Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila.

In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.

Novel Antennapedia and Ultrabithorax trimeric complexes with TBP and Exd regulate transcription.

BACKGROUND: Hox proteins interact with DNA and many other proteins, co-factors, transcriptional factors, chromatin remodeling components, non-coding RNAs and even the extracellular matrix that assembles the Hox complexes. The number of interacting partners continues to grow with diverse components and more transcriptional factors than initially thought. Hox complexes present many activities, but their molecular mechanisms to modulate their target genes remain unsolved. RESULTS: In this paper we showed the protein-protein interaction of Antp with Ubx through the homeodomain using BiFC in Drosophila. Analysis of Antp-deletional mutants showed that AntpHD helixes 1 and 2 are required for the interaction with Ubx. Also, we found a novel interaction of Ubx with TBP, in which the PolyQ domain of TBP is required for the interaction. Moreover, we also detected the formation of two new trimeric complexes of Antp with Ubx, TBP and Exd using BiFC-FRET; these proteins, however, do not form a trimeric interaction with BIP2 or TFIIEß. The novel trimeric complexes reduced Antp transcriptional activity, indicating that they could confer specificity for repression. CONCLUSIONS: Our results increase the number of transcriptional factors in the Antp and Ubx interactomes that form two novel trimeric complexes with TBP and Exd. We also report a new Ubx interaction with TBP. These novel interactions provide important clues of the dynamics of Hox-interacting complexes involved in transcriptional regulation, contributing to better understand Hox function.

Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch.

The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.

Trimeric complexes of Antp-TBP with TFIIEß or Exd modulate transcriptional activity.

BACKGROUND: Hox proteins finely coordinate antero-posterior axis during embryonic development and through their action specific target genes are expressed at the right time and space to determine the embryo body plan. As master transcriptional regulators, Hox proteins recognize DNA through the homeodomain (HD) and interact with a multitude of proteins, including general transcription factors and other cofactors. HD binding specificity increases by protein-protein interactions with a diversity of cofactors that outline the Hox interactome and determine the transcriptional landscape of the selected target genes. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but its precise mechanism remains to be elucidated. RESULTS: Here we report Antennapedia (Antp) Hox protein-protein interaction with the TATA-binding protein (TBP) and the formation of novel trimeric complexes with TFIIEß and Extradenticle (Exd), as well as its participation in transcriptional regulation. Using Bimolecular Fluorescence Complementation (BiFC), we detected the interaction of Antp-TBP and, in combination with Förster Resonance Energy Transfer (BiFC-FRET), the formation of the trimeric complex with TFIIEß and Exd in living cells. Mutational analysis showed that Antp interacts with TBP through their N-terminal polyglutamine-stretches. The trimeric complexes of Antp-TBP with TFIIEß and Exd were validated using different Antp mutations to disrupt the trimeric complexes. Interestingly, the trimeric complex Antp-TBP-TFIIEß significantly increased the transcriptional activity of Antp, whereas Exd diminished its transactivation. CONCLUSIONS: Our findings provide important insights into the Antp interactome with the direct interaction of Antp with TBP and the two new trimeric complexes with TFIIEß and Exd. These novel interactions open the possibility to analyze promoter function and gene expression to measure transcription factor binding dynamics at target sites throughout the genome.

Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy.

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

A Hox Transcription Factor Collective Binds a Highly Conserved Distal-less cis-Regulatory Module to Generate Robust Transcriptional Outcomes.

cis-regulatory modules (CRMs) generate precise expression patterns by integrating numerous transcription factors (TFs). Surprisingly, CRMs that control essential gene patterns can differ greatly in conservation, suggesting distinct constraints on TF binding sites. Here, we show that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle (Exd) and Homothorax (Hth) complexes to mediate dual outputs: thoracic activation and abdominal repression. Using reporter assays, we found that abdominal repression is particularly robust, as neither individual binding site mutations nor a DNA binding deficient Hth protein abolished cooperative DNA binding and in vivo repression. Moreover, a re-engineered DCRE containing a distinct configuration of Hox, Exd, and Hth sites also mediated abdominal Hox repression. However, the re-engineered DCRE failed to perform additional segment-specific functions such as thoracic activation. These findings are consistent with two emerging concepts in gene regulation: First, the abdominal Hox/Exd/Hth factors utilize protein-protein and protein-DNA interactions to form repression complexes on flexible combinations of sites, consistent with the TF collective model of CRM organization. Second, the conserved DCRE mediates multiple cell-type specific outputs, consistent with recent findings that pleiotropic CRMs are associated with conserved TF binding and added evolutionary constraints.

