Dissertations / Theses on the topic 'BRRF1'

Systemic Lupus Erythematosus (SLE) is an autoimmune disease affecting 20 to 250 individuals per 100,000 worldwide. Symptomology includes dermatological manifestations such as discoid lesions, acute cutaneous rashes, and oral and nasal ulcers, along with musculoskeletal, pulmonary, and renal complications. Abnormal T and B lymphocyte function and apoptosis, immune complex clearance, complement function, and nucleosome processing are typical of disease pathophysiology. SLE is the result of both environmental and genetic factors, which together create the conditions leading to disease onset and progression. Of these environmental factors, Epstein-Barr virus (EBV) infection is known to cause the genesis of cross-reactive antibodies in SLE prone individuals that can initiate disease activity. Viral infection and modulation of cellular genes is important in understanding the microenvironment that could lead to immune mis-regulation and the inception of lupus in those individuals at risk. During disease development, a variety of variables assist and detract from disease progression and the quality of life experienced by SLE patients. Research into EBV-infected naïve B lymphocytes revealed that EBV modulates the chemotactic receptor EBI2 during viral infection via the BRRF1 viral gene product Na. This likely changes B lymphocyte chemotaxis in secondary tissue in virally infected B cells. Current literature suggests this results in sequestration of cells to peripheral areas of the tissue and mis-regulation of the immune response. It is not uncommon for SLE patients to have neuropsychiatric disorders due to lupus disease activity. With SLE patients being up to 6 times more at risk for depression, recognition and treatment of depression and anxiety have been shown to improve quality of life, pain, and treatment outcomes. Two studies investigate both clinical laboratory and psychosocial assessment variables that we suspect to be correlated with depression in patients with SLE. Univariate and multivariate analysis from our first study identified an array of variables that show strong associations with depression, including: Body Mass Index, Pain, Total Complement, fatigue assessments, and SF-36 scores. The second study found similar associations, but further found that serum IL-10 levels demonstrated a strong correlation with depression in SLE patients. In this final study SLE patients are compared alongside healthy, clinically depressed, and rheumatoid arthritis patients to provide evidence that increased depression in SLE patients is due more to disease pathology than a result of chronic inflammation.

Tumors are composed of proliferating cells that invade healthy tissue and grow over time. Even though it is still unclear, it is a common opinion that the cells of origin should possess a proliferative capacity (Blanpain, 2013; Visvader, 2011). Particularly for brain cancers, the transition of neural progenitors to differentiated postmitotic neurons is considered irreversible in physiological and pathological conditions. Therefore, postmitotic neurons have not been considered as suitable cell of origin for brain cancer. Here, we show that neurons reprograming may occur upon Shh activation leading to medulloblastoma (MB) formation in vivo. Human SHH medulloblastoma (MB) is a brain tumor affecting adults and infants that is thought to originate from cerebellar granule neuron progenitors. Notably, several groups have shown that Shh pathway activation (SmoM2 overexpression) in mouse granule neuron progenitors is able to induce Shh MB (Schuller et al., 2008; Z.-J. Yang et al., 2008). These progenitors are present in infants and newborn mice, but they seem to be absent in adult humans and mice (Biran, Verney, & Ferriero, 2012; Marzban et al., 2014; Z.-J. Yang et al., 2008). Furthermore, it was recently discovered that the two different forms of SHH MB are distinguished by different transcriptome/methylome levels suggesting that the adult SHH MB may originate from a different cell of origin (Cavalli et al., 2017; Kool et al., 2014). Relying on these data, we take advantage of a conditional Cre-Lox recombination system to recapitulate the human adult medulloblastoma pathogenesis in mice, demonstrating that cerebellar post-migratory mature granule neurons upon SmoM2 overexpression can dedifferentiate and give rise to SHH MB in vivo. Moreover, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Notably, gene expression profiling on our samples allowed to associate “cerebellar granule progenitors-derived MB” with the human infant form while “truncated BRPF1-induced tumors” clustered with human adult SHH MB. Furthermore, as previously described by Kool et al., 2014, human adult SHH MB is characterised by the copresence of p-AKT and p-S6, compared to the human infant SHH MB that are positive for either p- AKT or p-S6 and always in a mutually exclusive way. Truncated BRPF1-induced tumors are double positive for p-AKT and p-S6, similarly to adult patients, while cerebellar granule progenitors derived MB present only p-S6. Furthermore, to define the contribution of chromatin changes in granule neurons dedifferentiation in response to Shh activation, we profiled changes in chromatin accessibility by ATAC-seq analysis on mice cerebella. SmoM2 overexpression changed the epigenetic landscape of granule neurons, enriching the number of open chromatin regions 12 associated with stem/progenitor-like genes. Moreover, the cooperation between truncated BRPF1 and SmoM2 in reshaping the chromatin arrangement of granule neurons was explored applying ATAC-seq on differentiated human cerebellar neurons derived from neuroepithelial cells. ATAC-seq analysis pointed out a synergistic mechanism between SmoM2 and truncated BRPF1 in modifying the epigenetic landscape of postmitotic neurons, increasing the chromatin accessibility of super-enhancers, associated with stemness and chromatin organization/modification genes. Our novel model of cancer development could explain the human SHH medulloblastoma onset in adult individuals where granule neuron progenitors are no more present. For these reasons, we strongly believe that our model configures as an important starting point for a new field in cancer and stem cell biology focusing on the study of mechanisms driving tumorigenesis in postmitotic cells.

