RESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Proteínas de Homeodominio , Proteína de Unión a TATA-Box , Factores de Transcripción , Animales , Proteína con Homeodominio Antennapedia/genética , Proteína con Homeodominio Antennapedia/metabolismo , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Unión Proteica , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Proteína con Homeodominio Antennapedia , Proteínas de Drosophila , Proteínas de Homeodominio , Proteína de Unión a TATA-Box , Factores de Transcripción TFII , Factores de Transcripción , Proteína con Homeodominio Antennapedia/genética , Proteína con Homeodominio Antennapedia/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
The positively-charged peptide antp derived from Antennapedia transcription protein is demonstrated to mediate the liposome translocation across the cell membrane. In the current investigation, we prepared a stable liposomal doxorubicin (Dox) formulation and targeted it with the antp peptide from 0 to 200 ligand/liposome. These antp-containing liposomes were investigated in terms of physical stability on storage in the refrigerator and upon incubation in blood. Also, other features like cell binding, uptake, biodistribution, and treatment efficiency were evaluated in C26 colon carcinoma BALB/c mice. The Antp in liposomes resulted in enhanced particle growth with the development of the enormously large liposomes from 2000 to 6000 nm. Upon incubation in blood, these large liposomes were removed. The antp also enhanced the cell binding affinity and cell uptake rate of the liposomes and resulted in the restriction of the cancer cell proliferation, but it failed to improve the chemotherapeutic property of the Dox-liposome. The i.v. injection of antp-liposomes (15 mg Dox/kg) caused severe body weight loss and early death incidence due to probably increased toxicity. The antp targeting offered no advantage to the Dox-liposome in the delivery of Dox to the tumor, and failed to enhance the treatment efficiency of the liposomes.
Asunto(s)
Proteína con Homeodominio Antennapedia , Doxorrubicina/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Péptidos , Animales , Proteína con Homeodominio Antennapedia/genética , Proteína con Homeodominio Antennapedia/toxicidad , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/toxicidad , Ratones , Ratones Endogámicos BALB C , Péptidos/genética , Péptidos/metabolismo , Péptidos/toxicidad , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Polietilenglicoles/toxicidad , Distribución Tisular , Pérdida de Peso/efectos de los fármacosRESUMEN
Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.
Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Proteínas de Drosophila/genética , Vuelo Animal , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Proteína con Homeodominio Antennapedia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dosificación de Gen , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Alas de Animales/anatomía & histología , Alas de Animales/metabolismoRESUMEN
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.
Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Complejos de Proteína Captadores de Luz/genética , Procesos Fototróficos/genética , Agregado de Proteínas/genética , Proteína con Homeodominio Antennapedia/química , Clorofila/química , Clorofila/genética , Clorofila/efectos de la radiación , Análisis por Conglomerados , Fluorescencia , Concentración de Iones de Hidrógeno , Luz/efectos adversos , Complejos de Proteína Captadores de Luz/química , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/efectos de la radiación , Espectrometría de Fluorescencia , Tilacoides/química , Tilacoides/genética , Tilacoides/efectos de la radiación , Zeaxantinas/genéticaRESUMEN
BACKGROUND: Radioresistance in tumors limits the curative effect of the radiotherapy. Mimetic compounds of second mitochondria-derived activator of caspase (Smac) are potential new tumor radiation-sensitizing drugs because they can increase radiation-induced tumor cell apoptosis. Here, we observed the radiosensitization effect of a new Smac mimetic Antennapedia protein (ANTP)-SmacN7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells. METHODS: The ANTP-SmacN7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells. A549 cells were divided into the control, radiation-only, ANTP-SmacN7-only and ANTP-SmacN7 + radiation groups. The cells were exposed to 0, 2, 4 and 6 Gy, with 20 µmol/L of ANTP-SmacN7. The radiation-sensitizing effects of the ANTP-SmacN7 fusion proteins were observed via clonogenic assay. Apoptosis was detected using flow cytometry. A comet assay was used to assess DNA damage. The levels and degrees of cytochrome-c, PARP, H2AX, caspase-8, caspase-3, and caspase-9 activation were detected via western blot assay. The radiation sensitization of the fusion peptide, expression of γ-H2AX and C-PARP were compared after adding the caspase inhibitor, Z-VAD. RESULTS: ANTP-SmacN7 fusion proteins entered the cells and promoted A549 cell radiosensitization. Treatment with ANTP-SmacN7 + radiation significantly reduced the A549 cell clone-forming rate, increased the cytochrome-c, cleaved caspase-8, cleaved caspase-3 and cleaved caspase-9 expression levels, promoted caspase activation, and increased the rate of radiation-induced apoptosis. The ANTP-SmacN7 fusion peptide significantly increased radiation-induced double-stranded DNA rupture in the A549 cells and increased DNA damage. Adding Z-VAD reduced the fusion peptide's proapoptotic effect but not the level of double-stranded DNA breakage. CONCLUSIONS: The ANTP-SmacN7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells. This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.
