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1
Bagci, Hakan. „Epigenetic reprogramming and DNA demethylation“. Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45352.
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Embryonic development begins with fertilization of the egg, a progressive process that gives rise to the zygote and subsequently to the formation of somatic tissues. Normally once cells acquire a fate, it is stably maintained. Conversion back to a multipotent state occurs rarely in-vivo, but can be achieved experimentally by inducing ‘reprogramming’. In this study I am looking at the epigenetic mechanisms that underlie reprogramming and, in particular, DNA methylation and demethylation. To address this I am taking advantage of the cellular fusion system. Fusion of pluripotent cells with differentiated cells results in the formation of transient heterokaryon and hybrid cells, where the somatic partner is efficiently reprogrammed. This gives me the opportunity to monitor early and late events in pluripotent conversion, in which global remodelling of chromatin and changes in DNA methylation occur. Here, I examine changes in DNA methylation that are induced at imprinted loci and pluripotency-associated genes when somatic cells are fused with either mouse embryonic stem (ES) or embryonic germ (EG) cells. I focus on defining the factors and order of events that accompany reprogramming. I show that acquisition of pluripotency is an early process occurring at the heterokaryon stage, and is followed by imprint erasure later in hybrids. However reprogramming of imprinting is only induced by EG, but not ES cells, and it requires sequential steps of 5-methylcytosine oxidation mediated by Tet proteins and nucleotide exchange upon several rounds of DNA synthesis. I provide evidence that Tet proteins are dispensable for pluripotent reprogramming using CRISPR-Cas9 genome editing to abrogate the expression of both Tet1 and Tet2. This result suggests that either DNA demethylation can occur without TET activity (implying a redundancy with other demethylating agents and routes), or that DNA demethylation is not required for inducing pluripotency. Finally, I describe how CRISPR/Cas9 approaches were used to demonstrate that non-canonical Wnt signalling components are downstream targets of JARID2 in ES cells.
2
Hajkova, Petra. „Epigenetic reprogramming in mouse germ cells“. [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=970526938.
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3
Rao, Venkata Lakshmi Prakruthi. „Epigenetic Reprogramming at the Th2 Locus“. University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543838686940608.
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4
Ribeiro, Lemos Pereira Carlos Filipe. „Epigenetic events underlying somatic cell reprogramming“. Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/4439.
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Although differentiated cells normally retain cell-type-specific gene expressionpatterns throughout their lifetime, cell identity can sometimes be modified or reversedin vivo by transdifferentiation, or experimentally through cell fusion or by nucleartransfer. Several studies have illustrated the importance of chromatin remodelling, DNAdemethylation and dominant transcriptional factor expression for changes in lineageidentity. Here the epigenetic mechanisms required to ?reset? genome function wereinvestigated using experimental heterokaryons.To examine the epigenetic changes that are required for the dominantconversion of lymphocytes to muscle, I generated stable heterokaryons betweenhuman B-lymphocytes and mouse C2C12 myotubes. I show that lymphocyte nucleiadopt an architecture resembling that of muscle and initiate the expression of musclespecificgenes in the same temporal order as developing muscle. The establishment ofthis novel gene expression program is coordinated with the shutdown of severallymphocyte-associated genes. Interestingly, inhibition of histone deacetylase (HDAC)activity during reprogramming selectively blocks the silencing of lymphocyte-specificgenes but does not prevent the establishment of muscle-specific gene expression.In order to reprogram somatic cells to pluripotency, I fused human Blymphocytesand mouse embryonic stem (ES) cells. The conversion of human cells isinitiated rapidly, occurring in heterokaryons before nuclear fusion. Reprogramming ofhuman lymphocytes by mouse ES cells elicits the expression of a human ES-specificgene expression profile in which endogenous hSSEA4, hFgf receptors and ligands areexpressed while factors that are characteristic of mouse ES cells, such as Bmp4 andLif receptor are not. Using genetically engineered mouse ES cells I demonstrate thatsuccessful reprogramming requires the expression of Oct4, but importantly, does notrequire Sox2, a factor implicated as critical for the induction of pluripotency. Followingreprogramming, mOct4 becomes dispensable for maintaining the multi-potent state ofhybrid cells. Finally, I have examined the reprogramming potential of embryonic germ(EG), embryonic carcinoma (EC) and ES cells deficient for the Polycomb repressivecomplex 2 (PRC2) proteins Eed, Suz12 and Ezh2. While EC and EG cells share theability to reprogram human lymphocytes with ES cells, the lack of Polycomb proteinsabolishes reprogramming. Thus, the repressive chromatin mark (H3K27 methylation)catalysed by PRC2 play a crucial role in keeping ES cells with full reprogrammingcapacity. Collectively my results underscore the importance of chromatin events duringcell fate reprogramming.
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Hajkova, Petra. „Epigenetic reprogramming in mouse germ cells“. Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2004. http://dx.doi.org/10.18452/15020.
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Bei Säugerkeimzellen, Zygoten und Embryos in frühen Stadien kommt der epigenetischen Neuprogammierung eine außergewöhnlich wichtige Rolle in der Regulation der Genomfunktionen in entscheidenden Entwicklungsstadien zu. Die epigenetische Neuprogrammierung in Keimzellen löscht zuerst die Imprinting-Markierungen und Epi-Mutationen und stellt dann geschlechtsspezifische Markierungen (genomische Prägung) wieder her. Die vorliegende Arbeit bezieht sich auf das Löschen epigenetischer Modifikationen in primordialen Mauskeimzellen (primordial germ cells (PGCs)) zwischen dem 10.5 bis 13.5 Tag nach der Befruchtung. Entgegen früheren Annahmen zeigen unsere Ergebnisse, daß primordiale Mauskeimzellen (PGCs) beim Eintritt in die embryonalen Keimdrüsen noch immer DNS Methylierungsmarker besitzen, die ähnlich dem Marker in somatischen Zellen sind. Kurz nach dem Eintritt in die Keimdrüsen werden die DNS Methylierungsmarker, die in Verbindung mit geprägten und nicht geprägten Genen stehen, gelöscht. Für die Mehrzahl der Gene beginnt die Löschung der Marker in männlichen und weiblichen Embryos gleichzeitig und ist innerhalb eines Entwicklungstages abgeschlossen. Diese Kinetik deutet auf einen aktiven Demethylierungsprozess hin, initiiert durch ein somatisches Signal, ausgehend von der embryonalen Keimdrüse. Der Zeitpunkt der Neuprogrammierung in den primordialen Keimzellen ist entscheidend, da er sicherstellt, daß Keimzellen beiden Geschlechts einen epigenetisch äquivalenten Status erhalten, bevor sie geschlechtsspezifisch ausdifferenzieren und anschließend neu elterlich geprägt werden. Vollständiges Verständnis des Prozesses der Neuprogrammierung der Keimzellen ist nicht nur im Hinblick auf genomisches Imprinting wichtig, sondern auch für die Erforschung von Mechanismen für die Wiederherstellung von omnipotenten Zellen bei Klonierung und Stammzellenerhaltung.Epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. Germ line epigenetic reprogramming assures erasure of all the imprinting marks and epi-mutations and establishment of new sex-specific gametic imprints. The presented work focuses on the erasure of epigenetic modifications that occur in mouse primordial germ cells (PGCs) between day 10.5 to 13.5 post coitum (dpc). Contrary to previous assumptions, our results show that as they enter the genital ridge the PGCs still possess DNA methylation marks comparable to those found in somatic cells. Shortly after the entry of PGCs into the gonadal anlagen the DNA methylation marks associated with imprinted and non-imprinted genes are erased. For most genes the erasure commences simultaneously in PGCs of both male and female embryos and is completed within only one day of development. The kinetics of this process indicates that is an active demethylation process initiated by a somatic signal emanating from the stroma of the genital ridge. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which, new parental imprints are established subsequently. Complete understanding of the germline reprogramming processes is important not only in the light of genomic imprinting but also for resolving other mechanisms connected with restoring cellular totipotency, such as cloning and stem cell derivation.
