Antennapedia

The Antennapedia and Abdominal-B homeodomains bind to TFIIEß, but the Even-skipped homeodomain does not. Using a two-hybrid assay performed in cultured cells, it can be shown that the interaction occurs in vivo. The Abdominal-B homeodomain is shown to activate transcription in vitro, and this activation can be blocked with anti-TFIIEß antibody without affecting basal transcription levels. How can an interaction between TFIIEß and the homeodomain contribute to transcriptionsal activation? At a biochemical level. TFIIE promotes the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II by TFIIH, but also inhibits a helicase activity shown by TFIIH that may be required to unwind the DNA prior to transcription initiation. Since the phosphorylated RNA polymerase II is the form of the enzyme that actively elongates transcripts, the ability of a homeodomain to attract TFIIE to the initiation complex would serve to stimulate transcription by enhancing the kinase activity of TFIIH resulting in a completely phosphorylated CTD. Alternatively, since TFIIE inhibits the helicase activity of TFIIH, it has been proposed that TFIIE might be removed from the complex following the CTD phosphorylation, and this be facilitated by interaction with a homeodomain (Zhu, 1996 and references).

The two Drosophila homeotic proteins encoded by Antennapedia and Sex combs reduced determine cell fates in the epidermis and internal tissues of the posterior head and thorax. Genes encoding chimeric ANTP/SCR proteins were introduced into flies and their effects on morphology and target gene regulation observed. The N-terminus of the homeodomain appears to be critical for determining the specific effects of these homeotic proteins in vivo, but other parts of the proteins have some influence as well. The N-terminal part of the homeodomain has been observed, in crystal structures and in NMR studies in solution, to contact the minor groove of the DNA. The different effects of Antennapedia and Sex combs reduced proteins in vivo may depend on differences in DNA binding, protein-protein interactions, or both (Zeng, 1993).

The secondary structure of an N-terminally elongated Antennapedia homeodomain (HD) polypeptide containing residues -14 to 67 (where residues 1-60 constitute the HD) has been determined by NMR in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met- (YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm (consisting of residues -14 to 6) is flexibly disordered, and the well-defined part of the HD structure (residues 7-59) is indistinguishable from that of the shorter ANTP HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter ANTP HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the ANTP protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the ANTP HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact ANTP protein (Qian, 1992).

The Ultrabithorax (UBX), abdominal-A (ABD-A), and ANTP homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress, whereas ANTP activates P1. Either of two regions of P1 can confer this pattern of differential regulation. One of the regions lies downstream and contains homeoprotein-binding sites flanking a 37-bp region called BetBS. ANTP protein activates transcription through the binding sites, whereas UBX and ABD-A both activate transcription through BetBS and use the flanking binding sites to prevent this effect. Thus, 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).

The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. The DNA sequence preferences were compared of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-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 Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed 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 Antennapedia homeodomain differs at only five amino acid positions from that of Sex combs reduced protein. In a chimeric Antp-Scr proteins expressed ectopically in Drosophila, the functional specificity of the ANTP protein is determined by the four specific amino acids located in the flexible N-terminal arm of the homeodomain. The three-dimensional structure of the ANTP homeodomain-DNA complex shows that this N-terminal arm is located in the minor groove of the DNA, suggesting that the functional specificity is determined either by slight differences in DNA binding and/or by selective interactions with other transcription factor(s) (Furukubo-Tokunaga, 1993).

One example of the role of Casein kinase II in modification of a Drosophila protein is found in the interaction of CkII with Antennapedia. The in vivo activity of this HOX protein is modified by phosphorylation due to CkII. Antp has four putative CkII target sites. Sites 1 and 2 are found in the amino-terminal portion of the protein, whereas sites 3 and 4 are clustered close to the homeodomain in the C-terminal tail. Antp with alanine substitutions at its CkII target sites produces altered thoracic and abdominal development. Ubiquitous expression of Antp in flies produces an inhibition of head involution, the elimination of dorsal head structures, a transformation of T1 into a second thoracic segment (T2), and the appearance of one to two partial T2 denticle belts in the head segment. Embryos that express Antp with altered CkII target sites (alanine replacing serine or threonine) exhibit additional phenotypes including an absence of Keilin's organs, shortened denticle belts, and a failure of germ-band retraction. Embryos that express altered Antp show a disorganized CNS with irregularly spaced or fused horizonal commissures and gaps in the longitudinal commissures. CkII sites 1 and 4 appear to be the most important in terms of the altered phenotypes produced (Jaffe, 1997).

