Protein Interactions

Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). Three results support a novel mechanism for Hox cofactor function. (1) The Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. (2) Either Dfd or a Dfd-VP16 hybrid (VP16 is a transcriptional activation domain) mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independent of Exd enhancement of Dfd-binding affinity on the composite site. (3) Transfection assays confirm that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family (Li, 1999a).

The neutral state of Dfd on simple binding sites indicates that additional regulatory steps and regulatory sequences are required for Dfd to activate gene expression. To test the hypothesis that Dfd binding per se is inherently neutral in embryos, a test was performed to see whether high levels of Dfd or Dfd-VP16 proteins could activate transcription through simple Dfd recognition sites. In vitro, a DNA sequence consisting of two tandem copies of the simple Deformed binding site (D site or 2×D), is bound by Dfd with high affinity but not detectably bound by Exd. The affinity of Dfd protein for the 2×D-site is not enhanced by the inclusion of Exd protein (Li, 1999a).

A test was performed of the embryonic function a varient of the D site reporter construct. This varient contains two tandem copies of a core sequence, to which Dfd and Exd bind together (2×ED2 sites). In vitro, the 2×ED2 site is bound weakly by Dfd protein alone, but is not bound detectably by Exd alone. Binding of Dfd to the 2×ED2 site is enhanced in the presence of Exd as shown by the formation of an abundant complex that contains Dfd, Exd and 2×ED2. The affinity of the Dfd-Exd heterodimer for the 2×ED2 site is approximately the same as the affinity of the Dfd protein alone for the 2×D site. Although the 2×D site and the 2×ED2 site have very similar in vitro affinity for Dfd in the presence of Exd, the 2×ED2 site is much more responsive than the 2×D site to either Dfd or Dfd-VP16 proteins in embryos. This strongly suggests that Exd is required to release the transcriptional activation function of Dfd in a way that is independent of the Exd enhancement of Dfd binding affinity on the 2×ED2 site. At present, the most widely accepted models propose Exd as a cofactor that has its effect on Hox specificity by acting to increase the binding affinity of different Hox proteins to different composite binding sites. The results presented here indicate that Exd has other regulatory effects on Hox proteins that may play a role in the diversification of function within the Hox family (Li, 1999a).

Dfd protein contains an autonomous activation domain that is functional in transfection assays when separated from the C-terminal half of the protein. On tandem repeats of simple Dfd-binding sites, the function of the Dfd transcription activation domain is suppressed both in cultured cells and in embryos. In embryos, this suppression can be partially relieved by the addition of Exd-binding sites to simple Dfd-binding sites. This is apparently due to the function of the Exd protein, since exd genetic function is required for the relief of the suppression of Dfd activation function on 2×ED2 sites. In cultured cells, the suppression of Dfd activation function can be conferred by the homeodomain regions from either Dfd or Ubx. Since no evidence is found that there is a direct intramolecular interaction between the Dfd homeodomain and its transcriptional activation region, a model is proposed that invokes a masking factor that suppresses the function of the activation domain by contacting the homeodomain region. In addition, it is speculated that Exd may be required to alleviate the suppressive effect of the proposed masking factor by competing for overlapping protein-protein interaction sites on the homeodomain (Li, 1999a).

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. The N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove (Draganescu, 1995).

The DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins were compared. 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).

Specific amino acid residues at the amino end of the Ultrabithorax homeodomain are required to specifically regulate Antennapedia transcription: 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).

Deformed possesses an acidic region just N-terminal to the homeodomain and a C-terminal sequence called the C-tail region, containing poly-glutamine and poly-asparagine tracts. Removal of the acidic domain and the C-tail region converts a chimeric Deformed/Abdominal-B protein, possessing the Abdominal-B homeodomain, from a strong activator to a repressor of a Distal-less promoter element, but has little effect on activation of an empty spiracles element. Constructs without a third domain, the N-terminal N domain, fail to show any regulatory activity. These results suggest transcriptional activation by the N domain can be modulated by acidic and C-tail domains (Zhu, 1996).

