京都大学 / 新制・課程博士 / 博士(医学) / 甲第23062号 / 医博第4689号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 妹尾 浩, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

Les cellules souches cancéreuses (CSC) sont à l’origine de la progression tumorale, des métastases et rechutes tardives. Elles ont été identifiées dans de nombreux cancers, comme le cancer du sein triple négatif (TNBC) et cancers de grade III-IV. Elles sont résistantes aux chimiothérapies et radiothérapie et résident dans une niche immuno-répressive. Cette étude vise à évaluer une stratégie d’immunothérapie qui cible sélectivement les CSC dans le modèle murin 4T1-GFP-Luc mimant le TNBC. Le phénotype/ génotype des mamosphères a été initialement caractérisé. Basée sur l’analyse génomique des CSC, nous avons développé une immunothérapie active associée à des agents immuno-modulateurs. Nous avons mesuré la taille des tumeurs et suivi l’apparition des métastases par bioluminescence. Une étude immunologique et analyse génomique de la tumeur a été réalisée. La combinaison thérapeutique provoque le recrutement dans la tumeur de lymphocytes T (CD4 +, CD8 +) et lymphocytes B par augmentation de CXCL13, une réduction des lymphocytes T reg et cellules myéloïdes suppressives. Cette induction de réponse immunitaire provoque la diminution de la taille de la tumeur et des métastases. Cette nouvelle immunothérapie active de type vaccinale pourra être utilisée en association avec les traitements actuels pour des mesures prophylactiques et curatives dans une grande variété de cancers. / Cancer stem cells (CSCs) are responsible for tumor progression, metastases, and late relapses. They have been identified in many cancers, such as triple negative breast cancer (TNBC) and grade III to IV cancers. They are resistant to chemotherapy and radiotherapy and reside in an immuno-repressive niche.This study aims to evaluate a immunotherapy strategy that selectively targets CSCs in the mouse model 4T1-GFP-Luc mimicking TNBC. The phenotype / genotype of mammosphere was initially characterized. Based on genomic analysis of CSC, we have developed an active immunotherapy associated with immunomodulatory agents. We measured the size of tumors and monitored the appearance of metastases by bioluminescence. We performed an immunological study and genomic tumor analysis. The therapeutic combination causes the recruitment of CD4 + and CD8 + T lymphocytes and B lymphocytes with increased CXCL13, the reduction of T reg cells and suppressive myeloid cells in the tumor. This induction of intra-tumor immune response leads to a decrease in tumor size and metastases.This new active immunotherapy can be used in combination with current treatments for prophylactic and curative measures in a wide variety of cancers.

No description available.

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells. / EPSRC, Biostatus / The full text was made available at the end of the re-embargo period, 1st September 2017.

No description available.

Notch receptors and ligands are single-pass transmembrane proteins which play important roles in cell-cell communication. Notch in ‘harmony’ with other signaling pathways regulate the entire diversity of metazoan life (Artavanis-Tsakonas & Muskavitch, 2010). These signaling pathways also play key roles in regulatingseveral developmental processes. Given the importance of Notch signaling in various developmental decisions, it is not surprising that aberrant gain or loss-of-function of Notch pathway leads to several human diseases including cancer (Ranganathan et al, 2011). Notch signaling has also been implicated in various human cancers, most notably in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al, 2004). In view of the importance of Notch signaling in cancers, therapeutic molecules targeting this pathway are making their way into clinical trials (Rizzo et al, 2008). This underscores the importance of understanding the mechanism of Notch receptor activation in normal and patho-physiological conditions. In this thesis, antibodies against different domains of human Notch1 receptor have been used as tools to understand the mechanism of receptor activation. This work has provided insights into the role of Notch1 extracellular domain in ligand-dependent receptor activation. Further, the mechanism of ligand-independent receptor activation