Los residuos 32 y 36 de la hélice 2 del homeodominio de Antennapedia son necesarios para la interacción con TFIIEβ y para su especificidad funcional en D. melanogaster.

RESUMEN: Las homeoproteínas constituyen una familia de factores de transcripción con un papel predominante para determinar el plan corporal de un embrión durante el desarrollo. Estas contienen el homeodominio (HD) de unión al DNA que aumenta su especificidad al interactuar con cofactores adicionales constituyendo el interactoma. En Drosophila, la primera liga de interacción funcional con un factor general de transcripción (GTF) se encontró entre Antennapedia (Antp) y BIP2 (complejo TFIID). Las proteínas Hox también interactúan con otros componentes de la maquinaria basal de la RNA Pol II, como la subunidad Med19 del complejo Mediador (MED), TFIIEβ y el factor de pausa de la transcripción M1BP. Todas estas interacciones muestran claramente que en la regulación transcripcional dirigida por Hox están implicadas diversas proteínas, sin embargo, el mecanismo molecular preciso no es del todo claro. En esta tesis, nos centramos en la interfase proteína-proteína de Antp-TFIIEβ para determinar los contactos específicos de interacción, así como su importancia funcional in vivo. La estrategia experimental consistió en analizar la interacción de las proteínas mediante el uso de Fluorescencia por Complementación Bimolecular (BiFC) in vivo en embriones y discos imaginales de Drosophila. Los resultados mostraron muy evidentemente que TFIIEβ interactúa con Antp a través de la hélice 2 del HD, específicamente las posiciones de aminoácidos I32 y H36 de Antp. Para determinar que estas dos posiciones en la hélice 2 son cruciales para la función homeótica de Antp en la involución de la cabeza y las transformaciones torácica y antena-tarso, se realizaron ensayos de expresión ectópica. De manera interesante, la sobreexpresión de Antp y TFIIEβ en el disco imaginal de antena mostró que esta interacción es indispensable para la transformación antena-tarso. También se determinó, mediante BiFC competitivo que los GTFs: TBP, Bip2, TFIIB y TFIIAγ, y las homeoproteínas EXD y AbdB afectaron la interacción dimérica Antp-TFIIEβ sugiriendo la formación probable de complejos triméricos. Nuestros resultados muestran claramente que los aminoácidos 32 y 36 en la hélice 2 de Antp HD son necesarios para la interacción con TFIIEβ y para la especificidad funcional de Antp, lo que abre la posibilidad para analizar su función en el control transcripcional de activación y/o represión de genes blanco en el interactoma Hox durante el desarrollo de Drosophila. ABSTRACT: 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, TFIIEβ and transcription-pausing factor M1BP. All these interactions clearly demonstrate Hox-driven transcriptional regulation, but the precise molecular mechanism remains unclear. In this thesis, we focused on the Antp-TFIIEβ protein-protein interface to establish the specific contacts as well as its functional role in vivo. Using Bimolecular Fluorescence Complementation (BiFC) in vivo we found that TFIIEβ 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 TFIIEβ in the antennal disc showed that this interaction is required for the antenna-to-tarsus transformation. We also determined by competitive BiFC in cell culture that the GTFs: TBP, Bip2, TFIIB y TFIIAγ, as well as homeoproteins EXD and AbdB interfere with the Antp-TFIIEβ dimeric interaction, suggesting the probable formation of trimeric complexes. In conclusion, interaction of Antp with TFIIEβ is important for the functional specificity of Antp, 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-TFIIEβ interaction on the transcriptional control for the activation and/or repression of target genes in the Hox interactome during Drosophila development

Interacciones moleculares proteína-proteína de ANTP con el factor TFIIE-β a través de los residuos 32 y 36 del homeodominio