A heat-shock promoter/selector gene was constructed that encodes a Deformed/Abdominal-B chimera in which the Abdominal-B homeodomain is substituted for that of Deformed. Expression of this chimeric protein throughout the embryo causes morphological transformation of anterior segments toward more posterior identities. A number of other homeotic selector genes, all normally repressed by Abdominal-B, are ectopically activated by the chimeric protein. These results support the hypothesis that the target specificity of similar homeodomain proteins is largely determined by the amino acid sequence of the homeodomain (Kuziora, 1990).

The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. Deformed and mouse Hoxa-5 binding to this neuronal enhancer is increased more than 10 fold by Pros. Pros reduces Eve's DNA binding to this enhancer, but does not modulate the binding of Engrailed. This interaction is unidirectional and specific, since neither Dfd, Eve nor En has an effect on Pros binding. The modulation by Pros does not require Pros binding to DNA. Pros protein modifies the trypsin sensitivity of Dfd protein, suggesting that Pros binds Dfd and is able to induce a conformation change in Dfd. Nevertheless, Pros is able to bind the Dfd neuronal autoregulatory enhancer and enhances Dfd binding to this DNA sequence. The DNA-binding and homeodomain protein-interacting activities of Prospero are localized to its highly conserved C-terminal region, and the two regulatory capacities are independent (Hassan, 1997).

Hox transcription factors, in combination with cofactors such as Exd protein and its mammalian Pbx homologs (PBC proteins), provide diverse developmental fates to cells on the anteroposterior body axis of animal embryos. However, the mechanisms by which the different Hox proteins and their cofactors generate those diverse fates remain unclear. Recent findings have provided support for a model where the DNA binding sites that directly interact with Hox-PBC heterodimers determine which member of the Hox protein family occupies and thereby regulates a given target element. In the experiments reported here, the function of chimeric Hox response elements is tested, and, surprisingly, evidence is found that runs counter to this view. A 21 bp cofactor binding sequence from an embryonic Deformed Hox response element (region 6), containing no Hox or Hox-PBC binding sites, was combined with single or multimeric sites that binds heterodimers of Labial-type Hox and PBC proteins (region 3). Normally, multimerized Labial-PBC binding sites are sufficient to trigger a Labial-specific activation response in either Drosophila or mouse embryos. The 21 bp sequence element plays an important role in Deformed specificity, because it is capable of switching a Labial-PBC binding site/response element to a Deformed response element. Thus, cofactor binding sites that are separate and distinct from homeodomain binding sites can dictate the regulatory specificity of a Hox response element (Li, 1999b).

The instructive role of factors bound to non-Hox binding sites in controlling Hox responses is probably a general mechanism by which different Hox proteins acquire distinct functions. Exd is a well-characterized example that is used in a subset of Hox-activated response elements. However, the influence of Exd on Hox specificity may be superseded in complex elements that contain sequences such as region 6. How the specificity code is generated in the average Dfd or Ubx response element is likely to vary depending on the cell type, the presence or absence of Exd in the cell, the stage of development, and the extracellular signals that are received by a given response element. The putative activating cofactor binding site(s) (GGC..AAAGC) in the region 6 element are present in other naturally derived Dfd response elements, so there may be an important subset of Dfd response elements that rely on these sites for maxillary specificity. At present, none of the known complex elements that respond to other Hox proteins contain good matches to the GGC..AAAGC motifs. The region 6 cofactor(s) that are required to elicit a Dfd-dependent activation response by interacting with the GGCnn(n)AAAGC motif are not yet known. The unknown region 6 cofactors might selectively release covert activation functions of Dfd, or interact with Dfd to form new activation functions. In this view, although multiple Hox proteins (e.g., Dfd and Lab) may bind to the region 3 Dfd binding site or the Lab-Exd composite site, only Dfd would functionally interact with the cofactors bound nearby on region 6 to activate transcription, while other Hox proteins would not (Li, 1999b).

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).

Deformed : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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