Tristetraprolin (TTP), butyrate response factor 1 (BRF1) and butyrate response factor 2 (BRF2) are members of a family of zinc finger containing ARE binding proteins known as the Zfp36 family. They all possess a conserved tandem zinc finger domain, which facilitates their binding to mRNAs that contain adenosine/uridine rich elements (ARE) in their 3' untranslated region (3'UTR). Binding to the target mRNA results in its destabilisation. Several mRNAs containing an ARE in their 3'UTR are stabilised by the mitogen activated protein kinase (MAPK) p38 pathway. Both the expression and the mRNA destabilising function of TTP are controlled by the p38 MAPK pathway. Much less is known about BRF1 and BRF2 functions, and it is not clear whether their expression or function is regulated by the p38 MAPK pathway. So far no difference has been seen in the binding specificities of the three proteins in vitro, however the phenotypes of knockout mice suggest that they have distinct functions, and may have different mRNA targets in vivo. Western blotting and quantitative PCR (qPCR) have been used to investigate the expression of members of the Zfp36 family. RNA interference was used to knock down the expression of BRF1 and BRF2 in HeLa cells, and the effects on p38-regulated inflammatory mediator expression were examined. BRF1-/- mouse embryonic fibroblast (MEF) cell lines were used to investigate the function of this family member. No evidence that BRF1 or BRF2 contributes to the post-transcriptional regulation of inflammatory mediators by the p38 MAPK pathway in HeLa cells or MEFs was found. BRF1 null MEFs over-expressed IL-6, protein, IL-6 mRNA and Cox-2 mRNA but did not over-express KC protein. The hypothesis that BRF1 is regulating IL-6 and Cox-2 by controlling mRNA stability was disproved. As a result investigation of the transcriptional regulation of these genes was researched. Primary transcript qPCR showed that both IL-6 and Cox-2 undergo more rapid transcription in BRF1-/- MEFs. This suggests that IL-6 and Cox-2 are indirect targets of BRF1 and that their regulation is through a transcription factor which is itself a target of BRF1.

La déficience intellectuelle (DI) est une trouble du neuro développement caractérisée par une extrême hétérogénéité génétique, avec plus de 700 gènes impliqués dans des formes monogéniques de DI. Cependant un nombre important de gènes restent encore à identifier et les mécanismes physiopathologiques de ces maladies neuro développementales restent encore à comprendre. Mon travail de doctorat a consisté à identifier de nouvelles causes génétiques impliquées dans la DI. En utilisant différentes techniques de séquençage de nouvelle génération, j’ai pu augmenter le taux de diagnostic chez les patients avec DI et identifié plusieurs nouvelles mutations (dans AUTS2, THOC6, etc) et nouveaux gènes (BRPF1, NOVA2, etc) impliqués dans la DI. Pour les moins caractérisés, j'ai effectué des investigations fonctionnelles pour valider leur pathogénicité, caractériser les mécanismes moléculaires qu'ils affectent et identifier leur rôle dans cette maladie. Mes travaux de doctorat permettront d’améliorer et d’accélérer la possibilité d’obtenir un diagnostic moléculaire qui donnera accès à un meilleur suivi et à une meilleure prise en charge pour les patients. Cela permettra également de mieux comprendre les mécanismes physiopathologiques impliqués dans ces troubles neuro développementaux. Ces connaissances aideront éventuellement à identifier de nouvelles cibles thérapeutiques
Intellectual disability (ID) is a group of neurodevelopmental disorders characterized by an extreme genetic heterogeneity, with more than 700 genes currently implicated in Mendelian forms of ID but still some are not yet identified. My PhD project investigates the genetic causes of these monogenic ID by using and combining different NGS techniques. By using this strategy, I reached a relative high diagnostic yield and identified several novel mutations (in AUTS2, THOC6) and genes (BRPF1, NOVA2, etc) involved in ID. For the less characterized ones, I performed functional investigations to prove their pathogenicity, delineate the molecular mechanisms altered and identify their role in this disease. Overall, this work improved and provided new strategies to increase the molecular diagnosis in patients with ID, which is important for their healthcare and better management. Furthermore, the identification and the characterization of novel mutations and genes implicated in ID better delineate the implicated pathophysiological mechanisms, opening the way to potential therapeutic targets