in T-ALL associated mutant Notch1 has also been investigated. This understanding of ligand-dependent and independent receptor activation facilitated development of mechanistic inhibitors of Notch signaling for therapeutic targeting of the cancer stem cells (CSCs) across the pectrum of cancers. The thesis is divived into two parts. Part-I focuses on understanding the role of Notch1 extracellular domain in receptor-ligand interactions using antibodies as a tool. In part-II, implications of these antibodies in therapeutic targeting of CSCs has been investigated. Part-I Unfolding the Mechanism of Notch1 Receptor Activation The extracellular domain of Notch1 receptor consists of 36 EGF-like repeats that contribute to ligand binding (Kopan & Ilagan, 2009). Despite extensive studies on the downstream consequences of Notch signaling, the initial events of ligandreceptor interactions have not been clearly elucidated. In the absence of structural insights into the receptor-ligand interactions, it was important to decipher the roles of various receptor domains in ligand-binding and consequent signaling. In this study, antibodies have been employed as tools for in-depth analyses of Notch receptorligand, interactions. Studies in Drosophila Notch receptor suggest that EGF-like repeats 11-12 are necessary and sufficient for ligand binding (Rebay et al, 1991). However, the role of these repeats in human Notch1 receptor-ligand interaction(s) was not clearly elucidated. Antibodies were generated against Notch1 EGF-like repeats 11-15. Further, these antibodies were characterized for their specificity for Notch1 receptor in various ligand-binding and signaling assays. The results suggest that the monoclonal antibodies (MAbs) against EGF-like repeats 11-12 were more potent inhibitors of ligand-binding compared to the antibodies against EGF-like repeats 13-15. As a part of these investigations, the Notch ligands Jagged1 and Jagged2, Delta-like1 and Delta-like4 were purified and characterized in various assays. Ability of these ligands to interact with Notch1 EGF-like repeat 11-15 was determined using Surface Plasmon Resonance. The Jagged family of ligands demonstrated higher affinity for this recept or fragment when compared to the Delta family of ligands. The relatively low affinities (μM) of all the ligands suggested possibile involvement of other EGF-like repeats in ligand-binding. This was further investigated using antibodies against other EGF-like repeats of Notch1. In Drosophila Notch EGF-like repeats 24-29 have been implicated in the ligand-dependent gain-of-function phenotype, suggesting a plausible involvement of this region in receptor activation (Pei & Baker, 2008). Therefore, role of human Notch1 EGF-like repeats 21-30 in ligand-binding and signaling was investigated. These EGF-like repeats demonstrated specific interaction with the ligand-binding domain (EGF-like repeats 11-15). This suggested that in the absence of the ligand, these inter-domain interactions keep the receptor in an auto-inhibited conformation. Further, ligand binding to EGF-like repeats 11-15 dissociated pre-formed interdomain interactions. These results suggested that, the binding of ligand to EGF-like repeat 11-12 overcomes the negative constraint imposed by the intra-domain interactions which might lead to receptor activation. Next, to understand the role of EGF-like repeats 21-30 in ligand binding, polyclonal antibodies were generated against the same and extensively characterized in various solid-phase and cell-based assays. These antibodies demonstrated partial inhibition of ligand-binding. Further, using immunoaffinity purified antibodies it was demonstrated that antibodies against EGF-like repeats 25-26 were most potent inhibitors of ligand-binding compared to antibodies against EGF-like repeats 21-24 and 27-30. These results provided novel insights into Notch1 receptor activation. The model proposed on the basis of these results suggested that ligand-binding to EGF-like repeats 11-12 competes with the inter-domain interaction, in turn dissociating EGF-like repeats 21-30 from the ligandbinding domain. It emerged that this altered conformation of the receptor creates a secondary ligand-binding site at EFG-like repeats 25-26. Overall these results provided novel insight into the mechanism of Notch receptor-ligand