Receptor-DNA interactions: EMSA and footprinting

Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.

Valoración de la Electrificadora del Meta S.A. ESP – EMSA ESP

El objetivo del presente estudio es establecer el valor de la Electrificadora del Meta SA ESP EMSA ESP, para ello se hace una revisión general del departamento del Meta, el sector eléctrico Colombiano, y la EMSA ESP, se revisa la forma como la actividad empresarial de EMSA ESP en su papel de distribuidor y comercializador de energía eléctrica ha influido en el desarrollo y crecimiento de las comunidades del departamento del Meta -- El análisis finalmente establece el valor económico de EMSA ESP mediante el uso de la metodología de flujos de caja descontados tomando como base la información pública disponible de la Compañía y las proyecciones de los estados financieros necesarios para realizar la valoración; dado que se trata de una valoración externa e independiente de algún interés particular se presenta este ejercicio para que sirva como herramienta para la toma decisiones de inversión, pues se concluye que EMSA ESP es atractiva para los inversionistas si se tiene en cuenta los desarrollos económicos que se están presentando en el departamento del Meta y la Orinoquia Colombiana que genera una expectativa positiva en cuanto a su viabilidad económica y financiera para los próximos 10 años

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

Gemini viral assembly and transport of viral DNA into nucleus for replication, ssentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton LeafCtirl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and Surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K-A, of 2.6 +/- 0.29 x 10(8) M-1 in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a Crucial role ill Virus assembly and in nuclear transport. (C) 2009 Elsevier Inc.

Genomic and functional profiling of gastric cancer

Helicobacter pylori infection is a risk factor for gastric cancer, which is a major health issue worldwide. Gastric cancer has a poor prognosis due to the unnoticeable progression of the disease and surgery is the only available treatment in gastric cancer. Therefore, gastric cancer patients would greatly benefit from identifying biomarker genes that would improve diagnostic and prognostic prediction and provide targets for molecular therapies. DNA copy number amplifications are the hallmarks of cancers in various anatomical locations. Mechanisms of amplification predict that DNA double-strand breaks occur at the margins of the amplified region. The first objective of this thesis was to identify the genes that were differentially expressed in H. pylori infection as well as the transcription factors and signal transduction pathways that were associated with the gene expression changes. The second objective was to identify putative biomarker genes in gastric cancer with correlated expression and copy number, and the last objective was to characterize cancers based on DNA copy number amplifications. DNA microarrays, an in vitro model and real-time polymerase chain reaction were used to measure gene expression changes in H. pylori infected AGS cells. In order to identify the transcription factors and signal transduction pathways that were activated after H. pylori infection, gene expression profiling data from the H. pylori experiments and a bioinformatics approach accompanied by experimental validation were used. Genome-wide expression and copy number microarray analysis of clinical gastric cancer samples and immunohistochemistry on tissue microarray were used to identify putative gastric cancer genes. Data mining and machine learning techniques were applied to study amplifications in a cross-section of cancers. FOS and various stress response genes were regulated by H. pylori infection. H. pylori regulated genes were enriched in the chromosomal regions that are frequently changed in gastric cancer, suggesting that molecular pathways of gastric cancer and premalignant H. pylori infection that induces gastritis are interconnected. 16 transcription factors were identified as being associated with H. pylori infection induced changes in gene expression. NF-κB transcription factor and p50 and p65 subunits were verified using elecrophoretic mobility shift assays. ERBB2 and other genes located in 17q12- q21 were found to be up-regulated in association with copy number amplification in gastric cancer. Cancers with similar cell type and origin clustered together based on the genomic localization of the amplifications. Cancer genes and large genes were co-localized with amplified regions and fragile sites, telomeres, centromeres and light chromosome bands were enriched at the amplification boundaries. H. pylori activated transcription factors and signal transduction pathways function in cellular mechanisms that might be capable of promoting carcinogenesis of the stomach. Intestinal and diffuse type gastric cancers showed distinct molecular genetic profiles. Integration of gene expression and copy number microarray data allowed the identification of genes that might be involved in gastric carcinogenesis and have clinical relevance. Gene amplifications were demonstrated to be non-random genomic instabilities. Cell lineage, properties of precursor stem cells, tissue microenvironment and genomic map localization of specific oncogenes define the site specificity of DNA amplifications, whereas labile genomic features define the structures of amplicons. These conclusions suggest that the definition of genomic changes in cancer is based on the interplay between the cancer cell and the tumor microenvironment.