Asunto(s)
Proteína con Homeodominio Antennapedia/química , Neoplasias Pulmonares/radioterapia , Oligopéptidos/química , Fragmentos de Péptidos/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Rayos gamma , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Discos Imaginales/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Proteína con Homeodominio Antennapedia/metabolismo , Sitios de Unión , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos , Femenino , Genes Homeobox , Genotipo , Homocigoto , Masculino , Modelos Biológicos , Modelos Teóricos , Fenotipo , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Espectrometría de Fluorescencia , Procesos Estocásticos , TransgenesRESUMEN
Homeoproteins contain the conserved homeodomain (HD) and have an important role determining embryo body plan during development. HDs increase their DNA-binding specificity by interacting with additional cofactors outlining a Hox interactome with a multiplicity of protein-protein interactions. In Drosophila, the first link of functional contact with a general transcription factor (GTF) was found between Antennapedia (Antp) and BIP2 (TFIID complex). Hox proteins also interact with other components of Pol II machinery such as the subunit Med19 from Mediator (MED) complex, TFIIEß and transcription-pausing factor M1BP. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but the precise molecular mechanism remains unclear. In this paper, we focused on the Antp-TFIIEß protein-protein interface to establish the specific contacts as well as its functional role. Using Bimolecular Fluorescence Complementation (BiFC) in cell culture and in vivo we found that TFIIEß interacts with Antp through the HD independently of the YPWM motif and the direct physical interaction is at helix 2, specifically aminoacidic positions I32 and H36 of Antp. We also found, through ectopic assays, that these two positions in helix 2 are crucial for Antp homeotic function in head involution, and thoracic and antenna-to tarsus transformations. Interestingly, overexpression of Antp and TFIIEß in the antennal disc showed that this interaction is required for the antenna-to-tarsus transformation. In conclusion, interaction of Antp with TFIIEß is important for the functional specificity of Antennapedia, and amino acids 32 and 36 in Antp HD helix 2 are key for this interaction. Our results open the possibility to more broadly analyze Antp-TFIIEß interaction on the transcriptional control for the activation and/or repression of target genes in the Hox interactome during Drosophila development.
Asunto(s)
Proteína con Homeodominio Antennapedia/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción TFII/metabolismo , Factores de Transcripción/metabolismo , Animales , Cruzamientos Genéticos , Colorantes Fluorescentes/química , Eliminación de Gen , Regulación de la Expresión Génica , Genes Homeobox , Células HEK293 , Humanos , Microscopía Fluorescente , Mutación , Plásmidos/metabolismo , Unión Proteica , Dominios Proteicos , Transcripción GenéticaRESUMEN
We previously showed that barley sprout extract (BSE) prevents chronic alcohol intake-induced liver injury in mice. BSE notably inhibited glutathione (GSH) depletion and increased inflammatory responses, revealing its mechanism of preventing alcohol-induced liver injury. In the present study we investigated whether the antioxidant effect of BSE involves enhancing nuclear factor-erythroid 2 related factor 2 (Nrf2) activity and GSH synthesis to inhibit alcohol-induced oxidative liver injury. Mice fed alcohol for four weeks exhibited significantly increased oxidative stress, evidenced by increased malondialdehyde (MDA) level and 4-hydroxynonenal (4-HNE) immunostaining in the liver, whereas treatment with BSE (100 mg/kg) prevented these effects. Similarly, exposure to BSE (0.1-1 mg/mL) significantly reduced oxidative cell death induced by t-butyl hydroperoxide (t-BHP, 300 µM) and stabilized the mitochondrial membrane potential (∆ψ). BSE dose-dependently increased the activity of Nrf2, a potential transcriptional regulator of antioxidant genes, in HepG2 cells. Therefore, increased expression of its target genes, heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase catalytic subunit (GCLC) was observed. Since GCLC is involved in the rate-limiting step of GSH synthesis, BSE increased the GSH level and decreased both cysteine dioxygenase (CDO) expression and taurine level. Because cysteine is a substrate for both taurine and GSH synthesis, a decrease in CDO expression would further contribute to increased cysteine availability for GSH synthesis. In conclusion, BSE protected the liver cells from oxidative stress by activating Nrf2 and increasing GSH synthesis.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/biosíntesis , Hordeum/química , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Extractos Vegetales/farmacología , Animales , Proteína con Homeodominio Antennapedia/farmacología , Supervivencia Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Proteínas de Drosophila/farmacología , Etanol/toxicidad , Células Hep G2 , Humanos , Peroxidación de Lípido , Masculino , Ratones , Subunidad p45 del Factor de Transcripción NF-E2/genética , Extractos Vegetales/química , Especies Reactivas de OxígenoRESUMEN
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.
