A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.

A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.

Os estresses abióticos impostos à planta no campo, tais como o estresse salino, toxidez por alumínio, frio, seca, entre outros, afetam seu crescimento, desenvolvimento e produtividade. Dentre as gramíneas cultivadas, o arroz (Oryza sativa) é uma das culturas de maior importância no Brasil, cuja oscilação na produção acarreta prejuízos consideráveis à economia brasileira. Dessa forma, o estudo das interações entre os estresses abióticos e as respostas dos vegetais frente a esses estímulos ambientais é fundamental para um conhecimento detalhado desses mecanismos. O arroz é um dos cereais mais tolerantes ao alumínio (Al), sendo um ótimo modelo para o estudo de mecanismos de tolerância. Neste trabalho, foi estudado o papel das proteínas ASR na tolerância ao Al. A expressão dos genes ASR (ABA, Stress and Ripening) é induzida por ABA, estresses e amadurecimento do fruto. Essas proteínas foram caracterizadas como chaperonas e fatores de transcrição. Os genes da família OsASR também têm o nível de transcritos aumentado em resposta ao Al, sendo OsASR1 e OsASR5 os genes com expressão mais abundante em arroz. No entanto, as regiões promotoras responsivas ao Al e os fatores de transcrição reguladores da expressão desses genes ainda não foram descritos. Assim, este trabalho tem como objetivo geral a identificação dos fatores que regulam a transcrição dos genes OsASR1 e OsASR5. Para tanto, construções contendo fragmentos dos promotores desses genes dirigindo a expressão do gene repórter GUS foram obtidas, visando ensaio de expressão transiente em protoplastos de arroz submetidos ao tratamento com Al. Os fragmentos das regiões promotoras de ambos os genes OsASR1 e OsASR5 foram todos responsivos ao Al. Também foi realizado ensaio de transativação em protoplastos de Arabidopsis thaliana para verificar a existência de auto-regulação nesses genes. Tanto ASR1 quanto ASR5 foram capazes de transativar seus próprios promotores. Além disso, foi realizada uma triagem de biblioteca de cDNAs por mono-híbrido em levedura com fragmento da região promotora de OsASR5. Com essa abordagem, foram identificados sete genes candidatos a codificadores de fatores capazes de interação DNA-proteína. / The abiotic stress that plants in the field are subjected to, such as salt, aluminum, cold, drought, among others, affect their growth, development and productivity. Among cultivated grasses, rice (Oryza sativa) is one of the most important crops in Brazil, whose oscillation in production causes considerable costs to the Brazilian economy. Thus, the study of interactions between abiotic stresses and plant responses to these environmental stimuli is essential to a detailed knowledge of these mechanisms. Rice is one of the most Al-tolerant crops, being a great model for studying Al-tolerance mechanisms. In this work, the role of ASR proteins in Al tolerance was studied. The expression of ASR (ABA, Stress and Ripening) genes is induced by ABA, stresses and fruit ripening. These proteins were characterized as chaperones and transcription factors. The OsASR genes also have increased transcript accumulation in response to Al, and OsASR1 and OsASR5 have the most abundant expression in rice. However, the Al-responsive promoter regions and the transcription factors that regulate these genes have not yet been described. Therefore, the goal of this work is to identify regulating factors of OsASR1 and OsASR5 gene transcription. For this, vectors containing promoter fragments of these genes driving the expression of the GUS gene were constructed and transient expression assays were performed in rice protoplasts subjected to Al treatment. All of the promoter fragments were Al-responsive for both OsASR1 and OsASR5 genes. Transactivation assays in Arabidopsis thaliana protoplasts were also conducted in order to verify the existence of auto-regulation in these genes. Both ASR1 and ASR5 were able to transactivate its own promoters. Furthermore, a library screening was performed by yeast one-hybrid using a promoter fragment from OsASR5. With this approach, seven candidate genes encoding transcription factors capable of DNA-protein interactions were identified.