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Dura, Mathilde. „Critical and different roles of DNA methylation in male germ cell development“. Electronic Thesis or Diss., Sorbonne université, 2021. http://www.theses.fr/2021SORUS187.
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La méthylation de l'ADN, associée à la répression des gènes et des éléments transposables (ET), joue un rôle essentiel dans la spermatogenèse. Le méthylome des futurs gamètes est reprogrammé : les profils de méthylation somatiques sont effacés, des profils spécifiques des cellules germinales sont établis. Trois de novo ADN méthyltransférases (DNMT) sont essentielles à la méthylation de l'ADN des cellules germinales mâles chez la souris : les enzymes DNMT3C et DNMT3A et leur cofacteur DNMT3L. Il a été montré que DNMT3C est l'enzyme qui méthyle sélectivement les ET les plus jeunes évolutivement. Cependant, les cibles et la fonction de DNMT3A étaient encore inconnues. Je me suis donc intéressée aux rôles de DNMT3A et DNMT3C dans la régulation épigénétique de la spermatogénèse. J'ai démontré (projet 1) une division de travail remarquable : alors que DNMT3C empêche les ET d'interférer avec la méiose, DNMT3A méthyle largement le génome, à l'exception des ET dépendants de DNMT3C. J'ai découvert que les cellules souches spermatogoniales (CSS) mutantes pour Dnmt3A ont perdu leur potentiel de différentiation à cause de l’activation erronée d’enhancers qui imposent un programme génétique de cellules souches. Ce travail révèle une nouvelle fonction de la méthylation de l'ADN dans la fertilité mâle. En parallèle (projet 2), j’ai étudié la nature de la spécificité de reconnaissance des jeunes ET par DNMT3C. Ces séquences présentent une dynamique chromatinienne unique: d’abord un profil bivalent de type H3K4me3-H3K9me3 qui évolue vers un enrichissement H3K9me3 exclusif. Mon travail a ainsi fourni des éléments nouveaux pour comprendre le rôle de la méthylation de l’ADN en reproductionDNA methylation, associated with gene or transposable element (TE) repression, plays a key role in spermatogenesis. During germ cell development, their methylome is reprogrammed: somatic patterns are erased and germ cell-specific patterns are established. Three de novo DNA methyltransferases (DNMTs) are essential for shaping male germ cell DNA methylation in mice: DNMT3C and DNMT3A enzymes and DNMT3L co-factor. DNMT3C was shown to selectively methylate young TEs. However, the targets and function of DNMT3A was still unknown. During my PhD, I investigated the interplay between DNMT3A and DNMT3C in the epigenetic regulation of spermatogenesis. First (project 1), I reported a striking division of labor: while DNMT3C prevents TEs from interfering with meiosis, DNMT3A broadly methylates the genome—except DNMT3C-dependent TEs—and controls spermatogonial stem cell (SSC) plasticity. By single-cell RNA-seq and chromatin states profiling, I found that Dnmt3A mutant SSCs cannot differentiate due to spurious enhancer activation that enforces a stem cell gene program. I thus demonstrated a novel function for DNA methylation for SSC differentiation and life-long spermatogenesis supply. Second (project 2), I investigated the chromatin determinants of DNMT3C specificity towards young TEs. I found that these sequences present unique dynamics: first a bivalent H3K4me3-H3K9me3 enrichment, followed by a switch to H3K9me3-only. H3K9me3-enrichment was also a hallmark of the sequences that gain DNA methylation upon ectopic DNMT3C expression in cultured embryonic stem cells. As a whole, my work provided novel insights into the complexity of DNA methylation-based control of reproduction
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Oksuz, Samet. „Targeting IL-4 locus for epigenetic reprogramming“. University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1423581203.
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8
Yong, Qian Yu. „A screen for modifiers of epigenetic reprogramming“. Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/50955/1/Qian_Yu_Yong_Thesis.pdf.
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Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development.
Changes in epigenetic modifiers have been shown be lethal, or cause diseases.
Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells.
During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32.
These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.
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Aguilar, Sanchez Cristina. „Epigenetic transitions in cardiovascular development and cell reprogramming“. Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28787.
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Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.
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Wanichnopparat, Wachiraporn [Verfasser]. „Epigenetic reprogramming of hepatocyte-like cells / Wachiraporn Wanichnopparat“. Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1239645333/34.
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11
Eggan, Kevin C. (Kevin Carl) 1974. „Cloning, stem cells and epigenetic reprogramming after nuclear transfer“. Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/29931.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.Includes bibliographical references (leaves 128-146).
The process by which a single totipotent cell becomes a complex organism is a unidirectional program, with each mitotic division generating new cells that gradually differentiate towards more specified fates and specialized functions. Nuclear transfer (NT) experiments have demonstrated the epigenetic nature of development and showed, that although differentiated cells have a very limited developmental potential, the nuclei of these cells retain the potency to direct embryogenesis after reintroduction into the unfertilized oocyte. Herein, we have used the mouse as a model system for understanding both the nature of epigenetic reprogramming that occurs after NT as well as the ramifications it has for the development of cloned animals. Specifically, we investigated how epigenetic states are reprogrammed after NT and demonstrated that the inactive X chromosome is reactivated in NT embryos, resulting in normal X inactivation in female clones. Additionally, investigations into the factors that influence the survival of cloned animals, indicate that there are considerable genetic influences on the cloning process. These genetic factors modify the survival of mice cloned from ES cells by influencing the developmental potential of the donor ES cells rather then the reprogramming process itself. This realization has subsequently led to the development of novel methods for the expedited production of complex mutant mice, which are also described. Finally, we have created cloned embryos by NT from both cortical and mature olfactory sensory neurons to address question of nuclear equivalence in the brain and to investigate whether generation of synaptic diversity or odorant receptor choice, are mediated by genetic as well as epigenetic events.
by Kevin C. Eggan.
Ph.D.
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Moley, Laura A. „Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos“. DigitalCommons@USU, 2019. https://digitalcommons.usu.edu/etd/7571.
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Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology with the birth of Dolly. By increasing efficiency by selecting the embryos early in development that are most likely to succeed following transfer into a surrogate mother, the technology could be more routinely utilized to enhance animal agriculture production. SCNT is believed to be highly inefficient as a result of incorrect DNA methylation and gene expression that are accumulated because of the SCNT technique. We proposed the use of a non-toxic, non-invasive detector of cell death, to quantitatively assess embryo competency prior to embryo transfer. We believed we could use SR-FLICA to identify the embryos with low levels of cell death as a result of proper DNA methylation and gene expression. By analyzing the whole embryo, differences in gene expression and DNA methylation were identified in embryos with high and low levels of cell death. However, the level of cell death did not prove to be a reliable indicator of embryo quality in predicting pregnancy outcome. This data supports the commonly held hypothesis that DNA methylation and gene expression after SCNT have random defects as a result of the random nature of resetting the DNA for embryo development. More research is required to identify the embryos which will prove to be successful following SCNT and embryo transfer.
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Mariano, Piero. „Epigenetic Regulation and Reprogramming of the H19 Imprinting Control Region“. Doctoral thesis, Uppsala universitet, Zoologisk utvecklingsbiologi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6299.
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The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.
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Molaro, Antoine. „Inheritance and evolution of epigenetic reprogramming in Mammalian germ cells“. Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00833274.
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During mammalian post-implantation development, germ cells are induced from the somatic tissues of the embryo. Following their induction, primordial germ cells undergo a genome-wide erasure and de novo re-establishment of DNA methylation marks. This epigenetic reprogramming re-instates pluripotency and allows parental imprints to be deposited. In the male germ line, a unique RNAi pathway involving PIWI proteins and their associated small RNAs (piRNAs) is necessary for proper de novo methylation. PIWI mutant mice are infertile and display methylation defects over transposon sequences. Using a transgenic approach, we investigated the signals necessary for piRNA production. We show that artificial piRNAs can be produced from reprogrammed loci outside of their native context. We then studied the genome-wide impact of piRNA loss on germ cell methylation. Whereas most of the genome is properly methylated, only a small group of transposons transiently reactivated in primordial germ cells is affected. Also we identified important structural differences in de novo methylation profiles between human sperm and ES cells. Finally, we compared sperm methylation profiles between human and chimpanzee and showed that the genome and the epigenome can evolve independently. Taken together, our results highlight the surprising plasticity of genome and epigenome interactions during development and evolution
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Choi, Inchul. „Effects of oocyte on epigenetic reprogramming of bovine SCNT embryos“. Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479364.
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Nguyen, Khoi Thien. „Epigenetic determinants of cellular differentiation, transcriptional reprogramming, and human disease“. Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130186.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 111-130).
Much of the diversity we observe in cellular and organismal phenotypes can be attributed to epigenetic and genetic variation. DNA provides the instructions for life, while epigenetic modifications regulate which parts of the genetic information contained in DNA can be read out in a given cell and how this information is interpreted. In recent years, epigenetic and genetic variation has been profiled on a large scale with sequencing-based assays, generating many datasets to be explored. In this thesis, I present three projects which apply computational techniques to identify and characterize epigenetic mechanisms that may contribute to the regulation of phenotypic variance. First, we mine a dataset charactering the epigenomes of diverse cell types in order to discover signatures of adult stem cell differentiation.
We identify a novel marker of the multipotent state, a chromatin state characterized by the histone marks H3K36me3 and H3K9me3, and describe biological processes that may be linked to the loss of this chromatin state in fully differentiated cell types. Next, I present what we learned from profiling the epigenetic state of cells before and after transplantation into Xenopus oocytes, a process that transcriptionally reprograms the cells. This analysis elucidates how the initial epigenetic state of a cell influences the success of cellular reprogramming and identifies transcription factors that help regulate this process. Finally, we integrate studies measuring the effects of genetic variants on disease with studies measuring the effects of genetic variants on transcriptional and epigenetic activity. This identifies specific mechanisms underlying disease processes, and demonstrates that transcriptional and epigenetic mechanisms may independently contribute to disease pathogenesis.
Together, these projects demonstrate the biological insights that can be gained from epigenetic profiling, and expand our understanding of the potential effects of epigenetic modifications.
by Khoi Thien Nguyen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering
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Adams, Kevin Douglas. „Innate Immune Cell Phenotypes Are Dictated by Distinct Epigenetic Reprogramming“. BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7700.
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The innate immune system is the first line of host defense against external exposures. During these initial encounters, antigen presenting cells - specifically monocytes and macrophages - modulate further inflammatory responses. Macrophages exist along a spectrum of phenotypic programs; on the inflammatory M1 end they enhance immune activity while on the anti-inflammatory M2 end they suppress further immune activation. Furthermore, within M2 macrophages there exist many subpopulations, namely M2a and M2d, each with specific roles during infection or exposure. We sought to compare the epigenetic profiles of these subpopulations of macrophages to determine key regulatory gene networks and factors that could be exploited for therapeutic benefit.While traditionally viewed as primitive and nonspecific, a growing body of clinical and experimental evidence argues the innate immune system develops memory as a result of previous exposures, allowing the innate system to respond with enhanced and broad immunological protection upon exposure to a secondary stimulus. This biological process of innate immunity has been termed trained immunity. Trained immunity shares many phenotypic and epigenetic characteristics with adaptive immune memory; however, one of the starkest distinctions is the propensity of trained immunity to develop against heterologous stimuli. Innate memory is not antigen specific, frequently protecting the host against unrelated organisms.
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Pérez, Camps Mireia. „Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish“. Doctoral thesis, Universitat Politècnica de València, 2010. http://hdl.handle.net/10251/6902.
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El estudio de los mecanismos de reprogramación nuclear tiene actualmente una notable importancia, dado que el dominio de estos procesos constituyen la clave para actuar eficazmente en cuestiones tan dispares como el cáncer o la medicina regenerativa. También resulta muy importante este tipo de estudios sobre reprogramación cuando se pretende la obtención de animales transgénicos múltiples y orientados.
Aunque para ello se pueden utilizar muy diversos modelos animales, en nuestro caso, se ha optado por el pez cebra, por sus características en el desarrollo, como la brevedad en la embriogénesis y transparencia de los embriones, su capacidad de regeneración y el conocimiento de su genoma, entre otras. Bien es cierto que a estas ventajas le acompañan cierto inconvenientes tales como no disponer hasta este momento de técnicas tales como el transplante nuclear y, a otro nivel, el quimerismo. Técnicas cuyos desarrollo se pretende en esta tesis, lo que justifica los objetivos aquí planteados.
Para ello se han realizado diferentes trabajos experimentales titulados: "Ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish", "Evaluation of presumptive caudal fin blastema cells as candidate donors in intraspecies zebrafish (Danio rerio) chimaeras", "Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization", "Transplant of adult fibroblast into the central region of metaphase II eggs resulted in mid blastula transition (MBT) embryos", "Electroactivation of zebrafish (Danio rerio) eggs", "Comparison of different activating stimuli efficiency in zebrafish nuclear transplant", "Reconstruction of heteroparental gynogenetic diploid condition by nuclear transplant in zebrafish".
Los dos primeros relativos al quimerismo, su eficiencia final se optimizó mediante la penalización con radiación UV del embrión receptor.Pérez Camps, M. (2009). Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/6902
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Wongtawan, Tuempong. „Epigenetic and chromatin reprogramming in mouse development and embryonic stem cells“. Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/8048.
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It is well established that epigenetics and chromatin modifications are important factors that can govern gene activity and nuclear architecture. They are also proven to be essential for normal embryonic development and cell differentiation. One important event during mouse development is the establishment of epigenetic reprogramming which is believed to be essential for normal growth and development, however; the mechanism is still poorly understood. The general objective of this PhD study was to investigate the profiles and mechanisms of epigenetic and chromatin modifications during normal mouse development and in embryonic stem cells. Mouse pre- and postimplantation embryos and ES cells were used in experiments employing a range of different methodologies. The dynamics of epigenetic DNA and histone methylation were captured using laser confocal immunofluorescent microscopy and western blotting. The activity of epigenetic modifiers was monitored by real-time PCR and candidate genes were validated using siRNA technology. The present studies demonstrate that heterochromatin markers H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. Demethylation of H3K9me2, H3K9me3 and H4K20me3 took place at two-cell stage and remethylation occurred at four-cell stage except for H4K20me3. The reestablishment of H4K20me3 was initially observed in early postimplantation embryos in extraembryonic tissue, specifically in the mural trophectoderm. In embryonic tissue, H4K20me3 was not clearly detected until in mid to late postimplantation development. The mechanism of H3K9me2 and H3K9me3 demethylation might be due to either an imbalance of epigenetic modifiers or the presence of Jmjd2a and Jmjd1a histone demethylase postfertilisation. We have also report evidence that HP1α and Suv4-20h are required in heterochromatin before the recruitment of H4K20me3 during mouse development and in ES cells. Therefore H4K20me3 removal was believed to involve the lack of prerequisite heterochromatin complexes such as HP1α and Suv4-20h enzymes. Furthermore, the presence and levels of H4K20me3 and HP1α might be strongly associated with cell differentiation and tissue maturation in mouse in vivo development but not in vitro early differentiated ES cells. Surprisingly, the results showed that chromatin modifications and their modifiers in ES cells are different from ICM and epiblast. Chromatin modifications H4K20me3 and HP1α were absent from ICM and epiblast, but were detected in ES cells. Notably, H4K20me3 and HP1α were established after early incubation of ICM into ES cell medium, but this change was not dependent on the presence of serum and leukaemia inhibiting factor. Epigenetic modifier Jmjd2a but not Jmjd1a was found in ICM. Conversely, Jmjd1a is highly expressed in ES cells while Jmjd2a was inactivated. In addition, the present studies revealed the substantial role of histone demethylases in development, as it may be important for epigenetic reprogramming. The results demonstrated that inhibition of demethylase Jmjd2a and Jmjd1a caused preimplantation embryos to arrest at the twocell stage while Jmjd2c deficient embryos failed to reach blastocyst. Thus it is possible that Jmjd2a and Jmjd1a were essential for epigenetic reprogramming while Jmjd2c is critical for cell fate establishment during blastocyst formation. In conclusion, the global chromatin signature in ES cells differs from ICM and epiblast; heterochromatin reprogramming occurs at two-cell stage; maturation of heterochromatin occurs at postimplantation; and histone demethylases Jmjd1a, Jmjd2a and Jmjd2c are important in preimplantation development. Results from the present studies could provide crucial information for developmental biology and stem cell research, and provide as a model for improvement of reproductive biotechnologies such as somatic cell reprogramming, and diagnosis of epigenetic abnormalities in early development.
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Gillich, Astrid. „Epigenetic reprogramming of epiblast stem cells to a naïve pluripotent state“. Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610321.
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Wang, Stan. „Reprogramming and epigenetic factors regulating pluripotency and the stem cell state“. Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709334.
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22
Zhang, Haixin. „Gene regulatory network and epigenetic reprogramming of pig primordial germ cells“. Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33452/.
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Primordial germ cells (PGC) are the precursors of the gametes. The mechanisms of PGC induction, specification and development are very well characterized in rodents, however recent investigations have demonstrated that the mechanisms of germ cell development differ significantly between mice and humans. Since the knowledge of PGC development in non-rodents is very limited, and early human embryos cannot be accessed it is important to establish a new model for PGC development with relevance to humans. In this thesis, I use pig embryo as a model for investigating PGC development in non-rodent mammals. The expression profile of key transcription factors, epigenetic reprograming and the role of signalling pathways were investigated during specification and development of pig PGCs. The key findings are: A- Specification of porcine PGC occurs after the onset of gastrulation, requiring BMP4 signalling. B- WNT signalling is required for the generation of precursors competent for germline commitment; however it is downregulated after PGCs are specified. WNT downregulation could be modulated by SOX17, the earliest gene expressed in pig PGCs. C- Epigenetic reprogramming of DNA and histone marks starts in pre-migratory porcine PGC. Furthermore, chromatin dynamics in pig gonadal PGCs resemble that of humans but differs to that of mice. D- The expression profile of transcription factors of porcine PGC is similar to that of humans, but different to mouse PGC. In conclusion, this study has highlighted critical differences between mice and humans/pigs during germ cell specification. I provide evidence that the pig embryo is a useful model for the study of human development, and future studies will need to be directed to re-evaluate concepts of cell differentiation and early lineage commitment established in mice that may not apply to humans.
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Gibson, Adrienne Rae. „Pharmacological ascorbate enhances oxygen consumption and epigenetic reprogramming in pancreatic cancer“. Thesis, University of Iowa, 2018. https://ir.uiowa.edu/etd/6424.
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Pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C) results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. We hypothesized that there may be a sustained effect (> 24 h) of P-AscH- that may contribute to cytotoxicity. P-AscH- significantly increased sustained oxygen consumption (OCR), DCFH-DA oxidation, and extracellular acidification (ECAR) in tumor lines with no change in non-tumorigenic cells. One possible source of this sustained ROS and OCR, the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX), which are epigenetically silenced by methylation in vitro and in vivo in PDAC, are up-regulated with P-AscH- treatment. Catalase pretreatment reversed the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate is unable to mediate the increase in DUOX expression. Together these results suggest that P-AscH--induced toxicity may be enhanced by late metabolic and epigenetic shifts in tumor cells resulting in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR. These data highlight a novel epigenetic mechanism of action for P-AscH-.
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Jouravleva, Karina. „Telomere-driven chromosome instability impacts the genetic program through genome-wide epigenetic reprogramming“. Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066286.
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Le raccourcissement télomérique est la source majeure de l'instabilité chromosomique (CIN) au cours de la progression tumorale. Nous avons montré que les cellules humaines embryonnaires de rein (cellules HEK) ayant traversé une période de CIN subissent des vastes changements dans l'expression des microARNs, ce qui induit une transition épithélio-mésenchymateuse (TEM), un processus permettant aux cellules cancéreuses épithéliales migrer et envahir de nouveaux tissus et former des métastases. Notre travail a aussi suggéré que les cellules ayant subi une TEM étaient capables de former des tumeurs dans un microenvironnement sénescent. De surcroît, cette évolution dans la capacité tumorale était associée à une dérégulation supplémentaire des microARNs et à l'acquisition des propriétés des cellules souches. Afin d'étudier comment ce potentiel est mis en place au cours de l'instabilité chromosomique et au contact avec le microenvironnement sénescent, nous avons modulé les niveaux d'expression de miR-145 et avons démontré que la répression de miR-145 était nécessaire pour le développement des caractéristiques des cellules souches. Afin de mieux comprendre l'impact de CIN sur le programme génétique des cellules épithéliales, nous avons utilisé des approches de haut débit et avons caractérisé les changements des paysages chromatiniens et leur mise en place dans les cellules ayant traversé une période de CIN. Nos résultats révèlent pour la première fois que l'instabilité télomérique modifie profondément la distribution des marques d'histones en conduisant aux changements d'expression des gènes et au processus de transformation des cellules épithéliales pré-tumoralesTelomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We have shown that human epithelial kidney (HEK) cells undergo massive microRNA deregulation upon CIN, in particular a miR-200-dependent epithelial-mesenchymal transition (EMT), which is thought to enable epithelial cancer cells to migrate and invade other tissues to form metastases. Our work also indicated that CIN+ cells that underwent EMT were able to form tumors in a senescent microenvironment. Notably, this progression in tumor capacity was associated with further microRNA deregulation and the manifestation of enhanced stem-like properties. To investigate how stem-like properties are acquired in CIN+ cells in the contact with senescent microenvironment we adapted knockdown and overexpression approaches to modulate miR-145 expression, and demonstrated that enhanced stem-like properties depended on miR-145 repression. To fully apprehend the impact of CIN on the genetic program of epithelial cells, we used an unbiased approach to characterize the chromatin state of HEK CIN+ cells and uncover genome wide redistributions that were in direct correlation with gene expression changes. Our results reveal for the first time that T-CIN profoundly modifies the chromatin landscape genome-wide thereby fueling the transformation process of pre-tumor epithelial cells
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Jouravleva, Karina. „Telomere-driven chromosome instability impacts the genetic program through genome-wide epigenetic reprogramming“. Electronic Thesis or Diss., Paris 6, 2015. http://www.theses.fr/2015PA066286.
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Le raccourcissement télomérique est la source majeure de l'instabilité chromosomique (CIN) au cours de la progression tumorale. Nous avons montré que les cellules humaines embryonnaires de rein (cellules HEK) ayant traversé une période de CIN subissent des vastes changements dans l'expression des microARNs, ce qui induit une transition épithélio-mésenchymateuse (TEM), un processus permettant aux cellules cancéreuses épithéliales migrer et envahir de nouveaux tissus et former des métastases. Notre travail a aussi suggéré que les cellules ayant subi une TEM étaient capables de former des tumeurs dans un microenvironnement sénescent. De surcroît, cette évolution dans la capacité tumorale était associée à une dérégulation supplémentaire des microARNs et à l'acquisition des propriétés des cellules souches. Afin d'étudier comment ce potentiel est mis en place au cours de l'instabilité chromosomique et au contact avec le microenvironnement sénescent, nous avons modulé les niveaux d'expression de miR-145 et avons démontré que la répression de miR-145 était nécessaire pour le développement des caractéristiques des cellules souches. Afin de mieux comprendre l'impact de CIN sur le programme génétique des cellules épithéliales, nous avons utilisé des approches de haut débit et avons caractérisé les changements des paysages chromatiniens et leur mise en place dans les cellules ayant traversé une période de CIN. Nos résultats révèlent pour la première fois que l'instabilité télomérique modifie profondément la distribution des marques d'histones en conduisant aux changements d'expression des gènes et au processus de transformation des cellules épithéliales pré-tumoralesTelomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We have shown that human epithelial kidney (HEK) cells undergo massive microRNA deregulation upon CIN, in particular a miR-200-dependent epithelial-mesenchymal transition (EMT), which is thought to enable epithelial cancer cells to migrate and invade other tissues to form metastases. Our work also indicated that CIN+ cells that underwent EMT were able to form tumors in a senescent microenvironment. Notably, this progression in tumor capacity was associated with further microRNA deregulation and the manifestation of enhanced stem-like properties. To investigate how stem-like properties are acquired in CIN+ cells in the contact with senescent microenvironment we adapted knockdown and overexpression approaches to modulate miR-145 expression, and demonstrated that enhanced stem-like properties depended on miR-145 repression. To fully apprehend the impact of CIN on the genetic program of epithelial cells, we used an unbiased approach to characterize the chromatin state of HEK CIN+ cells and uncover genome wide redistributions that were in direct correlation with gene expression changes. Our results reveal for the first time that T-CIN profoundly modifies the chromatin landscape genome-wide thereby fueling the transformation process of pre-tumor epithelial cells
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Berrens, Rebecca V. „Role of small RNAs and chromatin in transposable element silencing during global demethylation“. Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/275980.