Maintenance of Tissue Pluripotency by Epigenetic Factors Acting at Multiple Levels.

Pluripotent stem cells often adopt a unique developmental program while retaining certain flexibility. The molecular basis of such properties remains unclear. Using differentiation of pluripotent Drosophila imaginal tissues as assays, we examined the contribution of epigenetic factors in ectopic activation of Hox genes. We found that over-expression of Trithorax H3K4 methyltransferase can induce ectopic adult appendages by selectively activating the Hox genes Ultrabithorax and Sex comb reduced in wing and leg discs, respectively. This tissue-specific inducibility correlates with the presence of paused RNA polymerase II in the promoter-proximal region of these genes. Although the Antennapedia promoter is paused in eye-antenna discs, it cannot be induced by Trx without a reduction in histone variants or their chaperones, suggesting additional control by the nucleosomal architecture. Lineage tracing and pulse-chase experiments revealed that the active state of Hox genes is maintained substantially longer in mutants deficient for HIRA, a chaperone for the H3.3 variant. In addition, both HIRA and H3.3 appeared to act cooperatively with the Polycomb group of epigenetic repressors. These results support the involvement of H3.3-mediated nucleosome turnover in restoring the repressed state. We propose a regulatory framework integrating transcriptional pausing, histone modification, nucleosome architecture and turnover for cell lineage maintenance.

Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States.

Basic side chains play crucial roles in protein-DNA interactions. In this study, using NMR spectroscopy, we investigated the dynamics of Arg and Lys side chains of the fruit fly Antennapedia homeodomain in the free state and in the complex with target DNA. We measured 15N relaxation for Arg and Lys side chains at two magnetic fields, from which generalized order parameters for the cationic groups were determined. Mobility of the R5 side chain, which makes hydrogen bonds with a thymine base in the DNA minor groove, was greatly dampened. Several Lys and Arg side chains that form intermolecular ion pairs with DNA phosphates were found to retain high mobility with the order parameter being <0.6 in the DNA-bound state. Interestingly, some of the interfacial cationic groups in the complex were more mobile than in the free protein. The retained or enhanced mobility of the Arg and Lys side chains in the complex should mitigate the overall loss of conformational entropy in the protein-DNA association and allow dynamic molecular recognition.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes.

The homeodomain transcription factors antennapedia and POU-M2 regulate the transcription of the steroidogenic enzyme gene Phantom in the silkworm.

The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.

What makes a man a man? Prenatal antennapedia expression is involved in the formation of the male phenotype in Daphnia.

Cyclic parthenogenetic organisms show a switch in reproductive strategy from asexual to sexual reproduction upon the occurrence of unfavourable environmental conditions. The sexual reproductive mode involves the production of ameiotic diploid males and the fertilization of meiotic haploid eggs. One beautiful example for this switch between parthenogenesis and sexual reproduction is Daphnia. Male and female Daphnia from the same clone are genetically identical. Morphological differences should therefore only be due to differential gene expression. This differential gene expression leads to sexually dimorphic phenotypes with elongated and moveable (i.e. leg-like) first antennae in males in comparison to females. For other arthropods, it has been demonstrated that the formation of differential morphology of legs and antennae involves the regulation of the Hox gene antennapedia (antp). Here, we show that antp is expressed during the embryogenesis of Daphnia, and that adults contain much lower amounts of antp mRNA than eggs. The eggs of mothers that were treated with the juvenile hormone methyl farnesoate (responsible for the production of male offspring) showed lower expression of antp than parthenogenetically produced female eggs. We therefore conclude that differential antp expression is involved in the molecular pathways inducing the male phenotype of Daphnia.

Dual role for Hox genes and Hox co-factors in conferring leg motoneuron survival and identity in Drosophila.

Adult Drosophila walk using six multi-jointed legs, each controlled by ∼50 leg motoneurons (MNs). Although MNs have stereotyped morphologies, little is known about how they are specified. Here, we describe the function of Hox genes and homothorax (hth), which encodes a Hox co-factor, in Drosophila leg MN development. Removing either Hox or Hth function from a single neuroblast (NB) lineage results in MN apoptosis. A single Hox gene, Antennapedia (Antp), is primarily responsible for MN survival in all three thoracic segments. When cell death is blocked, partially penetrant axon branching errors are observed in Hox mutant MNs. When single MNs are mutant, errors in both dendritic and axon arborizations are observed. Our data also suggest that Antp levels in post-mitotic MNs are important for specifying their identities. Thus, in addition to being essential for survival, Hox and hth are required to specify accurate MN morphologies in a level-dependent manner.