RNA Polymerase III (Poll III) contributes to about 10% of nuclear transcription and is essential for the synthesis of short untranslated transcripts, including tRNA and 5S rRNA. Pol III deregulation has been implicated in driving cellular proliferation and transformation, along with increased expression of a number of Pol III specific transcription factors and transcripts. The significance of Pol III in clinical pathology, including that of human malignancies, remains to be formally tested. Using prostate cancer as a model, two key components of the Pol III complex, namely Brf1 and tRNAiMet, were investigated. The expression patterns of Brf1 and tRNAiMet were studied in this thesis using immunohistochemistry (IHC) and in situ hybridisation (ISH) respectively. Brf1, a subunit of transcription factor IIIB (TFIIIB), was detected predominantly in the nucleus with heterogenous staining intensity, its presence ranging from weak to strong. Examination across a wide range of tissue types and organs, both normal and tumour tissues revealed high levels of Brf1 expression in the epithelium. In addition, Brf1 expression could also be detected in connective tissues and, to a lesser extent, in muscular and nervous tissues. A number of tumour types exhibited elevated expression of Brf1 protein relative to their normal control tissues. These included prostate adenocarcinoma, B-cell lymphoma and, interestingly, tumours arising from connective tissues (sarcoma, fibrosarcoma and chondrosarcoma). As expected, tRNAiMet expression, as revealed by ISH analysis, was mostly observed in the cytoplasm, although some nuclear staining was also present. Similar to Brf1, tRNAiMet expression was detected predominantly in epithelial tissues such as the skin epidermis, prostate gland and epithelial lining of the cervix. A number of tumours were found to overexpress tRNAiMet. These included breast ductal carcinoma, oesophageal carcinoma and melanoma. The clinical impact of Brf1 and tRNAiMet overexpression was examined using tissue microarrays (TMA) containing tissue samples obtained from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH). Collectively, data from two independent patient cohorts, Glasgow TMA: BPH (n=21), PCa (n=151) and Newcastle TMA: BPH (n=113), PCa (n=365), showed that Brf1 expression was upregulated in prostate cancer relative to BPH (p=0.0034). Brf1 expression was not found to be associated with the following clinical and biologic parameters: Gleason sum score (indicative of tumour differentiation and morphology, p=0.653); prostate specific antigen (PSA level, indicative of tumour bulk or volume, p=0.381) and Ki-67 expression (signifying cellular proliferation, p=0.034). However, and importantly, within the prostate cancer patient cohorts studied, high Brf1 expression was associated with a significantly less favourable survival outcome (Kaplan Meier analysis, p<0.001). Together, the data presented in this study support the relevance of Brf1 and tRNAiMet overexpression as part of Pol III deregulation in tumours, especially of epithelial origin. This study also suggests the potential application of Brf1 as a prognostic marker in cancer, however, this warrants a further study.

La formation de nouveaux vaisseaux sanguins ou angiogenèse soutient la croissance tumorale en fournissant l'oxygène et les nutriments qui lui sont nécessaires. Le rôle clé du facteur de croissance de l'endothélium vasculaire VEGF dans ce processus a suscité le développement de stratégies anti-angiogéniques pour le traitement du cancer. Cependant, des travaux précliniques et des données cliniques suggèrent l'émergence de résistances aux anti-angiogéniques, en raison notamment de la redondance des facteurs de croissance pro-angiogéniques. Il est donc nécessaire de développer des stratégies alternatives plus efficaces. En 2010, notre laboratoire a apporté la preuve de concept d'une thérapie anti-tumorale et anti-angiogénique innovante basée la dégradation des ARNm à demi-vie courte par la protéine à doigts de zinc TIS11b. Néanmoins, l'instabilité de la protéine thérapeutique a entravé la caractérisation plus détaillée de cette stratégie. Dans ce contexte, l'objectif majeur de ma thèse était l'optimisation de la stabilité et de l'activité de TIS11b et l'évaluation de son efficacité thérapeutique. Pour cela, nous avons généré une nouvelle protéine TIS11b génétiquement modifiée sur la base d'études biochimiques et moléculaires. Notamment, nous avons observé que la phosphorylation de la sérine 334 située dans le domaine C-terminal de TIS11b augmente de façon très significative la stabilité de la protéine et potentialise son activité déstabilisatrice de l'ARNm du VEGF. De plus, la délétion du domaine N-terminal augmente également la stabilité de TIS11b sans altérer son activité. Nous avons alors généré deux nouvelles protéines thérapeutiques, la protéine ZnC et la protéine ZnC334D pour laquelle la troncation du domaine N-terminal et la substitution de la sérine S334 par un aspartate mimant une phosphorylation ont été combinées. Les nouvelles protéines ont été fusionnées à une étiquette polyarginine R9 leur permettant de traverser les membranes cellulaires (R9-ZnC et R9-ZnCS334D). Nous avons montré que R9-ZnC et R9-ZnCS334D inhibent l'expression de VEGF in vitro dans la lignée de cancer du sein murin 4T1. De plus, R9-ZnCS334D exerce une activité anti-proliférative, anti-migratoire et anti-invasive dans ces cellules. In vivo, l'injection intra-tumorale de R9-ZnCS334D dans des tumeurs 4T1 préétablies inhibe significativement l'expression du VEGF, la croissance et la vascularisation tumorales. De façon remarquable, l'analyse des extraits tumoraux indique que le traitement diminue l'expression de chimiokines clés dans les processus d'angiogenèse, d'inflammation et d'invasion (Fractalkine, MCP-1, NOV, SDF-1, Pentraxin…). Enfin, R9-ZnC et R9-ZnCS334D inhibent l'expression de marqueurs de la transition épithélio-mésenchymateuse, un processus impliqué dans la dissémination métastatique. L'ensemble de ces travaux indique que R9-ZnC et R9-ZnCS334D sont des molécules anti-tumorales multi-cibles, qui inhibent plusieurs étapes clés de la progression tumorale. Cette étude confirme que le ciblage de la stabilité des ARNm est une stratégie prometteuse et novatrice pour le développement de nouvelles thérapies anti-cancéreuses
One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms. In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9). In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells. In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9-ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies

En 2002, notre équipe a montré que la stimulation de cellules primaires cortico-surrénaliennes bovines en culture primaire par l'hormone hypophysaire ACTH induisait l'expression du VEGF de manière indépendante de sa transcription. En effet, en réponse à l'ACTH, l'expression du VEGF est régulée au niveau post-transcriptionnel par la stabilisation/déstabilisation de son transcrit via des séquences riches en AU (ARE) situées dans la région 3‘ non traduite (3'UTR) de son ARNm. Le laboratoire a mis en évidence une protéine, TIS11b, exprimée également en réponse à une stimulation des cellules par l'ACTH, mais de manière concomitante avec la phase de déstabilisation de l'ARNm du VEGF. TIS11b appartient à une famille de protéines déstabilisatrices des ARNm via les séquences ARE. L'interaction de TIS11b avec l'ARNm du VEGF a été confirmée par la suite, et le site de liaison de la protéine à l'ARNm a également été identifié.

Dans ce contexte, le premier objectif de ma thèse était d'évaluer la possibilité d'utiliser les propriétés déstabilisatrices de TIS11b sur l'ARNm du VEGF pour une thérapie anti-angiogène et anti-tumorale. Pour cela, la protéine a été vectorisée par la fusion de petits peptides ou PTD (protein transduction domain, Tat issu du VIH ou les polyarginines R7 et R9) lui permettant de traverser les membranes et d'atteindre sa cible intracellulaire, l'ARNm du VEGF. Nous avons pu montrer dans cette étude que 100 nM de protéines de fusion Flag-Tat-, Flag-R7- et Flag-R9-TIS11b sont capables d'induire une diminution du taux d'ARNm du VEGF ainsi que de la production de la protéine VEGF, dans des cellules en culture après 24 h d'incubation. De plus, nous avons pu montrer que l'injection de 100 nM de la protéine de fusion Flag-R9-TIS11b dans la glande corticosurrénalienne de souris, induisait une diminution d'environ 50 % de l'expression du VEGF par cette glande et que cette diminution était maintenue à 48 h. Des résultats préliminaires encourageants d'inhibition de croissance tumorale ont été obtenus avec l'injection de cette protéine de fusion dans des tumeurs pré-établies chez la souris nude, et doivent être confirmés.

L'ACTH étant une hormone qui active la voie de l'AMPc et de la protéine kinase A (PKA), le deuxième objectif de ma thèse était de caractériser la phosphorylation de TIS11b par la PKA en réponse à une stimulation par l'ACTH et d'étudier les conséquences de cette phosphorylation sur son activité déstabilisatrice des ARNm. Nous avons pu montrer pour la première fois, que la PKA phosphoryle la protéine TIS11b, in vitro, au niveau de la sérine 54. Un deuxième site de phosphorylation en réponse à une stimulation par l'ACTH, via probablement une autre kinase que la PKA, a été mis en évidence au niveau de la sérine 334. Il semblerait que la protéine TIS11b comporte un domaine d'activation et un domaine d'inhibition susceptibles d'être régulés en réponse à une stimulation par l'ACTH. L'attribution de ces domaines aux sérines 54 et 334 nécessite des expériences complémentaires.

Epstein-Barr Virus (EBV) is a ubiquitous human virus linked to several diseases, including cancers. CD8 T cells are important for controlling EBV replication. Generation and maintenance of virus-specific CD8 T cells is dependent on specific interaction between MHC-peptide complexes on the infected cell and the CD8 T cell receptor (TCR). Several lines of evidence suggest that the TCR repertoire is an essential component of the CD8 T-cell immune response. The current work focuses on delineating the features of the TCR repertoire that drive the selection of EBV-specific CD8 T cells into the memory phase. We used bulk and single-cell TCRαβ sequencing to analyze the TCR repertoire of human CD8 T cells specific for two immunodominant HLA-A02:01-restricted EBV-derived epitopes: BRLF1109-117 (YVLDHLIVV) and BMLF1280-288 (GLCTLVAML) during the acute and memory phases of primary EBV infection in humans. We showed that persistent EBV-specific clonotypes accounted for only 9% of unique clonotypes but were highly expanded in acute EBV infection and more commonly expressed identifiable features than non-persistent clonotypes. The other 91% of highly diverse unique clonotypes disappeared and were replaced in convalescence by equally diverse “de-novo” clonotypes. We provide evidence suggesting that recognition of BRLF1109-117may be driven by the TCRα. We identified a highly dominant and degenerate BRLF1109-117-specific TCRα sequence, AV8.1-CAVKDTDKLIF-AJ34, that was shared by all donors studied and identified conserved residues within this sequence that were important for antigen recognition. These findings are relevant to current efforts to develop or optimize the efficacy of T cell based therapies or vaccines.

碩士
長庚大學
基礎醫學研究所
95
Expression of Rta and Zta is critical to the activation of the Epstein-Barr virus (EBV) lytic cycle. In the immediate early stage of the lytic cycle, expression of Rta and Zta could be regulated by each other and could active many genes which express during lytic cycle. In the past, transcription of an EBV gene was usually studied by using the transient transfection system, which may not truly reflect how a gene is transcribed. This study uses a PCR-targeting method and generates mutations in the BRLF1 promoter on the EBV genome. Deletions in the promoter reveals the amounts of the BRLF1 mRNA, Rta, and the numbers of EBV particles are reduced as the deletions extends into the downstream regions of the promoter. The result also showed that the region between nucleotide -968 and -850 is crucial because without this fragment, activation of the promoter by Zta becomes inefficient. Additionally, mutating any one of the three Zta-response elements decreases the ability of Zta to activate the promoter by 50-80%, indicating the importance of these elements in the transcription of BRLF1. This study shows that specific DNA sequences can be mutated on the EBV genome to study how these mutations affect the expression of EBV genes.

碩士
長庚大學
基礎醫學研究所
92
Epstein-Barr virus (EBV) has two life cycles. After infecting B lymphocytes, the virus is normally maintained under latent conditions. However, the virus enters a lytic cycle to proliferate after exposing to specific chemical or physical stimuli. The mechanism underlying the lytic switch from latency is poorly understood. However, activation of Rta and Zta, two transcription factors expressed by EBV, is the key event leading to the activation. An earlier work demonstrated that transcription of BRLF1, the gene encoding Rta, is autoregulated by Rta itself and the cis element involved in this regulatory event associates with the two Sp1 sites in the BRLF1 promoter (Rp). This investigation demonstrates that an EBV immediate-early protein, Rta, interacts with MCAF and Sp1, this result leads to a hypothesis that MCAF facilitates the autoregulation of Rp by Rta. The binds are apparently important for Rp activation since expressing MCAF increases Rp activity. This study shows that MCAF brings Rta to the Sp1 binding to Rp to activate the transcription and MCAF may play a crucial role in activating the EBV lytic cycle.

BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 15-20% of all pediatric brain tumors. In patients with MB, prognosis depends heavily on the molecular makeup of the tumor. New genomic approaches over the last decade have enabled researchers to sub-classify MB based on differences in the transcriptome: WNT, Sonic hedgehog (SHH), Group 3 (MYC-amplified), and Group 4 (heterogeneous). SHH tumors represent a third of all MB cases, and small-molecule inhibitors have already been developed that target SHH signaling. Most notably, vismodegib has shown great promise in the treatment of MB and other SHH-driven cancers by targeting Smoothened (SMO), an upstream regulator of GLI activity. However, most patients who had initially responded to the drug quickly acquired point mutations in SMO that led to treatment resistance. In addition, patients who harbored mutations downstream of SMO had no response to treatment and were found to be intrinsically resistant. Although most patients with SHH-MB can be cured, current treatments often require broad base therapies, such as radiation and chemotherapy, which can have harmful and long-lasting side effects. These observations underscore the need for less toxic, more targeted therapies that act at the level of the GLI family of transcription factors themselves. However, as transcription factors are generally considered undruggable, Dr. Robbins’ group at the University of Miami Miller School of Medicine sought to address this need by using focused screens of siRNAs or small molecules that target epigenetic GLI regulators. They identified several candidates that act as readers, writers, and/or erasers of protein acetylation and methylation and showed that a subset of these candidates act downstream of SMO to attenuate GLI signaling (data not yet published). Bromodomain- and Plant Homeodomain-linked Zinc Finger-containing Protein 1 (BRPF1) was one of these candidates and further analysis revealed that its knockdown reduced Gli1 expression by more than 50%. Recent studies link BRPF1 to cerebellar development and tumor formation in SHH-MB and may be suggestive of its role as a negative regulator. OBJECTIVES: We sought to compare basal levels of Brpf1 expression in normal versus MB in mice; to characterize Brpf1 knockdown versus overexpression in SHH cell lines; and to determine if BRPF1 merits further investigation as a candidate for future drug targeting therapies in MB and other SHH-driven cancers. METHODS: We used RT-qPCR and immunoblotting analysis to look at Brpf1 expression in Ptch+/- and adult wild-type mice. cDNA and protein samples were donated by colleagues in the lab. We also grew and maintained SHH Light2 cells in culture and then used these cells to carry out siRNA and plasmid DNA transfections. RNA extraction, RT-PCR, and RT-qPCR were used to examine transfection efficiency and its effect on Gli1 expression. RESULTS: Brpf1 levels were higher in SHH-MB compared to normal cerebellum. However, BRPF1 proteins were not detected in either normal or tumor samples. Brpf1 knockdown in Light2 cells correlated with an overall decrease in Gli1 expression while overexpression had no obvious affect on Gli1 expression. CONCLUSIONS: Our findings suggest that BRPF1 may function as a positive regulator of GLI activity. Recent studies verify this claim at least partially stating that BRPF1 acts as both a positive and negative regulator of gene expression depending on the context. Thus, before we can draw any final conclusions, more research is needed to look at BRPF1 in the specific context of the SHH pathway and developing cerebellum.

博士
長庚大學
生物醫學研究所
104
Epstein-Barr virus (EBV) expresses a transcription factor, Rta, during its lytic cycle to activate the transcription of viral lytic genes. We find in a transient transfection study that upstream stimulating factor (USF) binds to E1, one of the five E boxes, which is located at -79 in Rp, to activate BRLF1 transcription. Furthermore, we show that Rta interacts with USF since these two proteins are coimmunoprecipitated from cell lysates. A GST-pulldown study using bacterially expressed proteins also shows these two proteins interact. Confocal laser-scanning microscopy further verifies that these two proteins colocalize in the nucleus. We also found that Rta binds to the E1 sequence in a biotin-labeled E1 probe only when USF1 is present, showing that these two proteins form a complex on E1. We also used a reporter plasmid, p188mSZ, which contains the sequence from -188 to +5 in Rp, within which the Sp1 site and Zta response element are mutated. We found that cotransfecting EBV-negative Akata cells with plasmids expressing USF1 and Rta synergistically activates Rp in p188mSZ. However, after mutating the E1 sequence in p188mSZ, USF1 and Rta no longer activate the promoter, showing that Rta autoregulates BRLF1 transcription via its interaction with USF1 on E1. This study shows that transfecting pUSF1 after EBV lytic induction of P3HR1 cells increases the expression of Rta, showing that USF1 activates Rta expression after the virus enters lytic cycle. This study reveals how USF promotes the expression of Rta to benefit viral lytic development.

碩士
長庚大學
基礎醫學研究所
96
Rta is a transcription factor expressed by the Epstein-Barr virus (EBV) during the immediate-early stage of the lytic cycle. This investigation finds that the promoter that transcribes the Rta gene, BRLF1, contains five putative E-Box sequences. Deletion and transient transfection analysis revealed that only the proximal E-Box (5’-CATGTG) located at between -84 and -79 is critical to the activation of the BRLF1 promoter by USF1. The binding of USF1 to this E-box sequence was confirmed by elelctrophoretic mobility shift assay (EMSA), DNA-affinity precipitation assay (DAPA)and chromatin immunoprecipitation (ChIP). Moreover, GST pull-down and immunoprecipitation demonstrated that USF1 forms a complex with Rta on this USF-1 binding site on Rp. The binding appears to autoregulate the transcription of BRLF1. The results from this study reveal how the transcription of BRLF1 is regulated by USF1 and Rta.

博士
長庚大學
基礎醫學研究所
89
Abstract Epstein-Barr virus (EBV) has two different life cycles. As generally known, when cells latently infected by EBV are exposed to ultraviolet light, heat, 12-O-tetradecanoylporbol-13-acetate (TPA), sodium butyrate, anti-immunoglobulin, or TGF-b, the virus immediately enters a lytic productive cycle. However, how EBV enters a lytic cycle under natural conditions remains unknown. BRLF1 and BZLF1 are the two genes expressed during the immediate-early stage of EBV lytic cycle. These two genes encode two transcription factors, Rta and Zta, respectively, which are required to activate the EBV early genes and lytic cascade. Both Rta and Zta are translated from a 3.3-kb mRNA transcribed from the promoter of BRLF1 (Rp). Furthermore, Zta is also translated from a 1-kb transcript transcribed from the promoter of BZLF1 (Zp). Therefore, the factors which activate Rp and Zp may also trigger EBV lytic cascade. Since chromosome structures are known to influence gene expression, this study treated P3HR1 cells, an EBV-containing B lymphocyte cell line, with a histone deacetylase inhibitor, trichostatin A (TSA), to examine how histone acetylation influence the expression of EBV lytic genes. Experimental results revealed that TSA treatment activates not only lytic cycle but also the transcription of EBV lytic genes, including BRLF1, BZLF1, BMLF1, and BcLF1. Furthermore, Rp is synergistically activated when the cells were treated with TSA and TPA, indicating that both histone acetylation and phosphorylation activate the promoter. A chromatin immunoprecipitation (CHIP) assay was performed to analyze if the nucleosomes at Rp on the EBV genome are acetylated during lytic cycle and revealed that H4 at Rp are acetylated after the cells were treated with TSA. A YY1 sequence on Rp, which is known to repress the transcription of Rp, was mutated by site-directed mutagenesis. This mutation increases the Rp activity 1.6-fold in P3HR1 cells and 3.3-fold in an epithelial cell line, C33A. CHIP assay also revealed that the mutation increases the degree of H4 acetylation at Rp 1.64-fold in P3HR1 cells and 3.08-fold in C33A cells. The correlation between Rp activation and histone acetylation suggests that histone acetylation at Rp influence the Rp activity. This study also found that transfecting a plasmid overexpressing Rta activates Rp and Zp in P3HR1 cells. Rp is activated 735-fold in P3HR1 cells, 86-fold in EBV-negative Akata cells, and 11.5-fold in C33A cells. Deletion of Rp revealed that a 80-bp PstI-DraI fragment located in the upstream region of Rp plays a crucial role in BRLF1 transcription. This fragment is also required for an autoregulation of Rp by Rta. Electrophoretic mobility shift assay revealed that proteins in C33A lysate binds to a 30-bp PD2 region of this DNA fragment. Furthermore, this study found that incubating P3HR1 cells at 44。 for 30 minutes activates EBV lytic cycle and transcription of immediate-early genes. Deletion analysis revealed presence of a 3 Zta-response element (ZRE) in Rp is required for the activation by heat. In EBV-negative Akata cells, heat shock activates Zp but does not activate Rp. My results also revealed that SB203580, an inhibitor of p38 signal transduction pathway, inhibits the activation of BMRF1 by heat shock. These results suggest that heat shock activates the p38 signal transduction pathway, ultimately activating Zp to induce EBV lytic cycle.

博士
國防醫學院
生命科學研究所
102
The TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open promoter formation in transcription initiation. We site-specifically incorporated non-natural amino acid cross-linker to Brf1 to map its protein interaction targets in the pre-initiation complex (PIC). My cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the polymerase active site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeats subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. Mutations in this region showed slow cells growth and defective transcription. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. We demonstrated the functional importance of Brf1c homology block I and Bdp1-binding domain in C-linker 1. Overall, the domain architecture of the PIC derived from the cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation.

碩士
國立臺灣大學
生化科技學系
106
Epstein-Barr virus (EBV) is a human herpesvirus, which infects more than 90% of the population. After infection, EBV immortalizes its host cells, establishing latent infection. Although the infection is usually asympotmatic, latent EBV infection is often associated with human cancers. The virus can be reactivated by specific environmental changes and enters the lytic cycle to produce virus particles. At the onset of the lytic cycle, the virus expresses two immediate-early transcription factors, Rta and Zta, which activate EBV’s lytic genes. The genes encoding Rta and Zta, BRLF1 and BZLF1, respectively, are situated adjacent on the viral genome with BRLF1 located upstream. The promoter of BRLF1 (Rp) transcribes a bicistronic BRLF1-BZLF1 mRNA (RZ-mRNA). Earlier study has established that Zta is translated efficiently from the bicistronic mRNA; Rta can elevate the translation of BZLF1 orf located downstream in cis via the intercistronic region (ICR) on the RZ-mRNA in P3HR1 B lymphocytes. This study finds that Rta can trans-activate the BZLF1 translation in 293T cells. By generating a bicistronic reporter plasmid (pEGFP-ICR-Luc), this study verified that Rta acts as an IRES trans-acting factor (ITAF) to activate downstream translation in a trans-acting manner and the N-terminal domain of Rta is essential in the scheme. Deletion analysis of ICR further indicates that the two regions within ICR, region I and region II, are important for the translational activation by Rta. Moreover, RNA-protein pulldown assay confirms that Rta binds to ICR in vitro. Altogether, this study demonstrated that the ICR in RZ-mRNA function as an IRES, while Rta serve as an ITAF that trans-activates the translation of BZLF1 from BRLF1-BZLF1 bicistronic mRNA, thereby promoting the expression of Zta.

Posttranscriptional gene expression is controlled and coordinated by RNA binding proteins (RBPs), many of which recognize specific RNAs through cis-regulatory RNA elements. One of the most highly studied classes of cis-regulatory RNA elements is the AU-rich elements (AREs). AREs are bound by a class of RBPs called ARE binding proteins (ARE-BPs), of which there are over a dozen in humans including HuR, tristetraprolin (TTP) and butyrate response factors 1 and 2 (BRF1 and BRF2). TTP, BRF1 and BRF2 belong to a family of tandem C3H zinc finger proteins that destabilize ARE-containing mRNAs. HuR acts to enhance the stability and translation of ARE-containing mRNAs, a function that is rare among ARE-BPs. While each of these ARE-BPs regulates the expression of ARE-containing mRNAs, some ARE-BPs themselves are also encoded by ARE-containing mRNAs, raising the possibility that each of these ARE-BPs may regulate one another's expression. In order to determine how these ARE-BPs influence each others expression and how this affects the regulation of global gene expression programs we have focused on three different aspects of these ARE-BP networks: control, response to stimuli, and global effects.

To address of network control of ARE-BPs we have focused on how HuR regulates a network of mRNAs including TTP, BRF1 and HuR's own mRNA. We demonstrate that HuR can bind to TTP's, BRF1's and its own mRNA. Furthermore, by employing overexpression and siRNA knockdown approaches we demonstrate that these mRNAs and their corresponding 3'UTR luciferase reporters are resilient to fluctuations in HuR levels and that the degree of this resiliency is cell type and condition specific.

To address the temporal responses within an ARE-BP network we focused on how each of the members of the TTP family of ARE-BPs reacts following the induction of the other family members by using epidermal growth factor (EGF) stimulation. Here we show that induction of TTP family member mRNAs during EGF stimulation is partially attributable to changes in mRNA stability. Furthermore, we also show that TTP and BRF1 are able to bind each of the TTP family member mRNAs and subsequently affect their expression by altering their mRNA degradation rates. In addition, we demonstrate that the unique temporal induction patterns of the TTP family member RBPs is correlated with the EGF stimulated induction of TTP-bound mRNAs, suggesting that a network comprised of TTP family members is able to influence the timing of complex gene expression patterns.

Finally, to address the influence of these networks on regulation of global gene expression programs we have focused on how HuR recognizes AREs and whether it can globally recognize multiple classes of ARE-containing mRNAs, including the canonical class of AREs recognized by the TTP family members. To investigate how the three RNA recognition motifs (RRMs) of HuR contribute to ARE recognition we generated a series of RRM point mutants and test their ability to disrupt RNA recognition of each of the RRMs. To identify different classes of ARE-containing mRNAs we examined these mutants with a global RNA binding site detection method called photoactivatable ribonucleoside crosslinking immunoprecipitation (PAR-CLIP). Together these techniques suggest that the RRMs of HuR cooperate to recognize mRNA targets and that HuR's ability to bind RNA is coupled to the cellular distribution of HuR, and thus, are important in its role for regulating expression of bound mRNAs.

Together these studies indicate that ARE-BP posttranscriptional networks are highly interconnected and display complex regulatory interactions depending on cell type and stimuli. Furthermore, these networks can create complex behaviors such as timing of expression events or resiliency to fluctuations in protein levels. Finally, the components of these ARE-BP networks target partially overlapping sets of mRNAs to impact global gene expression patterns that ultimately coordinate the cellular responses to external stimuli.

Dissertation