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DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.
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Michelatti, Daniela. „Oncogenic enhancer reprogramming in triple negative breast cancer tumour progression“. Doctoral thesis, Università degli studi di Trento, 2022. http://hdl.handle.net/11572/327998.
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Basal breast cancer is a heterogeneous disease whose unfavourable outcome is determined by a high risk of tumour relapse and metastasis formation. The potential of a cancer cell to adapt to foreign environments is favoured by oncogenic cell plasticity, which is supported by epigenetic reprogramming. It was previously demonstrated that MYC acts as an oncogenic reprogramming factor by inducing epigenetic rewiring at enhancers (Poli et al., 2018). This causes the activation of oncogenic pathways and pro-metastatic transcription factors such as SOX9, but scant pieces of evidence support a causal link between epigenetic alteration of oncogenic enhancers and cell plasticity. In the present work, we investigated the establishment of an alternative epigenetic program during tumorigenesis in a basal breast cancer xenograft derived model. We found that tumorigenic cells, primary tumour derived cells and metastasis derived cells showed intrinsically different phenotypic and epigenetic signatures, and that metastatic derived cells were characterized by the acquisition of pro-metastatic features, such as migration and invasion, that may increase their metastatic potential. Specifically, we provided data supporting the notion that changes of the chromatin landscape during tumour progression increased the responsiveness of cancer cells to environmental cues that they may encounter during dissemination and colonization of distant organs. We focused on investigating the role played by putative regulatory elements localized around the SOX9 locus, whose chromatin accessibility and interaction with the SOX9 promoter were increased in metastatic cells. We observed that SOX9 expression was responsive to the activation of the retinoic acid (ATRA) pathway, and our data suggests that this response may be strengthened by transcriptional memory priming SOX9 regulatory elements after a first exposure, so that the response is faster and more robust after the second one. SOX9 transcription modulation and ATRA response were also shown to be linked to the activation of a quiescence program specific of metastatic cells, which we hypothesise may favour cells during the dissemination steps of the metastatic cascade.
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Popp, Christian. „Investigating the role of the cytidine deaminase AID in epigenetic reprogramming in the germline“. Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608864.
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Loke, Justin Ching Ting. „Identification of common and distinct epigenetic reprogramming properties of core-binding factor fusion proteins“. Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7298/.
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RUNX1, also known as CBFa, is a master regulator of haematopoiesis. In Acute Myeloid Leukaemia (AML) it is frequently disrupted by translocations to different epigenetic regulators, resulting in the expression of core-binding factor fusion proteins. We compared the chromatin landscape of t(8;21) and t(3;21) AML which express RUNX1-ETO and RUNX1-EVI1, respectively. We found that the diverse clinical outcomes of patients with these two forms of AML are reflected in fundamental differences in gene expression and chromatin landscape. Despite both fusion proteins sharing a RUNT DNA binding domain, we show that RUNX1-EVI-1 targets a more immature stem cell-related gene expression program of genes as compared to RUNX1-ETO. Despite the differences in the epigenomic landscape of t(3;21) and t(8;21) leukaemia, knockdown of either core-binding factor fusion protein activates a common myeloid differentiation program involving up regulation of C/EBPa. By blocking C/EBPa DNA binding through a dominant negative partner, we showed that this factor is required for the downstream effects of RUNX1-EVI-1 knockdown. Even in the continued presence of RUNX1-EVI-1, ectopic expression of C/EBPa. is sufficient to initiate myeloid differentiation in t(3;21) cells. Overall, this suggests that deregulation of C/EBPa is a common pathway in the development of both t(8;21) and t(3;21) AML.
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Kutschat, Ana Patricia [Verfasser]. „Gemcitabine Resistance Elicits a Calcium Dependent Epigenetic Reprogramming in Pancreatic Cancer / Ana Patricia Kutschat“. Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1229692126/34.
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Perikala, V. „Understanding the relevance of epigenetic reprogramming for resistance to HDAC inhibitors in cancer cells“. Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004805/.
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Therapeutic responses to Histone deacetylase (HDAC) inhibitors (HDACi) in many cancers are well described but development of resistance to HDACi is a major stumbling block. Whether HDACis induce epigenetic reprogramming and how this contributes to relapse is not reported. A CTCL cell line HuT78, and a CLL cell line MEC1, were used to develop HDACi resistant clones (RHuT78 and RMEC1 respectively) that persistently grow in the presence of the clinically used HDAC inhibitor Romidepsin. RHuT78 cells show perturbed trimethylation of histone H3 lysine K4 on Romidepsin treatment which linked to higher protein expression levels of the implicated demethylase KDM5A. Following on from these experiments, a qRT-PCR epigenetic gene expression array was used to quantify levels of 84 epigenetic gene transcripts in RHuT78 cells and significantly altered genes were taken forward for further investigation. Studies of gene expression patterns in parental, resistant and ‘drug holiday’ cell lines of both HuT78 and MEC1 led to particular interest in HDAC8, DNMT3A and DNMT3B. Functional studies showed that HDAC8 overexpression increased proliferation and resistance of HuT78 cells to Romidepsin. Parallel observations suggested an increase in proliferation of resistant cell lines cultured in the presence of the HDACi. This increased proliferation was seen even with lower concentrations of Romidepsin and argues against prolonged monotherapy using HDACis. Significantly, inhibitors of DNA methyltransferases synergised with Romidepsin in a dose and schedule dependent manner, reversing the changes in epigenetic gene expression associated with resistance and causing increased apoptosis in RHuT78 cells. Taken together this thesis identifies and characterises an unacknowledged contribution of epigenetic reprogramming to drug resistance and provides insights into the effects of Romidepsin on the epigenome that could potentially contribute to HDACi resistance.
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Davis, Aaron Patrick. „Apoptotic and Epigenetic Induction of Embryo Failure Following Somatic Cell Nuclear Transfer“. DigitalCommons@USU, 2013. https://digitalcommons.usu.edu/etd/1941.