The novel functions that result from mutationally removing CkII sites suggest that altered Antp is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of Antp that contains acidic amino acid substitutions at its CkII target sites is greatly reduced, mimicking a constitutively phosphorylated Antp protein. This hypoactive form of Antp, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. These results suggest that phosphorylation of Antp by CkII is important for preventing inappropriate activities of this homeotic protein during embryogenesis. The information provided however does not address the mechanism by which phosphorylation alters Antp's properties. Thus phosphorylation appears to modulate Antp's properties, restricting its activity to an appropriate level (Jaffe, 1997).

The YPWM motif link Antennapedia to the basal transcriptional machinery

HOX genes specify segment identity along the anteroposterior axis of the embryo. They code for transcription factors harbouring the highly conserved homeodomain and a YPWM motif, situated amino terminally to it. Despite their highly diverse functions in vivo, HOX proteins display similar biochemical properties in vitro, raising the question of how this specificity is achieved. This study investigated the importance of the Antennapedia (Antp) YPWM motif for homeotic transformations in adult Drosophila. By ectopic overexpression, the head structures of the fly can be transformed into structures of the second thoracic segment, such as antenna into second leg, head capsule into thorax (notum) and eye into wing. This study found that the YPWM motif is absolutely required for the eye-to-wing transformation. Using the yeast two-hybrid system, a novel ANTP-interacting protein, Bric-a-brac interacting protein 2 (BIP2), was identified that specifically interacts with the YPWM motif of ANTP in vitro, as well as in vivo, transforming eye to wing tissue. BIP2 is a TATA-binding protein associated factor (also known as dTAFII3) that links ANTP to the basal transcriptional machinery (Prince, 2008).

This study used a gain-of-function approach to express the homeotic selector gene Antp in combination with a constitutively active form of the Notch receptor. In this context, N prevents Antp-induced apoptosis in the eye and allows the cells to adopt a new developmental fate of the dorsal second thoracic segment, the wing. This peculiar situation allows the study of two Antp-dependent functions at the same time: the ventral antenna-to-leg and the dorsal eye-to-wing transformation. Using this approach, a differential requirement was found for the YPWM motif of ANTP: the YPWM motif of Antp is strictly required for the eye-to-wing, but less stringently required for the antenna-to-second leg transformation. A similar differential requirement of peptide motifs was also found for the YPWM motif of ABD-A (Merabet, 2003) and for the QA motif of UBX (Hittinger, 2005) (Prince, 2008).

The addition of the well-known HOX co-factor exd, that has been shown to bind via the YPWM motif, antagonizes the eye-to-wing transformation, indicating a YPWM-motif-dependent Antp function, independent of exd. The possibility cannot be excluded that exd has an Antp-independent effect by repressing wing development, as is the case for leg development, but the overexpression of exd fused to a nuclear localization signal does not interfere with endogenous wing development. It cannot be distinguish whether deleting the YPWM motif of Antp changes its DNA-binding selectivity or whether Antp loses its transactivation potential, as the direct targets of Antp genes in the eye-to-wing transformation remain to be identified. Nevertheless, the later possibility is favored, although it has been shown that mutating the YPWM motif of HOXA5 does not interfere with the transcriptional activity of the protein (Prince, 2008).

bip2 was found to be acting as an Antp co-factor for ectopic wing formation, linking Antp to an activating TFIID complex and to the basal transcriptional machinery. Previously it has been shown that HOX gene activity regulation might play in important role in HOX-dependent gene regulation. bip2 might also provide target gene specificity by linking Antp to a specific TFIID complex, which might confer specificity through promoter selectivity, as was shown for other TAF-complexes. (Prince, 2008).

In summary, these data indicate that the YPWM motif is a more generally used protein-protein interaction interface interacting with at least two, but probably more protein co-factors, judging from the numerous exd-independent HOX functions that have been found (Prince, 2008).

The YPWM-motif dependence of the Antp-specific eye-to-wing transformation implies the existence of a novel YPWM-motif-interacting protein, as the YPWM motif is considered to be a protein-interaction domain. Using the yeast two-hybrid system, this study found a new ANTP-interacting protein, encoded by the bip2 gene (Gangloff, 2001), Drosophila TBP-associated factor 3 (dTafII3/dTAFII₁55; BIP2 - FlyBase). Several lines of evidence indicate that bip2 might be a novel Antp co-factor interacting with the YPWM motif. (1) In gain-of-function experiments, bip2 behaves as an Antp co-factor promoting ectopic wing development, and the bip2 loss-of-function mutation genetically interacts with the Antp allele Antp^Ctx, reducing the frequency of eye-to-wing transformations. (2) bip2 acts as a co-factor for an Antp function requiring the YPWM motif. (3) BIP2 interacts in vitro with the YPWM motif in a yeast two-hybrid assay and shows an in vivo requirement of the YPWM motif for the ANTP-BIP2 interaction in a co-immunoprecipitation assay (Prince, 2008).