Transcriptional Repression of Tumor Suppressor CDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation IMPLICATION FOR CANCER THERAPEUTICS

The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses beta-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

The TSC2 gene, mutated in patients with tuberous sclerosis complex (TSC), encodes a 200 kDa protein TSC2 (tuberin). The importance of TSC2 in the regulation of cell growth and proliferation is irrefutable. TSC2 in complex with TSC1 negatively regulates the mTOR complex 1 (mTORC1) via RHEB in the PI3K-AKT-mTOR pathway and in turn regulates cell proliferation. It shows nuclear as well as cytoplasmic localization. However, its nuclear function remains elusive. In order to identify the nuclear function of TSC2, a whole-genome expression profiling of TSC2 overexpressing cells was performed, and the results showed differential regulation of 266 genes. Interestingly, transcription was found to be the most populated functional category. EREG (Epiregulin), a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in TSC lesions, making its regulatory aspects intriguing. Using the luciferase reporter, ChIP and EMSA techniques, we show that TSC2 binds to the EREG promoter between -352 bp and -303 bp and negatively regulates its expression. This is the first evidence for the role of TSC2 as a transcription factor and of TSC2 binding to the promoter of any gene.

Estudio sobre helminthosporiosis en maiz efecto en rendimiento y epidemiología

Este estudio fue realizado para establecer una relación entre la severidad de Helminthosporium maydis y el rendimiento de maíz. Para alcanzar este objetivo se realizó un experimento de campo en el cual se trata de establecer una infección artificial del hongo; para la cual fue necesario desarrollar un método de reproducción masiva de conidios. Para reproducción masiva de H. maydis, un medio hecho a base de extracto de hoja de Maíz –Sacarosa –Agar (EMSA), resultó mejor que un medio papa-Dextrosa-Agar (P.DA) o Jugo V8, encontrándose que la reproducción repetida del hongo en un PDA y Jugo V8 produce conidios mal formados. Las condiciones climatológicas (4 horas de humedad relativa mayor de 95%, temperatura de 27ºc y 8 horas de sol durante el (día) que se determinaron después de la Inoculación artificial, no favorable para lograr una buena infección artificial del patógeno. Posteriormente en el cultivo de maíz surgió una infección natural: Mediante diferentes “tratamientos” se logró establecer diferentes niveles de severidad de H. maydis en las parcelas. Los tratamientos consistían en 6 aplicaciones de manzate (M&) (Maneb), tres aplicaciones de Manzate (M3) y una aplicación de Manzate (M1); seis aplicaciones de Difolatàn (D6) (Captalof). El tratamiento de seis aplicaciones de Manzate permitió alcanzar un rendimiento de 5886.3, el cual difiere de forma significativa del rendimiento obtenido de una aplicación de Manzate /4368.3 kg/ha). No hay diferencia entre una y tres aplicaciones de Manzate (5270.3 kg/ha). El tratamiento con difolatàn / produce conidios mal formados (5298.5 kg/ha) no difiere de forma significativa del rendimiento obtenido con una o tres aplicaciones de Manzate. En relación con la severidad de la enfermedad y el rendimiento, se elaboró en base a los datos obtenidos un modelo que describe el rendimiento en función de la severidad de H maydis y la altura de la planta. La ecuación que describe el rendimiento es: Y= 0.428 x 1 - 2.722 x 2 donde Y= rendimiento gramos/planta X1= altura de la planta en cm. A los 61 días después de la germinación. X2= porcentaje de la superficie foliar afectada por H. maydis a los 71 días después de la germinación. La constante es =0. De acuerdo a este modelo se conoce que por cada gramo en la severidad se pierde 181 kg/ha (en una densidad de siembra 66,500/ha. Este modelo es válido para condiciones con severidad de 4.3% de virus, 0.4% bacteria, 0.25 daño causado por psocóptera frugipterda y condiciones de clima y suelo similares a los de este experimento.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Efeito de ExoU na ativação de NF-κB e na secreção de IL-8 por células humanas infectadas por Pseudomonas aeruginosa

ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citossol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. Estudos anteriores realizados em nosso laboratório relataram a potente atividade pró-inflamatória de ExoU, responsável por um intenso recrutamento de neutrófilos para o sítio de infecção. No presente trabalho, o efeito de ExoU na modulação da ativação do fator transcricional NF-κB e na regulação da expressão e da secreção da quimiocina para neutrófilos IL-8 foi avaliado em culturas de células epiteliais respiratórias e endoteliais humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU) ou com a mutante deletada no gene exoU, PA103κexoU. Análises por RT-PCR semi-quantitativo mostraram que a infecção pela cepa produtora de ExoU levou ao aumento dos níveis de mRNA de IL-8, enquanto ensaios de alteração da mobilidade eletroforética (EMSA), supershift e com gene repórter mostraram que ExoU induziu a translocação nuclear do heterodímero transativador p65/p50 de NF-κB e a ativação da transcrição de genes dependente deste fator transcricional. Adicionalmente, o tratamento das culturas celulares com um inibidor de NF-κB antes da infecção bacteriana reduziu significativamente os níveis de mRNA de IL-8 e da secreção desta quimiocina. Em conjunto, estes resultados mostram que ExoU ativa NF-κB e, consequentemente, estimula a expressão e a secreção de IL-8 por células epiteliais respiratórias e células endoteliais infectadas com P. aeruginosa

Estudo da cromatina nos sítios de fixação à matriz nuclear no domínio do gene TP53 e das modificações epigenéticas e no modelo de progressão tumoral mamária 21T

Dentre os diversos tipos de câncer agressivos, o câncer de mama é o mais comum em mulheres. Mutações hereditárias e adquiridas, assim como alterações epigenéticas atuam em sinergia na carcinogênese mamária e na progressão tumoral. A proteína P53 é uma supressora de tumor e possui uma atuação fundamental na integridade genômica. Apesar do vasto conhecimento sobre o controle da P53 a nível de proteína, ainda pouco se sabe sobre o controle transcricional do gene TP53. A série 21T, uma série de 4 linhagens celulares originadas da mama da mesma paciente, representando diferentes estágios de progressão tumoral mamária, é um eficiente modelo para investigação das alterações epigenéticas e suas influências na expressão gênica ao longo da progressão do câncer de mama. Nós analisamos a organização do domínio do gene TP53 através da técnica de arranjo de DNA, em diversas linhagens celulares de câncer de mama e linhagens controle, e realizamos uma tentativa de caracterizar estes elementos de DNA nas linhagens controle não-tumorais HB2 e MCF10A e nas tumorais MCF-7, MDA-MB-231, T47D, através dos marcadores epigenéticos de eucromatina, H4Ac, e heterocromatina, H3K9me3. Ainda analisamos a ligação de proteínas à região associada à matriz nuclear (MAR), denominada MAR 2, e a possível ligação da proteína ligante à matriz nuclear (MARBP), PARP-1, através de ensaios de gel shift (EMSA). Detectamos que na linhagem controle epitelial mamária, HB2, o gene TP53 está posicionado num domínio de DNA relativamente pequeno, aproximadamente 50 kb, delimitado por dois sítios de fixação à matriz nuclear. Interessantemente, esta estrutura de domínio se apresentou radicalmente diferente nas linhagens de câncer de mama estudadas, MCF7, T47D, MDA-MB-231 e BT474, nos quais o tamanho do domínio estudado estava aumentado e a transcrição do TP53 diminuída. Os enriquecimentos com os marcadores epigenéticos de cromatina H4Ac e H3K9me3 estão diferentemente distribuídos nas MARs nas linhagens celulares. Surpreendentemente, a MAR 2 apresentou uma ligação altamente específica, o que poderia representar a atuação de fatores transcricionais envolvidos na organização da cromatina. Através de programas de bioinformática, detectamos putativos sítios para interessantes fatores de transcrição, tais como o c/EBP-beta e c-myb, que poderiam atuar em cis regulando a expressão do gene TP53 e outros flanqueadores. Nós propusemos um modelo para a organização da cromatina na região de domínio do gene TP53 com os genes flanqueadores. Através da série 21T, detectamos uma hipometilação global genômica, nas células cancerosas 21NT e 21MT1. Uma importante diminuição da expressão global do marcador H4Ac nas células metastáticas 21MT1, foi detectada em relação às outras linhagens. Os níveis de RNAm das principais enzimas relacionadas as modificações epigenéticas são consistentes com as observadas hipometilação genômica e hipoacetilação. Através de microscopia confocal, verificamos que o marcador H4Ac está localizado, na maior parte na periferia e o marcador H3K9me3, pericêntrico nos núcleos tumorais. Por fim, verificamos que o promotor P1 do gene TP53 apresenta um estado de cromatina aberta, e a expressão do gene TP53 é similar em todas as células da série 21T.