Asunto(s)
Proteína con Homeodominio Antennapedia/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Simulación de Dinámica Molecular , Movimiento (Física) , Animales , Proteína con Homeodominio Antennapedia/metabolismo , Arginina/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/química , Drosophila , Proteínas de Drosophila , Entropía , Enlace de Hidrógeno , Lisina/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear BiomolecularRESUMEN
Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.
Asunto(s)
Proteína con Homeodominio Antennapedia/inmunología , Proteínas Portadoras/inmunología , Células Dendríticas/inmunología , Ovalbúmina/inmunología , Vacunas/inmunología , Animales , Presentación de Antígeno , Artrópodos/inmunología , Proteínas Portadoras/síntesis química , Péptidos de Penetración Celular , Células Cultivadas , Sistemas de Liberación de Medicamentos , Epítopos de Linfocito T/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/síntesis químicaRESUMEN
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.
Asunto(s)
Drosophila melanogaster/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Elementos Reguladores de la Transcripción/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genética , Animales , Proteína con Homeodominio Antennapedia/genética , Sitios de Unión/genética , Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Homeodominio/metabolismo , Unión Proteica , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Animales , Animales Modificados Genéticamente , Proteína con Homeodominio Antennapedia/genética , Proteína con Homeodominio Antennapedia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Larva , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Daphnia/genética , Animales , Proteína con Homeodominio Antennapedia/metabolismo , Daphnia/crecimiento & desarrollo , Daphnia/fisiología , Femenino , Proteínas de Insectos/metabolismo , Masculino , Filogenia , Caracteres Sexuales , Procesos de Determinación del SexoRESUMEN
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.
Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Bombyx/genética , Regulación del Desarrollo de la Expresión Génica , Pleiotropía Genética , Proteínas de Insectos/genética , Sericinas/genética , Animales , Proteína con Homeodominio Antennapedia/metabolismo , Secuencia de Bases , Evolución Biológica , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sericinas/metabolismoRESUMEN
Chromatin boundary elements (CBEs) are widely distributed in the genome and mediate formation of chromatin loops, but their roles in gene regulation remain poorly understood. The complex expression pattern of the Drosophila homeotic gene Sex combs reduced (Scr) is directed by an unusually long regulatory sequence harboring diverse cis elements and an intervening neighbor gene fushi tarazu (ftz). Here we report the presence of a multitude of CBEs in the Scr regulatory region. Selective and dynamic pairing among these CBEs mediates developmentally regulated chromatin loops. In particular, the SF1 boundary plays a central role in organizing two subsets of chromatin loops: one subset encloses ftz, limiting its access by the surrounding Scr enhancers and compartmentalizing distinct histone modifications, and the other subset subdivides the Scr regulatory sequences into independent enhancer access domains. We show that these CBEs exhibit diverse enhancer-blocking activities that vary in strength and tissue distribution. Tandem pairing of SF1 and SF2, two strong CBEs that flank the ftz domain, allows the distal enhancers to bypass their block in transgenic Drosophila, providing a mechanism for the endogenous Scr enhancer to circumvent the ftz domain. Our study demonstrates how an endogenous CBE network, centrally orchestrated by SF1, could remodel the genomic environment to facilitate gene regulation during development.
