Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease that causes degeneration in both upper and lower motor neurons. ALS progresses relentlessly after the onset of the disease, with most patients die within 3-5 years of diagnosis, largely due to respiratory failure. Since SOD1 became the first gene whose mutations were associated with ALS in 1993, more than 17 ALS causative genes have been identified. Among them, TAR DNA-binding protein (TARDBP) lies in the central of ALS pathology mechanism study, because TDP43 proteinopathy is observed not only in familial ALS cases carrying TARDBP mutations, but also in most of the sporadic ALS cases, which account for 90% of the whole ALS population. Several TDP43 overexpression mouse models have been successfully generated to study the gain-of-toxicity mechanism of TDP43 in ALS development, while the investigation of loss-of-function mechanism which could also contribute to ALS still awaits a proper mouse model. The major difficulty in generating TARDBP knock out mouse model lies in the fact that TARDBP is a development essential gene and complete depletion of TDP43 function causes embryonic lethality. In chapter I, I reviewed the recent advances in ALS study. Emphasis was given to ALS mouse models, especially TARDBP ALS mouse model. In Chapter II, I made a Tet-responsive construct that contains mCherry, a fluorescent protein, as an indicator for the expression of the artificial miRNA (amiTDP) residing in the 3’UTR of mCherry and targeting TARDBP. The construct was tested in NSC34 cells and TRE-mCherry-amiTDP43 transgenic mouse was generated with this construct. Crossing TRE-mCherry-amiTDP43 mouse with mPrp-tTA mouse, mCherry expression was successfully induced in mouse forebrain and cerebellum, but not in other tissues including spinal cord. By quantitative real-time PCR, amiTDP43 expression was confirmed to be coupled with mCherry expression. Fluorescent immunostaining revealed that mCherry was expressed in neurons, but not in astrocytes or microglia cells, and that in mCherry positive cells, TDP43 was significantly knocked down. Results from Nissl staining and GFAP immunostaining suggested that decrease of TDP43 in forebrain neuron only was not sufficient to cause neurodegeneration and neuron loss. In chapter III, I investigated the function of Armadillo Containing Protein 4 (ARMC4), which was originally considered ALS causative gene. Our study of the function of CG5155, the possible homolog of ARMC4 in Drosophila, indicated that CG5155 is a male fertility gene that is involved in spermatogenesis. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with Bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 is a subfamily of Armadillo-repeat containing proteins with an evolutionarily conserved function in spermatogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. The nuclear factor κB (NF-κB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including CRC. Therefore, understanding the regulation of NF-κB is of ultimate importance for cancer therapy. Using a novel validation-based insertional mutagenesis (VBIM) strategy, our lab has identified the novel adenomatous polyposis coli (APC) like protein (ALP) gene as a negative regulator of NF-κB. Preliminary studies from our lab demonstrated that overexpression of ALP led to decreased NF-κB activity by κB reporter assay and electrophoresis mobility gel shift assay (EMSA). The current project aims to further evaluate the role of ALP in the regulation of NF-κB signaling in CRC cells. We found that overexpression of ALP in human CRC HT29 cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay. ALP overexpression also decreased the cell growth rate and cell migration ability, while shRNA mediated knockdown of ALP showed opposite effects, confirming that ALP is a tumor suppressor in CRC HT29 cells. Overexpression of ALP led to decreased NF-κB activity by κB reporter assay and condition media assay in CRC HT29 cells. Furthermore, immunohistochemical analysis with human colon vii tissues revealed that there is a gradual loss of ALP protein with tumor progression. We also found that ALP predominantly localizes in the cytoplasm, and binds to the p65 subunit of NF-κB, and might be functioning downstream of IκB kinase (IKK). In summary, in this study, we provide evidence regarding the tumor suppressor role of ALP in CRC by functioning as novel negative regulator of NF-κB. This discovery could lead to the establishment of ALP as a potential biomarker and therapeutic target in CRC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19174号 / 医博第4016号 / 新制||医||1010(附属図書館) / 32166 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 淳, 教授 村井 俊哉, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Durant le développement embryonnaire, une seule cellule, le zygote, est à l’origine de la formation de tous les types de cellules de l’organisme. L’information génétique nécessaire au destin d’une cellule y est gardé dans la chromatine condensée, d’abord inaccessible, et se déploie progressivement au moment opportun de la différenciation cellulaire grâce à des facteurs pionniers. Les facteurs de transcription pionniers sont des acteurs clés des cascades génétiques et épigénétiques déterminant le destin cellulaire. Les facteurs pionniers ont la propriété unique de reconnaître leurs cibles dans la chromatine fermée. Leur action permet d’augmenter l’accessibilité à un répertoire d’enhancers spécifique à un destin cellulaire, ouvrant la voie aux autres facteurs de transcription. Les aspects entourant la reconnaissance initiale des facteurs pionniers à leurs cibles dans la chromatine fermée sont mal compris. Dans l’hypophyse, le facteur pionnier Pax7 est spécifique aux cellules du lobe intermédiaire et met en place le programme génétique mélanotrope. Pour se faire, Pax7 repère les régions régulatrices clé de l’identité mélanotrope dans la chromatine fermée, afin d’accroître l’accessibilité à l’ADN permettant la liaison d’autres facteurs non-pionniers. Pax7 possède deux domaines de liaison à l’ADN, le domaine paired (PD) et l’homéodomaine (HD), chacun liant un motif de séquence caractéristique. De plus, une séquence