Os estresses abióticos impostos à planta no campo, tais como o estresse salino, toxidez por alumínio, frio, seca, entre outros, afetam seu crescimento, desenvolvimento e produtividade. Dentre as gramíneas cultivadas, o arroz (Oryza sativa) é uma das culturas de maior importância no Brasil, cuja oscilação na produção acarreta prejuízos consideráveis à economia brasileira. Dessa forma, o estudo das interações entre os estresses abióticos e as respostas dos vegetais frente a esses estímulos ambientais é fundamental para um conhecimento detalhado desses mecanismos. O arroz é um dos cereais mais tolerantes ao alumínio (Al), sendo um ótimo modelo para o estudo de mecanismos de tolerância. Neste trabalho, foi estudado o papel das proteínas ASR na tolerância ao Al. A expressão dos genes ASR (ABA, Stress and Ripening) é induzida por ABA, estresses e amadurecimento do fruto. Essas proteínas foram caracterizadas como chaperonas e fatores de transcrição. Os genes da família OsASR também têm o nível de transcritos aumentado em resposta ao Al, sendo OsASR1 e OsASR5 os genes com expressão mais abundante em arroz. No entanto, as regiões promotoras responsivas ao Al e os fatores de transcrição reguladores da expressão desses genes ainda não foram descritos. Assim, este trabalho tem como objetivo geral a identificação dos fatores que regulam a transcrição dos genes OsASR1 e OsASR5. Para tanto, construções contendo fragmentos dos promotores desses genes dirigindo a expressão do gene repórter GUS foram obtidas, visando ensaio de expressão transiente em protoplastos de arroz submetidos ao tratamento com Al. Os fragmentos das regiões promotoras de ambos os genes OsASR1 e OsASR5 foram todos responsivos ao Al. Também foi realizado ensaio de transativação em protoplastos de Arabidopsis thaliana para verificar a existência de auto-regulação nesses genes. Tanto ASR1 quanto ASR5 foram capazes de transativar seus próprios promotores. Além disso, foi realizada uma triagem de biblioteca de cDNAs por mono-híbrido em levedura com fragmento da região promotora de OsASR5. Com essa abordagem, foram identificados sete genes candidatos a codificadores de fatores capazes de interação DNA-proteína. / The abiotic stress that plants in the field are subjected to, such as salt, aluminum, cold, drought, among others, affect their growth, development and productivity. Among cultivated grasses, rice (Oryza sativa) is one of the most important crops in Brazil, whose oscillation in production causes considerable costs to the Brazilian economy. Thus, the study of interactions between abiotic stresses and plant responses to these environmental stimuli is essential to a detailed knowledge of these mechanisms. Rice is one of the most Al-tolerant crops, being a great model for studying Al-tolerance mechanisms. In this work, the role of ASR proteins in Al tolerance was studied. The expression of ASR (ABA, Stress and Ripening) genes is induced by ABA, stresses and fruit ripening. These proteins were characterized as chaperones and transcription factors. The OsASR genes also have increased transcript accumulation in response to Al, and OsASR1 and OsASR5 have the most abundant expression in rice. However, the Al-responsive promoter regions and the transcription factors that regulate these genes have not yet been described. Therefore, the goal of this work is to identify regulating factors of OsASR1 and OsASR5 gene transcription. For this, vectors containing promoter fragments of these genes driving the expression of the GUS gene were constructed and transient expression assays were performed in rice protoplasts subjected to Al treatment. All of the promoter fragments were Al-responsive for both OsASR1 and OsASR5 genes. Transactivation assays in Arabidopsis thaliana protoplasts were also conducted in order to verify the existence of auto-regulation in these genes. Both ASR1 and ASR5 were able to transactivate its own promoters. Furthermore, a library screening was performed by yeast one-hybrid using a promoter fragment from OsASR5. With this approach, seven candidate genes encoding transcription factors capable of DNA-protein interactions were identified.