bip2 (dTAFII3) is a member of the TBP-associated TFIID complex in the basal transcriptional machinery, and belongs to the class of histone-like TATA-binding protein (TBP)-associated factors (TAF) with two homologues in yeast, humans and mice (Gangloff, 2001). The bip2 gene codes for a protein with two distinct domains, a Histone Fold Domain (HFD) at the N terminus and a PHD finger at the C terminus. The HFD is a domain initially found in histones involved in the formation of histone dimers, whereas the PHD has been recently shown to specifically interact with three-methylated histone H3 at lysine 4. BIP2 forms a histone-like dimer with TAF10 (dTAFII24) (Gangloff, 2001). This dimer formation is conserved from yeast to humans. bip2 and its homologues have been identified as members of the TBP-containing TFIID complex, linking ANTP to the basal transcriptional machinery. But, BIP2 might also be a part of a TBP-free TAF-containing complex (TFTC), a histone acetyl transferase complex (HAT). The human homologue of BIP2 and TAF10, and TAF10 itself are found to co-immunoprecipitate with GCN5 (PCAF - FlyBase), the acetyl transferase of the TFTC HAT complex, and BIP2 harbours a PHD domain implicated in reading specific histone codes. Furthermore, Drosophila has two paralogous genes encoding TAF10 homologues, Taf10 and Taf10b, which are differentially expressed during development. BIP2 specifically forms a dimer with TAF10 and not with TAF10b (Gangloff, 2001); TAF10 was found to be present in both TFIID and TFTC-like complexes, whereas TAF10b was only identified in TFIID complexes. These results raise the possibility of ANTP being linked to a histone acetylase complex. The link unravelled between Antp and bip2 raises numerous questions, including which complex incorporating Antp is present to perform its wing promoting function? Interestingly, Katsuyama and co-workers found a novel gene winged eye (wge) implicated in the eye-to-wing transformation (Katsuyama, 2005). wge seems to be downstream of Antp in the developmental process of eye-to-wing transformation. wge codes for a bromo-adjacent homology domain (BAH)-containing protein (Katsuyama, 2005). The BAH domain has frequently been associated with other domains, such as bromodomains, PHD fingers, and Suppressor of variegation 3-9, Enhancer of zeste and Trithorax (SET) domains, in proteins that are suggested to be involved in the epigenetic regulation of gene expression. This indicates that epigenetic regulation of so far unknown genes is involved in eye-to-wing transformation (Prince, 2008).

It has previously shown that Antp in combination with N^act is able to induce ectopic wings by transforming eye-to-wing tissue. Although endogenous wing development is considered to be independent of Antp, this study found that Antp is the only HOX gene tested so far that is able to transform the eye into wing, which is in line with the fact that Antp specifies the entire second thoracic segment. Furthermore, Antp^Ctx is the only homeotic gain-of-function allele found that induces ectopic wings on the head (Prince, 2008).

Several lines of evidence indicate that N supports Antp in inducing ectopic wings, by preventing eye cells from undergoing apoptosis and in allowing them to adopt a new developmental fate. First, wings formed by ectopic expression of Antp in combination with N^act, or wings found on Antp^Ctx heads, show the same characteristic triple row of bristles at the wing margin. These bristles are found only when vg is ectopically co-expressed in combination with wingless (wg), not when in combination with N^act. Second, this study found that N alone does not induce ectopic wings, and that eye-to-wing transformation can also be achieved without the action of N, by using another eye-specific driver, OK-107-Gal4, indicating that N is not absolutely required for ectopic wing induction. Using different markers for different parts of the wing disc, parts of the eye disc were found to be transformed into most wing disc identities from wing pouch to notum, indicating an eye-to-dorsal T2 transformation, rather than the eye-to-wing pouch transformation seen in adult flies (Prince, 2008).

The known HOX co-factors exd and hth code for DNA-binding proteins that have been shown to increase DNA-binding specificity. bip2, however, encodes a member of the basal transcriptional machinery without any DNA-binding capacity, indicating a different mechanism of action, i.e. by linking Antp directly to the transcriptional machinery. In summary, it is proposed that ANTP interacts directly with BIP2, activating, in turn, a subset of genes that are implicated in wing development (Prince, 2008).

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).

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