interaction(s). Part-II Implication in Cancer Stem Cell Targeting Recent studies have suggested existence of the CSC population in various cancers (Clevers, 2011). Notch signaling plays an important role in maintenance of these CSCs (Pannuti et al, 2010). Thus, targeting Notch signaling may provide a potential therapeutic tool for CSC targeting. Several studies have indicated that Notch1 receptor and ligands are overexpressed in breast cancer cells compared to the normal breast epithelium (Mittal et al, 2009; Reedijk et al, 2005; Reedijk et al, 2008). Moreover, it has been suggested that Notch1 signaling plays a key role in breast carcinogenesis (Stylianou et al, 2006). Monoclonal antibodies (MAbs) were used as mechanistic inhibitors of aberrant Notch1 signaling for therapeutic targeting of CSCs. One such antibody, MAb 602.101, against Notch1 ligand-binding domain (EGF-like repeat 11-12) inhibited proliferation and depleted breast CSCs. This MAb also modulated genes associated with stemness and epithelial to mesenchymal transition (EMT). Furthermore, MAb 602.101 irreversibly inhibited the sphere-forming potential of breast cancer cells by modulating long-term self renewing capacity of breast CSCs. Inhibition of Notch1 signaling by the MAb also depleted the chemoresistant CD44Hi/CD24Low sub-population in breast cancer cells. Interestingly, antibody treatment led to elevated expression of genes associated with myoepithelial lineage, which suggested that inhibition of Notch1 signaling might induce a differentiation program leading to reduction in the CSC population. This study demonstrated the importance of Notch1 signaling in CSCs and effectiveness of antibodies as a tool for specific targeting of individual Notch receptors in cancer therapeutics. While aberrant expression of receptors and ligands leads to breast cancer (Reedijk et al, 2005), gain-of-function mutations are associated with 40-50% of TALL\ patients (Weng et al, 2004). These mutations lead to ligand-independent receptor activation (Malecki et al, 2006). Despite several attempts of successful antibodymediated therapeutic targeting of Notch1 (Aste-Amézaga et al, 2010; Wu et al, 2010), specific antibodies recognizing T-ALL associated mutant Notch1 remains elusive. Using homology modeling, the mutation induced conformational change in T-ALL associated mutant Notch1 was predicted. These results suggested that mutation led to conformational changes in the Notch1 negative regulatory region (NRR) This conformation change might result in the constitutive activation of Notch1 signaling leading to pathogenesis. Next, MAbs were generated against the wild-type Notch1 NRR and characterized in flow-cytometry based assays for identification of conformation specific antibodies. These antibodies were classified as either wild-type specific, mutant specific or unbiased to receptor conformations. One such mutant specific MAb 604.107 demonstrated higher binding to mutant Notch1 in flowcytometer and SPR based experiments. This MAb also demonstrated specific inhibition of T-ALL associated mutant Notch1 signaling without affecting the wildtype signaling. Moreover, antibody treatment also inhibited proliferation and depleted leukemia initiating sub-population in patient derived T-ALL cells. Taken together, this study provides a novel tool for specific targeting of mutant Notch1 receptors in TALL. CSCs are inherently chemo-resistant and lead to tumor relapse (Chen et al, 2012). Recent studies have demonstrated a strong correlation between Notch1 signaling in lung CSCs and chemotherapy resistance (Hassan et al, 2013). In this study, Notch1 heterogeneity in solid tumors viz. breast and colon cancers was investigated. Using the antibodies generated previously in this study, Notch1High and Notch1Low sub-populations from MDA-MB-231 (breast cancer) and HCT-116 (colon cancer) cell lines were flow-sorted. It was demonstrated that the Notch1High subpopulation represented the sphere-forming CSCs in breast and colon cancer. The Notch1High sub-population also demonstrated chemo-resistant properties and expressed higher level of EMT and stemness markers. These results suggested explicit involvement of Notch1 signaling in EMT and maintenance of CSCs subpopulation in these cancers. The anti-Notch1 MAb also inhibited proliferation of the chemo-resistant Notch1High