Os estresses abióticos impostos à planta no campo, tais como o estresse salino, toxidez por alumínio, frio, seca, entre outros, afetam seu crescimento, desenvolvimento e produtividade. Dentre as gramíneas cultivadas, o arroz (Oryza sativa) é uma das culturas de maior importância no Brasil, cuja oscilação na produção acarreta prejuízos consideráveis à economia brasileira. Dessa forma, o estudo das interações entre os estresses abióticos e as respostas dos vegetais frente a esses estímulos ambientais é fundamental para um conhecimento detalhado desses mecanismos. O arroz é um dos cereais mais tolerantes ao alumínio (Al), sendo um ótimo modelo para o estudo de mecanismos de tolerância. Neste trabalho, foi estudado o papel das proteínas ASR na tolerância ao Al. A expressão dos genes ASR (ABA, Stress and Ripening) é induzida por ABA, estresses e amadurecimento do fruto. Essas proteínas foram caracterizadas como chaperonas e fatores de transcrição. Os genes da família OsASR também têm o nível de transcritos aumentado em resposta ao Al, sendo OsASR1 e OsASR5 os genes com expressão mais abundante em arroz. No entanto, as regiões promotoras responsivas ao Al e os fatores de transcrição reguladores da expressão desses genes ainda não foram descritos. Assim, este trabalho tem como objetivo geral a identificação dos fatores que regulam a transcrição dos genes OsASR1 e OsASR5. Para tanto, construções contendo fragmentos dos promotores desses genes dirigindo a expressão do gene repórter GUS foram obtidas, visando ensaio de expressão transiente em protoplastos de arroz submetidos ao tratamento com Al. Os fragmentos das regiões promotoras de ambos os genes OsASR1 e OsASR5 foram todos responsivos ao Al. Também foi realizado ensaio de transativação em protoplastos de Arabidopsis thaliana para verificar a existência de auto-regulação nesses genes. Tanto ASR1 quanto ASR5 foram capazes de transativar seus próprios promotores. Além disso, foi realizada uma triagem de biblioteca de cDNAs por mono-híbrido em levedura com fragmento da região promotora de OsASR5. Com essa abordagem, foram identificados sete genes candidatos a codificadores de fatores capazes de interação DNA-proteína. / The abiotic stress that plants in the field are subjected to, such as salt, aluminum, cold, drought, among others, affect their growth, development and productivity. Among cultivated grasses, rice (Oryza sativa) is one of the most important crops in Brazil, whose oscillation in production causes considerable costs to the Brazilian economy. Thus, the study of interactions between abiotic stresses and plant responses to these environmental stimuli is essential to a detailed knowledge of these mechanisms. Rice is one of the most Al-tolerant crops, being a great model for studying Al-tolerance mechanisms. In this work, the role of ASR proteins in Al tolerance was studied. The expression of ASR (ABA, Stress and Ripening) genes is induced by ABA, stresses and fruit ripening. These proteins were characterized as chaperones and transcription factors. The OsASR genes also have increased transcript accumulation in response to Al, and OsASR1 and OsASR5 have the most abundant expression in rice. However, the Al-responsive promoter regions and the transcription factors that regulate these genes have not yet been described. Therefore, the goal of this work is to identify regulating factors of OsASR1 and OsASR5 gene transcription. For this, vectors containing promoter fragments of these genes driving the expression of the GUS gene were constructed and transient expression assays were performed in rice protoplasts subjected to Al treatment. All of the promoter fragments were Al-responsive for both OsASR1 and OsASR5 genes. Transactivation assays in Arabidopsis thaliana protoplasts were also conducted in order to verify the existence of auto-regulation in these genes. Both ASR1 and ASR5 were able to transactivate its own promoters. Furthermore, a library screening was performed by yeast one-hybrid using a promoter fragment from OsASR5. With this approach, seven candidate genes encoding transcription factors capable of DNA-protein interactions were identified.

Gene regulatory networks (GRNs) are central to every biological process from development to disease. GRNs are mediated through the activities of transcription factors (TFs), which interact in a sequence-specific manner with their target DNA elements to drive gene expression. In this thesis, two main aspects of GRNs are studied: (1) rewiring of GRNs by alternative TF isoforms, and (2) immune GRNs and strategies to modulate gene expression in immune diseases. TF isoforms resulting from alternative splicing, alternative transcription start sites, or alternative transcription termination sites, are prevalent and can have profound changes in GRNs. However, the extent to which differences in TF isoforms affect global GRNs and how such regulatory network rewiring leads to altered gene expression programs remain unclear. In this thesis, a large clone collection of ~800 human TF isoforms was generated, and then used in high-throughput systematic experimental strategies to investigate the extent to which TF isoforms differ at the level of molecular protein-DNA interactions (PDIs) and transcriptional regulatory activities. The findings show that at least half of alternative TF isoforms exhibit functional differences and tend to behave like distinct proteins with different molecular capabilities. In the context of global GRNs, these findings reveal a widespread expansion of PDI and transcriptional regulatory capabilities through alternative TF isoforms. Altogether, this work constitutes an important step towards the long-term goal of contextualizing and functionalizing large numbers of TF isoforms in rewiring GRNs. GRNs provide a wealth of information that can be leveraged in myriad ways including therapeutics. In particular, immune GRNs provide a framework for modulating cytokine gene expression, which are dysregulated in many human diseases. Proper cytokine gene expression is essential in development, homeostasis and immune responses. However, studies on the transcriptional control of cytokine genes over the last three decades have mostly focused on highly researched TFs and cytokines, resulting in an incomplete portrait of cytokine gene regulation. In this thesis, high-throughput assays were used to derive a comprehensive network that greatly expands the known repertoire of TF–cytokine gene PDIs and the set of TFs known to regulate cytokine genes. An enrichment of nuclear receptors was found and their role in cytokine regulation in primary macrophages was confirmed. Additionally, the network was used as a framework to identify TFs and synergistic TF pairs that can be targeted with FDA-approved drugs to modulate cytokine production. Finally, the PDI data was integrated with single cell RNA-seq datasets to identify druggable TF targets in cytokine-associated immune diseases (i.e., inflammatory bowel disease and COVID-19). Overall, this comprehensive cytokine GRN provides a rich resource to interrogate cytokine regulation in a variety of physiological and disease contexts. Altogether, the work in this thesis accomplishes the following: (1) identifies alternative TF isoforms as a major driver of GRN rewiring, (2) delineates a comprehensive cytokine GRN that greatly expands three decades of research, and (3) leverages the cytokine GRN to identify candidate therapeutic TF targets in diseases associated with dysregulated cytokine gene expression. These findings contribute a significant step in the effort to understand mechanisms of GRN rewiring and to generate comprehensive GRNs that provide a framework for modulating gene expression, particularly in diseases. / 2023-11-01T00:00:00Z