Variation and constraint in Hox gene evolution.

Despite enormous body plan variation, genes regulating embryonic development are highly conserved. Here, we probe the mechanisms that predispose ancient regulatory genes to reutilization and diversification rather than evolutionary loss. The Hox gene fushi tarazu (ftz) arose as a homeotic gene but functions as a pair-rule segmentation gene in Drosophila. ftz shows extensive variation in expression and protein coding regions but has managed to elude loss from arthropod genomes. We asked what properties prevent this loss by testing the importance of different protein motifs and partners in the developing CNS, where ftz expression is conserved. Drosophila Ftz proteins with mutated protein motifs were expressed under the control of a neurogenic-specific ftz cis-regulatory element (CRE) in a ftz mutant background rescued for segmentation defects. Ftz CNS function did not require the variable motifs that mediate differential cofactor interactions involved in homeosis or segmentation, which vary in arthropods. Rather, CNS function did require the shared DNA-binding homeodomain, which plays less of a role in Ftz segmentation activity. The Antennapedia homeodomain substituted for Ftz homeodomain function in the Drosophila CNS, but full-length Antennapedia did not rescue CNS defects. These results suggest that a core CNS function retains ftz in arthropod genomes. Acquisition of a neurogenic CRE led to ftz expression in unique CNS cells, differentiating its role from neighboring Hox genes, rendering it nonredundant. The inherent flexibility of modular CREs and protein domains allows for stepwise acquisition of new functions, explaining broad retention of regulatory genes during animal evolution.

The role of the histone H2A ubiquitinase Sce in Polycomb repression.

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. Here, we created an Sce knockout allele and show that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression.

Development of protein mimics for intracellular delivery.

Designing delivery agents for therapeutics is an ongoing challenge. As treatments and desired cargoes become more complex, the need for improved delivery vehicles becomes critical. Excellent delivery vehicles must ensure the stability of the cargo, maintain the cargo's solubility, and promote efficient delivery and release. In order to address these issues, many research groups have looked to nature for design inspiration. Proteins, such as HIV-1 trans-activator of transcription (TAT) and Antennapedia homeodomain protein, are capable of crossing cellular membranes. However, due to the complexities of their structures, they are synthetically challenging to reproduce in the laboratory setting. Being able to incorporate the key features of these proteins that enable cell entry into simpler scaffolds opens up a wide range of opportunities for the development of new delivery reagents with improved performance. This review charts the development of protein mimics based on cell-penetrating peptides (CPPs) and how structure-activity relationships (SARs) with these molecules and their protein counterparts ultimately led to the use of polymeric scaffolds. These scaffolds deviate from the normal peptide backbone, allowing for simpler, synthetic procedures to make carriers and tune chemical compositions for application specific needs. Successful design of polymeric protein mimics would allow researchers to further understand the key features in proteins and peptides necessary for efficient delivery and to design the next generation of more efficient delivery reagents.

Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression.

Evolution often results in morphologically similar solutions in different organisms, a phenomenon known as convergence. However, there is little knowledge of the processes that lead to convergence at the genetic level. The genes of the Hox cluster control morphology in animals. They may also be central to the convergence of morphological traits, but whether morphological similarities also require similar changes in Hox gene function is disputed. In arthropods, body subdivision into a region with locomotory appendages ("thorax") and a region with reduced appendages ("abdomen") has evolved convergently in several groups, e.g., spiders and insects. In insects, legs develop in the expression domain of the Hox gene Antennapedia (Antp), whereas the Hox genes Ultrabithorax (Ubx) and abdominal-A mediate leg repression in the abdomen. Here, we show that, unlike Antp in insects, the Antp gene in the spider Achaearanea tepidariorum represses legs in the first segment of the abdomen (opisthosoma), and that Antp and Ubx are redundant in the following segment. The down-regulation of Antp in A. tepidariorum leads to a striking 10-legged phenotype. We present evidence from ectopic expression of the spider Antp gene in Drosophila embryos and imaginal tissue that this unique function of Antp is not due to changes in the Antp protein, but likely due to divergent evolution of cofactors, Hox collaborators or target genes in spiders and flies. Our results illustrate an interesting example of convergent evolution of abdominal leg repression in arthropods by altering the role of distinct Hox genes at different levels of their action.

Analysis of central Hox protein types across bilaterian clades: on the diversification of central Hox proteins from an Antennapedia/Hox7-like protein.