Asunto(s)
Proteína con Homeodominio Antennapedia/genética , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción Fushi Tarazu/genética , Genes de Insecto , Elementos Aisladores , Regiones Promotoras Genéticas , Factores de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The efficiency of antigen capture, processing, and presentation by antigen-presenting cells is the key to induce an effective immune response. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargoes and have an attractive potential for vaccine delivery. In this study, the Drosophila Antennapedia homeoprotein (Antp) and the human immunodeficiency virus-1 transactivator of transcription (TAT) peptides were fused to the N- or C-terminus of Sia10, a protective antigen of Streptococcus iniae, resulting in four recombinant fusion proteins, i.e., rAntp-Sia10, rSia10-Antp, rTAT-Sia10, and rSia10-TAT. All fusion proteins were expressed and purified, and their ability to penetrate into cells was examined. The results showed that rTAT-Sia10 had the strongest ability to translocate through the cellular membrane into cells. Immunofluorescence microscopy and Western blot assay confirmed that rTAT-Sia10 could penetrate into the head kidney lymphocytes and gill cells of Japanese flounder (Paralichthys olivaceus). Immunological analysis showed that rTAT-Sia10 significantly enhanced macrophage activation and peripheral blood leukocyte proliferation, and induced production of specific serum antibodies at 2-8 weeks post-vaccination. Transcriptional analysis showed that vaccination with rTAT-Sia10 up-regulated the expression of the genes encoding IL-1ß, IL-8, NKEF, Mx, IgD, IgM, TNFα, MHC I α, MHC IIα, and CD8α. Fish vaccinated with rTAT-Sia10 exhibited significantly higher levels of survival rates (98% at 1 month and 92% at 2 months) compared to fish vaccinated with rSia10 (57% at 1 month and 53% at 2 months). Taken together, these results indicate that TAT-derived peptide has a great potential in the application of bacterial vaccines.
Asunto(s)
Vacunas Bacterianas/inmunología , Péptidos de Penetración Celular/inmunología , Enfermedades de los Peces/prevención & control , Lenguado/inmunología , Infecciones Estreptocócicas/veterinaria , Animales , Proteína con Homeodominio Antennapedia/genética , Proteína con Homeodominio Antennapedia/inmunología , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/genética , Péptidos de Penetración Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/inmunología , Enfermedades de los Peces/inmunología , Lenguado/genética , Lenguado/microbiología , Expresión Génica , Humanos , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/prevención & control , Streptococcus/inmunología , Streptococcus/patogenicidad , Vacunación/veterinaria , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
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.
Asunto(s)
Proteína con Homeodominio Antennapedia/metabolismo , Proteínas del Helminto/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Insectos/metabolismo , Metamorfosis Biológica , Oxigenasas de Función Mixta/metabolismo , Factores del Dominio POU/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Proteína con Homeodominio Antennapedia/genética , Bombyx , Biología Computacional , Ecdisona/química , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Oxigenasas de Función Mixta/genética , Muda , Mutación , Nucleopoliedrovirus/genética , Factores del Dominio POU/genética , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
Magnetic nanoparticles (MNPs) represent one of the most promising materials as they can act as a versatile platform in the field of bionanotechnology for enhanced imaging, diagnosis, and treatment of various diseases. Silica is the most common compound for preparing coated iron oxide NPs since it improves colloidal stability and the binding affinity for various organic molecules. Biomolecules such as cell penetrating peptides (CPPs) might be employed to decorate MNPs, combining their promising physicochemical properties with a cell penetrating ability. In this work, a computational investigation on adsorption of Antennapedia homeodomain-derived penetrating peptide (pAntp) on silica and magnetite (MAG) surfaces is presented. By employing umbrella sampling molecular dynamics, we provided a quantitative estimation of the pAntp-surface adsorption free energy to highlight the influence of surface hydroxylation state on the adsorption mechanism. The interaction between peptide and surface has shown to be mainly driven by electrostatics. In case of MAG surface, also an important contribution of van der Waals (VdW) attraction was observed. Our data suggest that a competitive mechanism between MNPs and cell membrane might partially inhibit the CPP to carry out its membrane penetrating function.
Asunto(s)
Proteína con Homeodominio Antennapedia/química , Péptidos de Penetración Celular/química , Adsorción , Secuencia de Aminoácidos , Nanopartículas de Magnetita/química , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Dióxido de Silicio/químicaRESUMEN
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.