation domains, thereby preventing recruitment of co-activators (including MED) necessary for activated transcription. The number of targets remains too small to be sure Med19 is consecrated to activation. Nonetheless, it will be of interest to determine whether Groucho can play a role in blocking MED/Ubx interactions that could provide an economical means for distinguishing gene activation from repression (Boube, 2014).

The conserved Hox proteins and the gene complexes that encode them are well-known and widely used to study development and evolution. As to the evolutionary conservation of the Mediator transcription complex, the presence of MED constituents in far-flung eukaryotic species from unicellular parasites to humans indicates that this complex existed well before the emergence of the modern animal Hox protein complexes. The DNA-binding domains are often the most conserved elements of TF primary sequence, and in the case of the Hox HD, recent forays into 'synthetic biology' agree that this was the functional heart of the ancestral proto-Hox proteins. Indeed, Scr, Antp and Ubx mini-Hox peptides containing HX, linker and HD motifs behave to a good approximation like the full-length forms, directing appropriate gene activation and repression resulting in genetic transformations. The current results showing direct HD binding to Med19 HIM, and thus access to the PolII machinery, allow the activity of these mini-Hox proteins to be rationalized. It is surmised that at the time when the Hox HD emerged to become a major developmental transcription player, its capacity to connect with MED through specific existing sequences was a prerequisite for functional success. One expected consequence of this presumed initial encounter with Med19 (a selective pressure on both partners and subsequent refinement of binding sequences) is in agreement with the well-known conservation of Hox homeodomains, and with the observed conservation of the newly-identified HIM element in Hox-containing eumetazoans. It is imagined that subsequent evolution over the several hundred million years separating flies and mammals will have allowed this initial contact to be consolidated through subsequent binding to other MED subunits, ensuring versatile but reliable interactions at the MED-TF interface (Boube, 2014).

Hox homeodomain proteins are traditionally referred to as selector or 'master' genes that determine developmental transcription programs. The low sequence specificity of Hox HD transcription factors is enhanced by their joint action with other TFs, of which prominent examples, the TALE homeodomain proteins Extradenticle/Pbx and Homothorax/Meis are considered to be Hox co-factors. However, a Hox TF in the company of Exd and Hth could still not be expected to shoulder all the regulatory tasks necessary to make a segment with all the coordinated cell-types it is made up of, and collaboration with cell-type specific TFs appears to be requisite. A useful alternative conception visualizes Hox proteins not as 'master-selectors' that act with co-factors, but as highly versatile co-factors in their own right that can act with diverse cell-specific identity factors to generate the cell types of a functional segment. A model is envisaged where a Hox protein would be central to assembling cell-specific transcription factors into TF complexes that interface with MED (Boube, 2014).

Such Hox-anchored TF complexes could make use of selective HD binding to Med19 as a beach-head for more extensive access to MED, such that loss of the Hox protein would incapacitate the complex: in the case of Ubx^- cells, inactivating bab2 or de-repressing sal. Accordingly, three observations suggest that binding of Hox-centered TF complexes involves additional MED subunits surrounding Med19: (1) bab2 target gene expression is entirely lost in Ubx-deficient cells but can persist in some Med19^- cells; (2) edge-GFP in Med19^- cells expressing Med19ΔHIM-VC was not altogether refractory to Ubx-activated edge-GFP expression; and (3) Med19ΔHIM-VC is not entirely impaired for Ubx binding, as seen in co-immunoprecipitations. Thus Hox protein input conveyed through Med19-HIM at the head-middle-Cdk8 module hinge might provide an economical contribution toward organizing TF complexes that influence overall MED conformation and hence transcriptional output. Decoding how the information-rich MED interface including Med19 accomplishes this will be an important part of understanding transcriptional specificity in evolution, development and pathology (Boube, 2014).

flexible extension of the Drosophila Ultrabithorax homeodomain defines a novel Hox/PBC interaction mode

The patterning function of Hox proteins relies on assembling protein complexes with PBC proteins (the homeodomain proteins encoded by the Drosophila extradenticle (exd) and vertebrate pbx genes), which often involves a protein motif found in most Hox proteins, the so-called Hexapeptide (HX). Hox/PBC complexes likely gained functional diversity by acquiring additional modes of interaction. This study structurally characterized the first HX alternative interaction mode of the Hox protein Ultrabithorax based on the paralogue-specific UbdA motif and further functionally validates structure-based predictions. The UbdA motif folds as a flexible extension of the homeodomain recognition helix and defines Hox/PBC contacts that occur, compared with those mediated by the HX motif, on the opposing side of the DNA double helix. This provides a new molecular facet to Hox/PBC complex assembly and suggests possible mechanisms for the diversification of Hox protein function (Foos, 2015).

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