Las homeoproteínas contienen el homeodominio (HD) que actúa como dominio de unión al DNA y así mismo participa en interacciones proteína-proteína que intensifican la especificidad de unión. La homeoproteína Antennapedia (Antp) es responsable de la formación de segmentos torácicos y de la cabeza. El motivo de unión proteína-proteína YPWM liga a Antp a la maquinaria basal transcripcional ya que interactúa con el factor transcripcional BIP2. Estudios realizados en el laboratorio mostraron que Antp interacciona con el factor transcripcional TFIIE-β y que la hélice II del HD está involucrada en esta interacción (Cárdenas-Chávez 2012). Como los residuos 32 y 36 de la hélice II del HD se encuentran expuestos en la hélice II y están conservados en Antp y otras homeoproteínas, el objetivo de ésta tesis consistió en determinar si éstas posiciones están involucradas en la interacción molecular proteína-proteína Antp-TFIIEβ. Para ello, se realizó mutagénesis sitio-dirigida de los residuos 32 y 36 de la hélice II de Antp y se determinó su efecto en la interacción proteína-proteína con TFIIE-β mediante Fluorescencia por Complementación Bimolecular (BiFC) y activación de la transcripción. Los resultados de BiFC mostraron una considerable disminución de la interacción molecular en células transfectadas con las mutantes AntpI32L (29%), AntpI32A (31%), AntpH36R (23%) y AntpH36A (25%) con TFIIE-β. Las mutantes dobles de los residuos 32 y 36 mostraron una drástica reducción del porcentaje de interacción con TFIIE-β correspondientes al 11% con AntpI32L-H36R y 12% con AntpI32A-H36A. La falta de interacción de Antp con TFIIE-β debido a la mutación de los residuos I32 y H36 en la hélice II recuperó la actividad transactivadora de Antp en cultivo celular ya que no mostró efecto significativo en ausencia y presencia de TFIIE-β. Estos resultados en conjunto indican que los residuos 32 y 36 de la hélice II del HD de Antp están involucrados directamente en la interacción proteína-proteína con TFIIEβ y rescataron la actividad transcripcional de Antp en los sitios blanco de unión al DNA. El análisis de interacción molecular contribuye al conocimiento de los procesos de regulación génica de homeoproteínas mediante interacciones proteína-proteína con factores generales de transcripción como TFIIE-β que requieren ser verificados in vivo en Drosophila para determinar cómo contribuyen a la especificidad funcional. - 7 - 2. ABSTRAC

Conditioned medium derived from murine BM-MSCs cultured as spheroids exhibit in vitro immunomodulatory capacity

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Trimeric interaction of Antp-Ubx with TBP and homeoprotein EXD in the genetic control of development in D. melanogaster

Homeoproteins specify body segments along the anteroposterior axis during embryo development; they bind DNA through the homeodomain (HD). Homeoproteins bind to similar and repetitive target sequences on DNA, raising the question of how they achieve functional specificity. Homeoproteins form complexes with proteins through the HDs or other regions. Antennapedia (Antp) and Ultrabithorax (Ubx) have an important role in conferring thorax identity; also they are involved in a repression transcriptional mechanism: Ubx binds to Antp promoter repressing its expression. We analyzed if Ubx and Antp perform protein-protein interaction as a regulation mechanism and if the complex Antp-Ubx are involved in trimeric interaction with other transcription factors. Using Bimolecular Fluorescence Complementation (BIFC) we showed Antp-Ubx interaction in cell culture and in vivo in embryos and imaginal discs of Drosophila melanogaster. Also, we detected the Antp HD importance as well as the E19G residue in the interaction. We next analyzed if dimer Antp-Ubx affected Antp function in head involution on larvae, showing 80% of larvae with homeosis. In adult flies dimer Antp-Ubx caused a partial antenna to leg transformation. Additionally, using BiFC-based FRET, we showed that Antp-Ubx form trimeric complexes with TBP and EXD in cell culture and we test the trimeric complexes function in vivo in head involution on larvae, showing 80% of homeosis expressing Antp-Ubx/TBP complex and 66% of homeosis expressing Antp-Ubx/EXD complex. We conclude that Antp-Ubx is involved in trimeric interaction with TBP and EXD and both trimeric complexes are important for Antp function in vivo

Development of a dynamic SH-SY5Y 3D culture model for biological evaluation of Alzheimer-induced pathology

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Antp transcriptional activity is modulated by the formation of the trimeric Antp-TBP complexes with TFIIEβ, Exd and BIP2