sub-population. Further, treatment with MAb inhibited expression of ABCC1 transporters in these drug-resistant cells leading to augmentation of chemotherapeutic response. Using mouse xenograft assays, it was demonstrated that Notch1 signaling plays an important role in the maintenacne of tumor-initiating sub-population in breast and colon cancer cells. Prior exposure of breast and colon cancer cells to MAb inhibited the tumor forming potential of these cells in xenotransplantation assays. Treatment with MAb alone or in combination with chemotherapy led to regression of pre-formed tumors in breast and colon xenograft models. These results demonstrated existence of Notch1 heterogeneity in breast and colon cancer cells and emphasised the importance of targeting Notch1 signaling to overcome drug-resistance in these cancers. The results described above have provided important insights into Notch1 receptor activation and this understanding was translated into therapeutic targeting of CSCs. This “proof-of-principle” demonstration has significant mechanistic and applied implications in Notch and cancer biology.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in both men and women, and one of the leading causes of cancer-related deaths worldwide. One frequently mutated pathway involved in oncogenesis in CRC is the RAS/RAF/MAP kinase pathway. Oncogenic activation of KRAS and BRAF occur in 30‒40% and 5‒15% of all CRCs, respectively, and the mutations are mutually exclusive. Even though KRAS and BRAF are known to act in the same pathway, KRAS- and BRAF-mutated CRCs have different clinical and histopathological features. For example, BRAF mutation in CRC is tightly linked to microsatellite instability (MSI) and a CpG island methylator phenotype (CIMP), which is not seen in KRAS-mutated tumours. BRAF-mutated CRCs are also more often found in right-sided tumours. However, the underlying molecular reasons for these differences have not yet been defined. The overall aim of this thesis was to investigate molecular differences between KRAS- and BRAF-mutated CRCs to understand how KRAS and BRAF mutations differentially affect tumour progression. We used an in vitro cell culture system to explore molecular differences between KRAS- and BRAF-mutated CRCs and verified our findings using CRC tissue specimens from the Colorectal Cancer in Umeå Study (CRUMS). We found that BRAF mutation, but not KRAS mutation, was associated with expression of the stem cell factor SOX2. Furthermore, SOX2 was found to be correlated to a poor patient prognosis, especially in BRAF-mutated cancers. We further investigated the role of BRAF in regulation of SOX2 expression and found that SOX2 is at least partly regulated by BRAF in vitro. We continued by investigating the functional role of SOX2 in CRC and found that SOX2-expressing cells shared several characteristics with cancer stem cells, and also had down-regulated expression of the intestinal epithelial marker CDX2. There was a strong correlation between loss of CDX2 expression and poor patient prognosis, and patients with SOX2 expression were found to have a particularly poor prognosis when CDX2 levels were down-regulated. In conclusion, in these studies we identified a subgroup of BRAF-mutated CRCs with a particularly poor prognosis, and having a cancer stem cell-like appearance with increased expression of SOX2 and decreased expression of CDX2. Tumour progression is regulated by interactions with cells of the immune system. We found that BRAF-mutated CRCs were more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours, while the opposite was true for KRAS-mutated CRCs. Interestingly, we found that part of this difference is probably caused by differences in secreted chemokines and cytokines between KRAS- and BRAF-mutated CRCs, stimulating different arms of the immune response. Altered levels of expression of miRNAs have been seen in several malignancies, including CRC. We found that BRAF- and KRAS-mutated CRCs showed miRNA signatures different from those of wild-type CRCs, but the expression of miRNAs did not distinguish KRAS-mutated tumours from BRAF-mutated tumours. In summary, our findings have revealed possible molecular differences between KRAS- and BRAF-mutated CRCs that may explain some of the differences in their clinical and histopathological behaviour.