Hox proteins are among the most intensively studied transcription factors and represent key factors in establishing morphological differences along the anterior-posterior axis of animals. They are generally regarded as highly conserved in function, a view predominantly based on experiments comparing a few (anterior) Hox proteins. However, the extent to which central or abdominal Hox proteins share conserved functions and sequence signatures remains largely unexplored. To shed light on the functional divergence of the central Hox proteins, we present an easy to use resource aimed at predicting the functional similarities of central Hox proteins using sequence elements known to be relevant to Hox protein functions. We provide this resource both as a stand-alone download, including all information, as well as via a simplified web-interface that facilitates an accurate and fine-tuned annotation of novel Hox sequences. The method used in the manuscript is, so far, the only published sequence-based method capable of differentiating between the functionally distinct central Hox proteins with near-identical homeodomains (such as the Drosophila Antp, Ubx and Abd-A Hox proteins). In this manuscript, a pairwise-sequence-similarity based approach (using the bioinformatics tool CLANS) is used to analyze all available central Hox protein sequences. The results are combined with a large-scale species phylogeny to depict the presence/absence of central Hox sequence-types across the bilaterian lineage. The obtained pattern of distribution of the Hox sequence-types throughout the species tree enables us to infer at which branching point a specific type of central Hox protein was present. Based on the Hox sequences currently available in public databases, seven sequence-similarity groups could be identified for the central Hox proteins, two of which have never been described before (Echi/Hemi7 and Echi/Hemi8). Our work also shows, for the first time, that Antp/Hox7-like sequences are present throughout all bilaterian clades and that all other central Hox protein groups are specific to sub-lineages in the protostome or deuterostome branches only.

Cellular uptake of the Antennapedia homeodomain polypeptide by macropinocytosis.

Antennapedia homeodomain has been shown to be able to translocate from extracellular space into the cytoplasm of cells in a receptor-independent manner. Its third α-helix domain, designated as "Penetratin", was proposed to be the functional transduction domain that is responsible for the translocation, and it is widely used for intracellular delivery of various exogenous proteins. Although Penetratin has been regarded to be the only element conferring the capacity on its parent polypeptide to penetrate through the plasma membrane, we found that the complete Antennapedia homeodomain exhibits an appreciably higher level of translocation efficiency as compared to Penetratin. Pharmacological analysis demonstrated that macropinocytic endocytosis plays a significant role underlying the process of the homeodomain internalization, and this is consistent with the observation that internalized polypeptide co-localizes with a fluid phase dye. Our results identify macropinocytosis as a major mechanism by which Antennapedia homeodomain obtains the access to the interior of cells, providing a novel perspective in the field of protein translocation and transduction.

Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide.

BACKGROUND: Heat shock protein (Hsp) 90 and Hsp70 are indispensable for cell survival under conditions of stress. They bind to client proteins to assist in protein stabilization, translocation of polypeptides across the cell membrane, and recovery of proteins from aggregates in the cell. Therefore, these proteins have recently emerged as important targets in the treatment of cancer. We previously reported that the newly designed Antp-TPR hybrid peptide targeting Hsp90 induced cytotoxic activity to cancer cells both in vitro and in vivo. METHODS: To further improve the cytotoxic activity of Antp-TPR toward cancer cells, we investigated the effect of a Hsp70-targeted peptide, which was made cell-permeable by adding the polyarginine with a linker sequence, on the cytotoxic activity of Antp-TPR in breast cancer cell lines. RESULTS: It was revealed that Antp-TPR in the presence of a Hsp70-targeted peptide induced effective cytotoxic activity toward breast cancer cells through the descrease of Hsp90 client proteins such as p53, Akt, and cRaf. Moreover, the combined treatment with these peptides did not induce the up-regulation of Hsp70 protein, as determined by western blotting, a promoter assay using a luminometer, and single-cell level imaging with the LV200 system, although a small-molecule inhibitor of Hsp90, 17-allylamino-demethoxygeldanamycin (17-AAG), did induce the up-regulation of this protein. We also found that treatment with Antp-TPR, Hsp70-targeted peptide, or a combination of the two did not induce an increase in the glutathione concentrations in the cancer cells. CONCLUSION: These findings suggest that targeting both Hsp90 and Hsp70 with Antp-TPR and Hsp70-targeted peptide is an attractive approach for selective cancer cell killing that might provide potent and selective therapeutic options for the treatment of cancer.

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression.

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary--whereas the entire N terminus of the protein is dispensable--for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors.