ERRα est un récepteur nucléaire dont l’activité est controlée par des co-régulateurs transcriptionnels. La forte expression de ERRα dans les cancers est corrélée à un mauvais pronostic. Les mécanismes par lesquels ERRα régule la migration des cellules cancéreuses sont mal compris, tout comme les co-régulateurs impliqués. Nous avons identifié deux enzymes modificatrices d’histone, LSD1 et SET7, agissant comme régulateurs positifs de ERRα.I. ERRα modifie les activités biochimiques de la déméthylase LSD1 vers la déméthylation (activatrice) de H3K9me2. L’activation des cibles de ERRa-LSD1 (identifiées par RNA-Seq) requiert le recrutement de ce complexe aux sites d’initiation de la transcription (TSSs), réalisé par le facteur de transcription NRF1 qui, lui, ne régule pas l’activité enzymatique de LSD1.II. Un autre groupe de cibles de ERRα (identifié par RNA-Seq) est sous le contrôle de l’histone méthyltransférase SET7 qui mono-méthyle H3K4. Le recrutement de SET7 aux TSSs est contrôlé par le facteur de transcription ETS1, qui promeut les interactions entre SET7 et ERRα, conduisant à l’activation de l’expression des gènes en aval.Des analyses par Gene Ontology ont montré que les cibles communes de ERRα/LSD1 et de ERRα/SET7 sont fortement enrichies en termes d’invasion cellulaire. De manière cohérente, la déplétion individuelle de chacun de ces facteurs (et également celle de NRF1 ou ETS1) réduit les capacités d’invasion, observée en tests in vitro (transwell) ou in vivo par xénogreffe sur embryons de poisson-zèbre.En résumé, nos résultats montrent deux réseaux de régulation impliquant des modifications d’histone induites par ERRα, conduisant à l’invasion cellulaire. / ERRα is a nuclear receptor whose activity mainly depends on its interaction with transcription co-regulators. High levels of ERRα are found in various cancer types and correlate with poor prognosis. However, the mechanisms linking ERRα to cancer cell migration as well as the coregulators involved are unclear. In our study, we found two histone-modifying enzymes, LSD1 and SET7, acting as positive regulators of ERRα.I. ERRα impacts the biochemical activities of the LSD1 demethylase. Activation of ERRα -LSD1 targets (identified by RNA-Seq) requires the recruitment of this complex at Transcriptional Start Sites (TSSs), which is achieved by the NRF1 transcription factor. In our study, we have shown several points: NRF1, but not ERRα , is involved in positioning LSD1 to TSS, whereas ERRα , but not NRF1, regulates LSD1 enzymatic activities towards demethylating H3K9me2.II. A distinct group of ERRa target genes (identified by RNA-Seq) is under the control of the histone methyltransferase SET7 which mono-methylates H3K4. Appropriate recruitment of SET7 at TSSs is controlled by the ETS1 transcription factor, promoting the interaction between SET7-ERRa, leading to target gene expression.Gene Ontology analysis revealed that ERRa-LSD1 co-targets, as well as ERRa-SET7 co-targets, are enriched in terms of cell invasion. Consistently, depletion of each of these factors, as well as depletion of NRF1 or ETS1, leads to reduced cell invasion capacities as observed in transwell assays or in vivo, using xenotransplantation in the zebrafish embryo.Altogether, our results show two regulatory networks involving histone modifications induced by nuclear receptors, leading to increased cell invasion.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E–Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

Das Ubiquitin-Proteasom-System (UPS) stellt eines der wichtigsten zellulären Abbausysteme dar. Es vermittelt die Degradation fehlgefalteter, beschädigter sowie regulatorischer Proteine. Folglich ist es essentiell für die Proteinqualitätskontrolle und für eine Vielzahl zellulärer Prozesse. Eine Störung des UPS steht im engen Zusammenhang mit neurodegenerativen Erkrankungen und malignen Tumoren. Adaptive Mechanismen ermöglichen es der Zelle das UPS an den stetig schwankenden Bedarf proteolytischer Aktivität anzupassen. So wirkt eine erhöhte Expression proteasomaler Gene einem Abfall der proteasomalen Aktivität entgegen. Der Transkriptionsfaktor TCF11/Nrf1 wurde hierbei als Hauptregulator identifiziert. Unter physiologischen Bedingungen ist TCF11/Nrf1 in der ER-Membran lokalisiert und wird über das ER-assoziierte Degradationssystem (ERAD) abgebaut. In Antwort auf Proteasominhibition wird der Transkriptionsfaktor aktiviert und in den Nukleus transferiert. Hier vermittelt er durch Bindung der regulatorischen antioxidative response elements die Genexpression proteasomaler Untereinheiten. Die Ergebnisse dieser Arbeit zeigten, dass es sich bei diesem autoregulatorischen Rückkopplungsmechanismus um einen generellen adaptiven Regulationsmechanismus in Mammalia handelt. Zudem ergaben weitere Untersuchungen, dass der durch Proteasominhibition hervorgerufene oxidative Stress, die TCF11/Nrf1-vermittelte Aktivierung der Genexpression fördert. Die induzierende Wirkung von oxidativem Stress wurde ferner unter Verwendung des Pro-Oxidans Rotenon bekräftigt. Dieses Neurotoxin induziert die TCF11/Nrf1-abhängige Transkription proteasomaler Untereinheiten und folglich die Neubildung aktiver Proteasomkomplexe. Der Transkriptionsfaktor förderte ferner die Zellviabilität Rotenon-behandelter SH-SY5Y Zellen. Diese Ergebnisse demonstrieren, dass die TCF11/Nrf1-vermittelte Genexpression proteasomaler Untereinheiten bedeutend für die Aufrechterhaltung der Redox- sowie der Protein Homöostase ist. / The ubiquitin proteasome system (UPS) represents a major protein degradation machinery. It facilitates the degradation of misfolded and damaged as well as regulatory proteins, thereby ensuring protein quality control and regulation of various cellular processes. Disturbances of the UPS are strongly associated with neurodegeneration and cancer. Adaptive mechanisms enable the cell to deal with changing demand in proteolytic activity. A rise in proteasomal gene expression compensates for decreased proteasomal activity. This adaption is mainly regulated by the transcription factor TCF11/Nrf1. Under unstressed conditions TCF11/Nrf1 resides in the ER-membrane where it is degraded via the ER-associated protein degradation system (ERAD). Proteasome inhibition causes the nuclear translocation of TCF11/Nrf1. In the nucleus, it mediates the gene expression of proteasomal subunits by interacting with their regulatory antioxidant response elements. Within this thesis, it was shown, that this autoregulatory feedback loop represents a general adaptive mechanism in mammalian cells. Moreover, experiments using antioxidative compounds revealed, that the oxidative stress induced by proteasomal inhibition promotes the TCF11/Nrf1-dependent proteasomal gene expression. The inducing effect of oxidative stress was verified using the pro-oxidant rotenone. This neurotoxin activates the transcription of the proteasomal genes resulting in the formation of newly synthesised, active proteasome complexes. Thus, TCF11/Nrf1 exerts a cytoprotective function in response to oxidative and proteotoxic stress in SH-SY5Y cells. In conclusion, this thesis revealed that TCF11/Nrf1-dependent induction of the proteasome expression promotes the maintenance of the redox as well as protein homeostasis.

漢厚朴酚是從木蘭科植物中萃取之天然化合物，已知具有抗氧化、抗發炎及神經保護之生理活性功能。先前的研究證明漢厚朴酚可以保護多巴胺神經元對抗6-OHDA所引起的細胞傷害，並且可以減緩6-OHDA 動物模式由apomorphine所誘發的旋轉行為，但漢厚朴酚對於神經保護之分子機制的相關研究尚未釐清。蛋白激酶CK2是具有多功能的絲氨酸/蘇氨酸激酶，高度表現在大腦紋狀體中，先前的研究證實蛋白激酶CK2參與調節神經系統功能和具有神經保護之作用。先前研究也指出轉錄因子Nrf1（Nuclear factor E2-related factor 1）是蛋白激酶CK2下游磷酸化受質，會調控小鼠胚胎纖維細胞中蛋白酶體基因的表現。抗細胞凋亡蛋白Mcl-1 (myeloid cell leukemia 1) 屬於Bcl-2蛋白家族的成員之一，在細胞凋亡的過程中，其蛋白含量減少與細胞凋亡有密切關聯性，抑制Mcl-1蛋白的降解可以延遲細胞死亡。因此本論文主要探討漢厚朴酚的神經細胞保護機制是否透過CK2-Nrf1細胞訊息路徑調控蛋白酶體活性，進而減少Mcl-1的降解速率。實驗結果顯示，轉染CK2α-EGFP DNA質體會增加Nrf1磷酸化並抑制蛋白酶體活性，泛素化之Mcl-1蛋白含量亦伴隨增加；轉染CK2α siRNA則會降低Nrf1磷酸化並促進蛋白酶體活性，導致naive Mcl-1蛋白質含量減少24小時的漢厚朴酚後處理（post-treatment）可以部份恢復因轉染CK2α siRNA所造成之CK2蛋白、Phosphoserine蛋白和Mcl-1蛋白質含量減少，在設計縮短間隔5小時漢厚朴酚後處理（post-treatment）的實驗結果雖然仍無法有效恢復CK2蛋白含量，但對於Phosphoserine和Mcl-1蛋白含量以及蛋白酶體活性則具有部份恢復的功效。利用過氧化氫造成細胞氧化壓力環境下，實驗發現間隔3小時的漢厚朴酚後處理才能有效恢復細胞存活率，間隔5小時的漢厚朴酚後處理則無法恢復細胞存活率。在大白鼠紋狀體腦區給予漢厚朴酚微量注射則對pTH、TH和GAD蛋白質含量皆有促進增加的作用，乙醯化的Histone H3蛋白含量也有顯著增加。綜合以上結果，推測漢厚朴酚對細胞保護作用的其中一個機制是參與調控CK2-Nrf1路徑而抑制蛋白酶體活性，減少Mcl-1蛋白質降解速率和提升氧化壓力下之細胞存活能力；此外，從活體動物的實驗結果顯示漢厚朴酚亦可能參與調控多巴胺和γ-氨基丁酸神經細胞功能的機制之中。 / Honokiol is a natural compound, extracted from the Magnolia officinalis, and is known as its anti-oxidative, anti-inflammatory and neuroprotective effects. The previous study has been demonstrated that the honokiol can protect striatal dopamine neuron against 6-OHDA induced damage and reverse the apomorphine-induced rotational behavior in Parkinson’s disease model of rats. However, the cellular mechanisms for its neuroprotective effects are not fully investigated. Protein kinase Casine kinase 2 (CK2) is a serine/threonine kinase has a highly abundant expression in the striatum compared with other brain areas. Further, CK2 is shown to regulate many neuronal functions including neuroprotection. The nuclear factor E2-related factor 1 (Nrf1) has been identified as one of the substrate proteins for CK2 and is indicated to involve in the induction of proteasome subunits gene expressions in mouse embryonic fibroblasts. The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) is shown to play a critical initiation role during the apoptosis process due to its synthesis blockage and proteasome degradation. The present study is aimed to investigate whether one of protective effects of honokiol is through CK2-mediated Nrf1 signaling pathway to regulate the proteasome activity in the mouse N2a neuroblastoma cell line. In the current results, transfection of the CK2α-EGFP plasmid DNA increased Nrf1 phosphorylation accompanied with the decrease in the proteasome activity but increased the ubiquitinated Mcl-1 protein. Whereas, transfection of CK2α siRNA decreased Nrf1 phosphorylation leading to the increase in proteasome activity and Mcl-1 protein degradation. The 24 hr duration of honokiol post-treatment only slightly reversed the knock-down effect of CK2α siRNA on CK2α and Mcl-1 protein levels. However, 5 hr duration of honokiol post-treatment could partially reverse the Mcl-1 protein level and proteasome activity but no effect on CK2α protein levels. In the H2O2-induced oxidative stress condition, only 3 hr duration of honokiol post-treatment could protect cells against H2O2-induced cell death. In the experiments of in vivo rat animal model, local administration of honokiol was found to increase phospho-TH, naive TH, GAD as well as acetylated Histone H3 protein levels. These above results suggest one of the protective mechanisms of honokiol might be through CK2-mediated Nrf1 signaling to inhibit the proteasome activity. and to promote cell survival under oxidative stress. Beside these functions, honokiol might also involve in the regulation of nurophysiological functions of dopamine and GABA neurons.

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1(+/+) than in KEAP1(-/-) isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1(-/-) cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.

Despite tremendous progress in the understanding of breast cancer (BC), gaps remain in our knowledge of the molecular basis underlying the aggressiveness of BC and BC disparities. Nuclear respiratory factor 1 (NRF1) is a transcription factor (TF) known to control breast cancer cell cycle progression. DNA response elements bound by NRF1 positively correlate with the progression of malignant breast cancer. Mechanistic aspects by which NRF1 contributes to susceptibility to different breast tumor subtypes are still not fully understood. Therefore, the primary objective of this dissertation was to decipher mechanisms by which NRF1 coordinates changes in the transcriptional and chromatin landscape affecting development and progression of invasive breast cancer. Our hypothesis was that NRF1 reprogramming the transcription of tumor initiating gene(s) and tumor suppressor gene(s) contribute in the development and progression of invasive breast cancer. To test this hypothesis, we proposed three specific aims: (a) Decipher regulatory landscape of NRF1 networks in breast cancer. (b) Determine the role of NRF1 gene networks in different subtypes of breast cancer. (c) Determine differential NRF1 gene network sensitivity contributing to breast cancer disparities. Our approach to test these aims consisted of a systematic integration of ChIP DNA-seq, RNA-Seq, NRF1 protein-DNA motif binding, signal pathway analysis, and Bayesian machine learning. We uncovered a novel oncogenic role for NRF1. This discovery strongly supported the supposition that NRF1 overexpression is sufficient to derive breast tumorigenesis. We also observed new roles for NRF1 in the acquisition of breast tumor initiating cells, regulation of epithelial to mesenchymal transition (EMT), and invasiveness of BC stem cells. Furthermore, through the use of Bayesian network structure learning we found that the NRF1 motif was enriched in 14 associated with HER2 amplified breast cancer. Three genes—GSK3B, E2F3, and PIK3CA—were able to predict HER2 breast tumor status with 96% to100% confidence. The findings of this study also showed the roles of NRF1 sensitivity to development of lobular A, Her2+, and TNBC in different racial/ethnic groups of breast cancer patients. In summary, our study revealed for the first time the role of NRF1 in the pathogenesis of invasive BC and BC disparities.

Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...