Homeoproteins are transcriptional factors that bind to DNA through a highly conserved binding domain known as the homeodomain (HD) which recognizes short regions rich in AT to control the development of the body appendages of organisms. However, their structural and recognition similarities make it difficult to explain how homeoproteins are capable of carrying out their function. Previous results have shown that Antp homeoprotein can establish dimeric interactions with TBP, TFIIEβ, Exd, BIP2 and more recently through BiFC-FRET we confirmed that Antp and TBP can form trimeric complexes with TFIIEβ/Exd/BIP2. Therefore, is important to show how these trimeric complexes modulate Antp transcriptional activity. The experimental approach selected for this project was to perform in vitro transactivation assays in HEK-293 cells transfected with the combinations of Antp and TBP-producing plasmids with TFIIEβ / Exd / BIP2 plasmids using a Luciferase reporter plasmid. Our results show that trimeric interaction of Antp-TBP-TFIIEβ induced a significant increase of 138% in the Antp transcriptional activity. By contrast, trimeric complexes of Antp-TBP-Exd and Antp-TBP-BIP2 modified the transactivating capacity of Antp, decreasing transcription by 20 and 26% respectively. According to this, we were able to confirm that the trimeric complexes Antp-TBP/TFIIEβ/Exd/BIP2 are involved in the modulation of Antp transcriptional activity. So, now it is interesting to analyze how these trimeric complexes are involved in the activation and/or repression of target genes of Antp during genetic control of development in Drosophila melanogaster

Trimeric complex interactions of Antp-TBP with TFIIEB and Exd are involved in the genetic control of Drosophila melanogaster

Background: Homeoproteins are transcriptional factors (TFs) that shape animal body axes during development. These TFs are highly conserved and represent one of the most fascinating groups of regulatory molecules. Reports shown the multiplicity of interactions in hox proteins, as complexes trimeric involved to transcriptional activity. The study of trimeric complexes in Hox interactome will allow the better understanding of Hox genetic regulation during embryonic development. Methodology: Using a new combination BiFCFRET approach performed in HEK293, the quantification was performed by FRETTY of ImageJ. Fly crosses were incubated at 25ºC on standard yeast-agar-cornmeal medium. Embryo cuticle preparations were carried out according to (Gibson & Gehring 1998) and mounted on slides with Visicol. For adult imaging, the heads and antennae were dissected and directly transferred to microscopic slides without coverslips. The images were merged using the software HeliconFocus. Results: We found the presence of trimeric interactions of Antp-TBP-TFIIEβ and Antp-TBP-Exd in cell culture. Further were confirmed these trimeric interactions using Antp mutants. Interestingly, the trimeric Antp-TBP-TFIIEB shown diminish of homeotic effect caused by Antp in larvae. To analyze the effect in antenna-leg transformation we drive the trimeric complex and found inhibition of antenna-leg in head of D. melanogaster. Antp-TBP-TFIIEB shown homeotic reduction of 77% in larvae and found inhibition of 100% of antenna-leg transformation. Conclusions: Our results show that these trimeric interactions are involved in the genetic control of Drosophila melanogaster

Simultaneous genetic engineering and culture media manipulation improve rh-EPO productivity through lactate re-metabolization on CHO cell culture

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Transcription factor TFIIEβ interacts with two exposed positions in helix 2 of the Antennapedia homeodomain to control homeotic function in Drosophila

Abstract 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, TFIIEβand 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-TFIIEβ 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 TFIIEβinteracts 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 TFIIEβin the antennal disc showed that this interaction is required for the antenna-to-tarsus transformation. In conclusion, interaction of Antp with TFIIEβis 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-TFIIEβinteraction on the transcriptional control for the activation and/ or repression of target genes in the Hox interactome during Drosophila development

Centros de escritura universitarios: una estrategia para la permanencia estudiantil

PublishedLas universidades colombianas han sido precursoras en la creación de centros y programas de escritura en Latinoamérica, así como en la producción y difusión de estudios sobre su labor. No es casual, entonces, que la iniciativa para la publicación de un nuevo libro haya surgido en el ámbito académico colombiano. Gracias a la convocatoria del Centro de Escritura, Lectura y Oralidad Académica de la Universidad Santiago de Cali (CELOA) nació Centros de escritura universitarios: Una estrategia para la permanencia estudiantil, un libro que reúne investigaciones llevadas a cabo por cinco centros de escritura de universidades colombianas. El título se nutre de la experiencia y de los trabajos de investigadores que desde hace décadas vienen aportando y reflexionando sobre la importancia de la escritura y la lectura para la construcción del saber y la comunicación de ideas. Entre las contribuciones más citadas por los autores de los capítulos se encuentran las de Paula Carlino, quien en su libro Escribir, leer y aprender en la universidad (Fondo de Cultura Económica, 2005) introdujo el concepto de “alfabetización académica”, que ha sido fundamental para desarrollar prácticas y profundizar en los estudios sobre la enseñanza terciaria