cible composite, formé de la juxtaposition des motifs reconnus par PD et HD, est enrichie aux sites pionniers de Pax7. Dans le but de définir les propriétés de liaison à l’ADN de Pax7, nous avons caractérisé les interactions de Pax7 avec ses différentes séquences cibles. Nous avons démontré par des expériences de liaison à l’ADN in vitro (retard sur gel) que l’interaction de Pax7 avec le motif composite est d’affinité supérieure et sous forme de monomère où le PD est principalement impliqué. Comme les sites cibles de Pax7 dans l’hétérochromatine sont marqués par la méthylation de l’ADN, nous avons montré que la méthylcytosine dans le motif de liaison n’interfère pas avec la liaison à l’ADN par Pax7. De plus, des mutations ponctuelles aux domaines de liaison à l’ADN de Pax7 ont montré que les deux domaines de liaison à l’ADN sont nécessaires pour le recrutement aux sites dans la chromatine fermée, a fortiori pour l’ouverture de la chromatine. Des études antérieures ayant identifié des transcrits alternatifs de Pax7, nous avons étudié l’impact fonctionnel des variants Pax7. Les quatre isoformes Pax7 comportent des résidus d’acides aminés alternatifs au niveau du PD : elles ont des propriétés de liaison à l’ADN in vitro ainsi qu’un potentiel de transactivation très similaires. Bien que les isoformes de Pax7 aient des activités pionnières variables, leur action différentielle n’est pas dû à leurs propriétés intrinsèques de liaison à l’ADN ou de transactivation. Nos travaux ont identifié les particularités de Pax7 par rapport à d’autres facteurs pionniers dans les mécanismes de reconnaissance des sites dans l’hétérochromatine. Ainsi, les enhancers sujet à l’action pionnière de Pax7 contiennent typiquement plus de motifs cibles que les cibles d’action transcriptionnelle et l’action pionnière de Pax7 un recrutement fort aux sites pionniers ; de plus, les deux domaines de liaison à l’ADN intacts sont essentiels pour ce recrutement et l’action pionnière. Nos travaux ont défini les paramètres requis pour le reprogrammage cellulaire par un pionnier, Pax7, tout particulièrement en regard des sites de recrutement dans la chromatine fermée. La reprogrammation de cellules souches pour la production de cellules différenciées présente un potentiel thérapeutique unique en médecine régénérative et nos travaux supportent le rôle critique des pionniers dans ce contexte. / During embryonic development, a single cell, the zygote, is responsible for the formation of all cell types in the body. The genetic information necessary for cell fates is stored there in condensed chromatin, initially inaccessible, and is gradually deployed at the opportune moment of cell differentiation by the pioneer factors. Pioneer transcription factors are key determinants in the genetic and epigenetic cascades leading to cell fate. Pioneer factors have the unique property of recognizing their DNA targets in closed chromatin. Their action provides accessibility to a repertoire of enhancers specific to a cell fate, opening the way for other transcription factors. We poorly understand initial recognition of pioneer factors at their targets in closed chromatin. In the pituitary gland, the pioneer factor Pax7 is specific to the intermediate lobe and implements the melanotrope genetic program. To do so, Pax7 identifies key regulatory regions of melanotrope identity in closed chromatin to provide DNA accessibility allowing the binding of other non-pioneer factors. Pax7 has two DNA binding domains (DBD), the paired domain (PD) and the homeodomain (HD), each binding a characteristic sequence motif. In addition, a composite target sequence, formed from the juxtaposition of PD and HD motifs, is enriched at the pioneer sites of Pax7. In order to define the DNA binding properties of the pioneer factor Pax7, we characterized the interactions of Pax7 with its different target sequences. We have demonstrated by in vitro DNA binding experiments (gel shift) that the interaction of Pax7 with the composite motif is of higher affinity and as a monomeric form in which the PD is primarily involved. As Pax7 target sites in heterochromatin are marked by DNA methylation, we showed that methylcytosine within the binding motif does not interfere with Pax7 DNA binding. Using point mutations in the Pax7 DBDs, we showed that both DBDs are required for the recruitment to sites in closed chromatin, furthermore for chromatin opening. Since previous studies identified alternative Pax7 transcripts, we investigated the functional impact of Pax7 variants. The four Pax7 isoforms contain alternative amino acid residues in the PD: they have similar in vitro DNA binding properties and transactivation potential. Although Pax7 isoforms have varying pioneering activities, their differential action is not due to their intrinsic DNA-binding or transactivation properties. Our work has identified the particularities of Pax7 compared to other pioneering factors in the mechanisms of site recognition in heterochromatin. Thus, the enhancers subject to the pioneer action of Pax7 typically contain more target motifs than the targets of transcriptional action and the pioneer action of Pax7 a strong recruitment to the pioneer sites; moreover, the two intact DNA-binding domains are essential for this recruitment and pioneering action. Our work has defined the parameters required for cellular reprogramming by a pioneer, Pax7, particularly with regard to recruitment to sites in closed chromatin. The reprogramming of stem cells for the production of differentiated cells has unique therapeutic potential in regenerative medicine and our work supports the critical role of pioneers in this context.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor (TNF) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNF-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNF to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNF-mediated signaling, supporting the functionality with an NF-B luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNF to its receptors and influencing TNF-mediated signaling via its interaction with progranulin.

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