L'objectif de cette thèse est d'étudier la sensibilité des lignes cellulaires du cancer du sein BTCL aux agents stabilisants des microtubules (taxanes et ixabépilone) afin de: 1 - identifier la pharmaco-génomique prédictif de la réponse (résistance / sensibilité) comme une signature moleculaire, et de valider cette signature sur d'autres études dont les données génomiques sont disponibles en ligne, donc mis l'expression des gènes prédictifs de GES pour Tax- sensibilité (333 gènes ) et Ixa-sensibilité (79 gènes) ont été définis, et les Taxanes prédicateurs GES a considérablement prédit Pac-sensibilité dans BTCL, et pathologiques réponse complète à base de Pac-chimiothérapie néoadjuvante chez les patients du cancer du sein. 2 - étudier le rôle des cellules souches du cancer (ALDH +) sur la réponse thérapeutique aux Taxanes et donc, Nous identifions quatre lignes BTCL qui présentent un enrichissement significative dans le pourcentage et le nombre absolu de ALDELFUOR cellules positives dans chacun de ces quatre BTCLs après 5 jours de traitement par le paclitaxel, en contraste avec les résultats précédents, nous avons constaté que dans ces autres 3 BTCLs le phénomène est inversé avec la diminution significative du pourcentage et le nombre absolu de cellules positives ALDELFUOR trouve dans chacun de ces trois BTCLs après 5 jours du traitement par le paclitaxel. Une signature moléculaire de SCC résistant / sensible de 243 pb avec 179 gènes dont 152 gènes sont régulés à la hausse et 27 gènes régulés à la baisse au CSC résistantes au paclitaxel, une sorte prédicteurs génomiques pour Tax - sensibilité au CSC résistantes au paclitaxel peut être dérivée à partir BTCL et peut être utile pour mieux comprendre les mécanismes de résistance aux taxanes et de l'implication de la CSC dans cette résistance, afin de mieux sélectionner des traitements cytotoxiques chez les patients du cancer du sein et l'identification des d'autres marqueurs potentiels de thérapies ciblées dans l'avenir. 3 - Nous avons testé l'impact de l'altération des paramètres génomiques et protéiques ou les mutations de certains gènes comme tau (MAPT), K-alpha tubuline (TUB A1B) tubuline alpha-6 (A1C TUB) tubuline beta 3 (TUBB3) et stathmine (STMN1), malheureusement nous n'avions jamais identifier une mutation pour être corrélée à la réponse des BTCL aux Taxanes. 4 - Nous essayons d'étudier au niveau de protéines par immunohistochimie sur le tissu de micro-array et cyto-micro-array pour certains paramètres qui ont été déjà prouvé (in vitro) pour être corrélée à la réponse aux Taxanes, (cette partie est en fait en cours). / The aim of this thesis is to study the sensitivity of breast cancer cell lines BTCL to microtubule-stabilizing agents (Taxanes and ixabepilone) in order to:1- identify pharmaco-genomic predictor of response (resistance /sensitivity) as a molecular signature, and to validate this signature on others studies of which the genomic data are available on line, so gene expression set GES predictors for Tax-sensitivity (333 genes) and Ixa-sensitivity (79 genes) were defined, and the Taxanes GES predictors has significantly predicted Pac-sensitivity in BTCL, and pathological complete response to Pac-based neo-adjuvant chemotherapy in BC patients.2- study the role of cancer stem cell (ALDH+) on the therapeutic response to Taxanes and their we identify 4 BTCLs which present a significant enrichment in the percentage and the absolute numbers of ALDELFUOR-positive cells found in each of these 4 BTCLs after 5 days of treatment by Paclitaxel , In contrast to the previous results we found that in others 3 BTCLs these phenomenon is inversed with the significant decrease of the percentage, and the absolute numbers of ALDELFUOR-positive cells found in each of these 3 BTCLs after 5 days of treatment by Paclitaxel.A molecular signature of CSC resistant /sensitive of 243 pb with 179 genes of which 152 genes are up- regulated and 27 genes down-regulated in CSC resistant to Paclitaxel, so a genomic predictors for Tax-sensitivity in CSC resistant to Paclitaxel can be derived from BTCL and may be helpful for better understanding the mechanisms of resistance to Taxanes and the implication of CSC in this resistance in order to better select of cytotoxic treatment in breast cancer patients and identification of others potential markers for targeted therapies in the future .3- we tested the impact of the alteration of genomic and proteic parameters or the mutations of some genes like tau (MAPT),Tubulin K- ALPHA (TUB A1B) Tubulin alpha-6 (TUB A1C) Tubulin beta 3 (TUBB3) and Stathmin (STMN1), unfortunately we did'nt identify a mutations to be correlated to BTCL response to Taxanes .4- we try to study at the level of proteins by immunohistochemistry on the tissue micro- array and cyto-micro-array for some parameters which have been already proved (in vitro) to be correlated with response to Taxanes , ( this part is actually ongoing).

<p>Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity. </p> <p>As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation. </p> <p>Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.</p>