水稻OsMYB3R-2 基因对逆境响应的功能研究

低温威胁水稻的生产，其中苗期和生殖阶段对寒害是最敏感的时期。在苗期，阶段性冷害使水稻幼苗生长延迟，甚至造成烂秧现象；在生殖阶段，无法预测的突然降温会导致水稻花粉不育，并致使水稻大幅减产。因此，对水稻逆境胁迫调控的分子机制的深入研究在理论和实践上具有重要的意义。本研究从东乡野生稻、栽培稻及其杂交后代的低温芯片中筛选对低温响应基因的分析着手，对其中一个受低温诱导上调的基因OsMYB3R-2 作进一步研究。生物信息学的分析表明OsMYB3R-2 编码一个R1R2R3 MYB 蛋白，利用基因枪瞬时转化法、酵母GAL4 系统和电泳迁移率变动分析发现OsMYB3R-2 蛋白能够定位在细胞核中、具有转录激活和DNA 结合特性，表现为MYB 转录因子的典型特征。 超表达OsMYB3R-2 的转基因水稻呈现幼苗的矮化和生长相对滞后的表型，对低温胁迫具有耐受性。盐抑制水稻种子的萌发，与野生型和反义的株系相比，OsMYB3R-2 超表达株系的萌发对盐敏感，表现为萌发过程及萌发之后幼苗的生长更加滞后。而OsMYB3R-2 转基因株系对干旱处理敏感。为了进一步寻找OsMYB3R-2 蛋白的靶序列及其调控的靶基因，我们利用电泳迁移率变动分析发现OsMYB3R-2 能够与有丝分裂特异的激活子（mitosis-specific activator）元件特异结合。在低温条件下，OsMYB3R-2 超表达能够激活水稻G2/M 期特异基因的表达，主要包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1 等。另一方面，OsMYB3R-2 超表达能够增加根尖细胞的有丝分裂指数，这进一步说明OsMYB3R-2 参与了水稻细胞周期调控。EMSA、RT-PCR 和流式细胞仪分析的结果表明OsMYB3R-2 通过激活其靶基因OsCycB1;1 的表达参与水稻对低温胁迫的调控，该过程由细胞周期介导。 为了研究OsMYB3R-2 与水稻DREB/CBF 途径的关系，我们分析了转基因水稻中DREB/CBF 类基因及其可能调控的下游基因与OsMYB3R-2 的关系，RT-PCR 的结果表明超表达转基因植物中DREB 表达未见明显变化，而其下游基因OsCPT1 在低温条件下被激活表达。同时，转基因植物在低温条件下脯氨酸水平显著提高。这说明OsMYB3R-2 可能在水稻DREB/CBF 途径的下游参与调控。 总之，OsMYB3R-2 基因的超表达赋予转基因水稻在苗期对低温胁迫具有耐受性，并呈现矮化和生长滞后的表型。OsMYB3R-2 蛋白行使R1R2R3 MYB 转录因子的功能，在体外能够结合OsCycB1;1 和OsKNOLLE2 基因启动子中有丝分裂特异的激活子元件，在低温条件下激活了G2/M 期特异基因的表达，这些基因包括OsCycB1;1、OsCycB2;1、OsCycB2;2 和OsCDC20.1。低温条件下，在OsMYB3R-2 转基因超表达株系中OsCPT1 基因的转录被激活，细胞的游离脯氨酸的含量也显著增高。这些结果都表明OsMYB3R-2 基因在水稻的冷胁迫信号途径中起重要的作用，该过程受细胞周期及DREB/CBF 途径介导。 我们的实验结果暗示水稻对低温的耐受是通过分生组织细胞周期调控完成的，这个过程由OsMYB3R-2 等关键基因控制。

Glutathione-Mediated Release of Functional Plasmid DNA from Positively Charged Quantum Dots

DNA was efficiently bound to water-soluble positively charged CdTe quantum dots (QDs) through complementary electrostatic interaction. These QDs-DNA complexes were disrupted and DNA was released by glutathione (GSH) at intracellular concentrations. Interestingly, there was almost no detectable DNA released by extracellular concentration of GSH. The formation of QDs-DNA complexes and GSH-mediated DNA release from the complexes were confirmed by dye displacement assay, electrophoretic mobility shift assay (EMSA), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) experiments.