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Somatic cell nuclear transfer (SCNT) is a useful tool for selective breeding, conservation, and production of transgenic animals. Despite the successful cloning of several species, high rates of embryo failure following SCNT prevent the wide-scale use of the technique. Embryos produced through cloning have a higher incidence of developmental arrest, decreased developmental potential, frequent implantation failures, and increased incidence of abortion. The objective of this dissertation research was to characterize the factors that lead to SCNT failures by examining epigenetic and apoptotic pathways that can negatively influence the development of cloned preimplantation embryos. Aberrant genome reprogramming is generally considered to be a key factor in the failure of SCNT embryo development. Therefore, we used bisulfite pyrosequencing technology to compare DNA methylation patterns of several genes critical for embryonic development (POU5F1, NANOG, SOX2, and KLF4) in SCNT and in vitro fertilized (IVF) blastocyst stage embryos. The methylation profiles obtained from these experiments indicate that methylation patterns of the POU5F1 gene were undermethylated compared to IVF embryos, suggesting reprogramming did occur, but that the reduced methylation was inappropriate for the blastocyst stage. Furthermore, aberrant methylation profiles were detected for SOX2 and NANOG, suggesting that problems of genome reprogramming following SCNT can be gene-specific or localized. Because high rates of apoptosis are associated with failure of preimplantation embryos, we compared the activation of the P53-mediated apoptosis pathway in individual IVF and SCNT preimplantation embryos at multiple developmental stages. This pathway is activated in response to cell stress and genomic instability, and in response to the expression of genes associated with somatic cell reprogramming. Evidence from gene expression and immunohistochemistry analyses suggests that the P53 pathway is frequently active in SCNT embryos. Also, we detected expression of several factors known to induce apoptosis more frequently and at higher levels in SCNT embryos. Collectively, the work presented here illuminates some of the molecular consequences of incomplete or inappropriate genome reprogramming in cloned embryos. The identification of these factors may lead to interventions that target the apoptosis pathway during preimplantation development and increase SCNT success rates.
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XIERAILI, AOBULI. „IDENTIFICATION OF EPIGENETIC INHIBITORS OF PHYSIOLOGICAL CELLULAR PLASTICITY AS NOVEL TUMOR SUPPRESSOR“. Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/609578.
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Cellular plasticity, the inter-conversion of cells between differentiated cells and stem cells (SCs), can lead to tissue regeneration and restoration of homeostasis after injury. Conversely, inappropriate induction of cellular plasticity could be involved in tumor initiation and progression, through de-novo generation of cancer stem cells (CSCs) by de-differentiation of normal or non-tumorigenic bulk tumor cells. This intrinsically dangerous potential must be tightly controlled by genetic and epigenetic mechanisms, to prevent unscheduled de-differentiation. However, their systematic identification by large-scale screenings is just beginning to be exploited.
In order to identify physiological inhibitors of cell plasticity, that could play a function as tumor suppressors, we performed short-hairpin RNA (shRNA) screens. In the screens, we used pooled lentiviral shRNA libraries targeting 234 epigenetic regulators to identify shRNAs endowing mouse mammary progenitors with the SC-specific ability to regenerate mammary gland tissue upon in vivo orthotopic transplantation. Sequencing of genomic DNA extracted from the regenerated mammary glands led to the identification of 38 hits/genes. We individually validated 7 hits by in vivo regeneration assays, showing that their down-regulation leads to conversion of mammary progenitors into stem cells able to regenerate mammary glands. We performed also in vitro mammosphere and phenotypic cell conversion assays to examine the hits’ function in self-renewal and cell plasticity, respectively. Next, among the validated hits, we showed that the inhibition of Cbx5 and Kmt2d induced efficient reprogramming of mammary progenitors. Therefore, we focused to investigate their mechanistic function in reprogramming at the transcriptome level, by RNA sequencing. We identified a specific enrichment for pro-inflammatory signalling pathways as an early transcriptional reprogramming response (ETRR), followed by up-regulation of Myc target genes.
Finally, in order to set the basis for further characterization of reprogramming mechanisms induced by the validated shRNAs, we established an organoid assay using MycER, our positive control for mammary progenitors reprogramming, showing that MycER over-expression bestows mouse luminal cells with enhanced self-renewal ability and differentiation capacity. Moreover, we performed a single cell transcriptome analysis in mouse primary mammospheres that revealed considerable heterogeneity as 20 clusters identified and led to the identification of Cd36 (glycoprotein, collagen type I receptor) as a putative mammary SC-specific surface marker. We expect that further analyses of these data, together with single cell transcriptomic analysis of mammospheres interfered for the validated hits, will shed light on the mechanisms involved in physiological cell plasticity.
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Köhler, Daniela. „Cloning in cattle : nuclear architecture and epigenetic status of chromatin during reprogramming of donor cell nuclei“. kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/9915/.
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Hajkova, Petra [Verfasser], Harald [Gutachter] Saumweber, Jörn E. [Gutachter] Walter und Wolf [Gutachter] Reik. „Epigenetic reprogramming in mouse germ cells / Petra Hajkova ; Gutachter: Harald Saumweber, Jörn E. Walter, Wolf Reik“. Berlin : Humboldt-Universität zu Berlin, 2004. http://d-nb.info/1207678422/34.
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POLI, VITTORIA. „MYC agisce da fattore di riprogrammazione tumorale tramite induzione di uno stato di staminalità cellulare in cellule epiteliali umane della ghiandola mammaria“. Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/117389.