No description available.

Secondary xylem, commonly known as wood, is a valuable commercial commodity. Among the major components of wood are the elongated, thick-walled water-conducting cells known as tracheary elements. Understanding tracheary element differentiation and maturation is of scientific and commercial importance as it may lead to broad understanding of cellular differentiation processes as well as ways to increase both the quality and quantity of wood produced by economically important tree species. One way to begin to understand the regulation of tracheary element differentiation is to identify elements that control expression of genes associated with tracheary elements. In Arabidopsis thaliana, Xylem Cysteine Protease 1 (XCP1) is specifically expressed in tracheary elements where it catalyzes microautolysis. Thus XCP1 can serve as a useful model for identifying factors that regulate tracheary element-specific gene expression. A deletion analysis of the XCP1 promoter was conducted to identify promoter elements that are necessary and sufficient for tracheary element-restricted gene expression. Two regions required for tracheary element-specific gene expression were identified. One of these was assembled as a multimeric bait construct and used in yeast one-hybrid assays to identify candidate transcription factors that bind to the XCP1 promoter region. Subsequently, a southwestern blot analysis was used to identify transcription factors displaying specific binding to a previously reported cis-element, CTTCAAAGCCA, found in the XCP1 promoter and other tracheary element-associated genes from multiple species. / Master of Science

The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. / Ph. D. / Trees are unique among plants since they have extremely long life spans and the ability to generate large quantities of woody biomass. The woody biomass derived from forest trees can function to provide renewable energy in the form of biofuels. The process of wood formation is complex and requires coordinated activation of genes involved in multiple metabolic pathways. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA. These protein-DNA interactions regulate gene expression during plant growth and development. In this study, new regulators of genes known to be involved in wood formation were identified using the Yeast One-Hybrid (Y1H) assay. One of the proteins identified, PtrRAD1 had not been previously linked to wood formation and was a candidate for further characterization. Further testing of PtrRAD1 by the Yeast Two-Hybrid (Y2H) assay resulted in identification of a protein-protein interaction with Populus trichocarpa DIVARICATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay to identify numerous interacting proteins, in addition to those reported previously in other species. Further characterization of PtrDRIF1, identified an N-terminal region capable of forming interactions with MYB/SANT domain proteins, and C-terminal region that interacted with homeodomain proteins. PtrRAD1, PtrDRIF1, and the homeodomain containing PtrWOX13c were able to form a complex in an Y2H-bridge assay. Combined, these findings support a potential role for PtrDRIF1 in regulating wood polarity, wood formation, and stem cell proliferation.

PIN-FORMED (PIN) proteins are plant-specific secondary transporters acting in the efflux of plant signaling molecule auxin from cells. Their asymmetrical localization within cells determines the directionality of auxin flow and thereby influences plant development. The activity of PIN proteins is regulated at multiple levels; however the primary step in the regulation of PIN proteins takes place at the level of gene transcription. Therefore the main focus of this diploma thesis is the characterization of the transcriptional regulation of PIN proteins, namely PIN4 protein. The observation of plants carrying transcriptional fusion consisting of various lengths of PIN4 promoter and green fluorescent protein (GFP) showed which part of PIN4 promoter is essential for binding transcription factors and for the start of transcription. This part of PIN4 promoter was used as bait for transcription factors in yeast one hybrid screens. Altogether, 24 transcription factors were identified in which the most numerous were transcription factors from GATA and APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) families. To verify the interactions between identified transcription factors and PIN4 promoter, the protoplast transient expression assay was used. Protoplasts isolated from Arabidopsis thaliana leaves and tobacco BY-2 cell...