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Cancer is both a genetic and an epigenetic disease whose outcome is influenced by tumor microenvironment. These three determinants represent the major driving forces of tumorigenesis and cause the functional heterogeneity observed in most of the cancer types. Both normal and neoplastic cell populations are known to harbor subpopulations of Stem Cells (SCs) that can both self-renew and spawn more differentiated progeny that, in the case of cancer, forms the tumor bulk. For what concerns Cancer Stem Cells (CSCs), they are proposed to held most of the tumor initiating potential and a higher proportion of CSCs within a tumor often correlates with poorer prognosis. Accumulating evidences indicate that SCs transcriptional program of both normal and neoplastic tissues rely on common molecular regulators including important developmental signals, such as the WNT pathway, whose de-regulation is causative of tumor initiation. Given their importance in the maintenance and propagation of cancer, identifying the cell of origin of CSCs would represent a great opportunity for understanding the molecular mechanisms underlying the onset of tumorigenesis, thereby offering new therapeutic strategies. A likely scenario of how multistep tumorigenesis may proceeds is that pools of transit amplifying progenitor cells, that are mitotically more active and more numerous than SCs, may serve as targets of somatic and epigenetic alterations that would cause their de-differentiation, pushing them back to the SCs compartment. This emerging model is based upon recent findings that hierarchically organized cell populations are, at least in epithelial tissues such as the mammary gland, more plastic than previously imagined. In this view, cell transformation can be described as a cell reprogramming process toward a SC-like state, in which a committed cell has to over-come a number of epigenetic barriers, that normally stabilize it, in order to alter its identity. Although promising, the mechanisms that should drive to the epigenetic reprogramming of committed cells are largely undefined and the putative role of developmental signals in this process has not been elucidated so far. We hypothesize that the proto-oncogene c-MYC could represent a bona fide tumor reprogramming factor. c-MYC over-expression in cancer cells can result from constitutive activation of a number of developmental pathways that lay up-stream c-MYC expression, such as the WNT/β-Catenin pathway constitutive activation is a rate-limiting step in tumorigenesis. On the basis of the findings obtained in our laboratory, MYC is able to sustain embryonic stem cells self-renewal by activating the WNT/β-Catenin pathway, through direct recruitment of Polycomb Repressive Complex 2 (PRC2) on the promoter of the two major inhibitors DKK1 and SFRP1. We hypothesize that a similar mechanism could explain what happens in the early events of tumorigenesis. More specifically, MYC over-expression in committed cells could induce their reprogramming to a SC-like state, through induction of autocrine WNT pathway hyper-activation, therefore favoring the acquisition of further genetic and epigenetic insults, that would induce the uprising of a CSC phenotype. Our results indicate that MYC over-expression in human Immortalized Mammary Epithelial Cells (IMECs) induces a cellular reprogramming towards a luminal progenitor cell-like state and that such condition is associated to hyper-activation of the WNT/β-Catenin pathway. Unlikely wild type cells, MYC-enriched pool of progenitor cells is prone to originate CSCs, since an additional genetic insult, such as over-expression of PIK3CAH1047R, is sufficient to endow IMEC MYC with tumorigenic capacity, while has no effect on wild type cells.
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VECELLIO, MATTEO LUCA. „Differentiation and reprogrammig of human mesenchymal stromal cells: insights from epigenetic assessments and pre-clinical studies“. Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/30253.
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Mesenchymal stromal cells (StC) are cells with plastic properties virtually present in every adult tissue. Recently, StC have also been isolated from adult human cardiac tissue (CStC) and the hypothesis has been raised that StC deriving from the heart may be genetically committed to cardiovascular differentiation. In this light, the enhancement of CStC cardiovascular precursor properties may represent a potentially successful strategy for cardiac regeneration purposes. Although of adult origin, CStC exhibit Islet1 expression and respond to chemically-determined cardiogenic epigenetic stimuli. Specifically we created an epigenetic chemical cocktail (EpiC)that is able to up-regulate the expression of cardiac resident stem cell markers c-Kit and MDR-1, together with the expression of a large number of cardiovascular-associated genes and regulatory RNAs including c-Kit, MDR-1, KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, ALPHA-MHC, Alpha-sarcomeric actin, miR-1 and miR-499. Remarkably, EpiC-treated CStC also exhibited immature electrophysiological properties. Mechanistically, the EpiC treatment determined genome-wide histone modifications associated with a transcriptionally competent chromatin. Chromatin immunoprecipitation experiments (Chip) revealed that permissive histone modification H3K4Me3 was present in c-Kit, MDR-1 and Nkx2.5 promoter regions, possibly contributing to their expression. Altogether these data indicate that Isl1+ CStC may be epigenetically reprogrammed to acquire functionally competent cardiovascular precursor properties. CStC therefore appear as a potentially useful cell type for potential cardiac and vascular reconstructive therapies
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BIANCHI, ENRICA. „Controllo traduzionale dell'espressione genica all'inizio dell'embriogenesi“. Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/762.
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L’embriogenesi iniziale è un ottimo esempio di acquisizione della totipotenza da parte di una cellula staminale a partire da cellule terminalmente differenziate. I gameti sono cellule aploidi non più in grado di dividersi, che dopo la fecondazione uniscono il loro genoma convertendolo in uno embrionale. Lo zigote deve riprendere il ciclo cellulare mitotico, rimodellare la cromatina, attivare la trascrizione e iniziare il programma di sviluppo embrionale, che richiede l’acquisizione della totipotenza dei blastomeri.
In diversi organismi modello l’attivazione traduzionale di RNA messaggeri materni quiescenti è importante per innescare i meccanismi molecolari dello sviluppo. Per studiare tali meccanismi, abbiamo focalizzato l’attenzione su due aspetti: a) l’identificazione di RNA materni reclutati per la traduzione all’inizio dell’embriogenesi; b) il ruolo svolto in questo processo dalla RNA-binding protein Sam68.
a) L’ovocita è trascrizionalmente inattivo durante le divisioni meiotiche e nel primo ciclo cellulare embrionale. Per identificare gli mRNA materni importanti per l’embriogenesi iniziale, abbiamo isolato i polisomi da due diversi stadi di sviluppo: ovociti maturi ovulati, fermi allo stadio di metafase II (MII) e zigoti allo stadio di pronuclei (PN). Gli mRNA purificati dalle frazioni polisomali sono stati analizzati tramite microarray. Abbiamo effettuato un’analisi comparativa utilizzando sia il database DAVID che un’analisi manuale utilizzando il database NCBI . Queste analisi ci hanno permesso di raggruppare gli RNA in specifiche categorie funzionali e hanno evidenziato alcune interessanti differenze tra i due stadi differenziativi esaminati. Negli MII i polisomi sono maggiormente arricchiti in RNA codificanti per proteine importanti per il metabolismo cellulare e la biogenesi dei ribosomi, suggerendo che gli ovociti ovulati sono prevalentemente impegnati nel mantenimento del loro stato energetico, in attesa della fecondazione. Al contrario, dopo la fecondazione, i polisomi sono arricchiti in RNA codificanti per proteine coinvolte nel ciclo cellulare, nel rimodellamento della cromatina e nella trascrizione. In particolare, la categoria “rimodellamento della cromatina” comprende geni quali Epc1 ed Epc2, Nalp9b Suv39h1 e Metll8 che potrebbero svolgere un ruolo importante nell’embriogenesi iniziale e nell’acquisizione della totipotenza.
b) Sam68 fa parte della famiglia delle RNA binding protein chiamate STAR, le quali svolgono funzioni durante lo sviluppo che si sono conservate nel corso dell’evoluzione. Abbiamo studiato l’espressione e la funzione di Sam68 durante l’embriogenesi iniziale. Essa localizza nella vescicola germinale negli ovociti immaturi (GV) ed è rilasciata nel citoplasma durante la prima divisione meiotica, dove permane fino all’arresto citostatico. Dopo la fecondazione Sam68 localizza nel citoplasma fino alla completa formazione dei pronuclei. Durante ciascuna divisione mitotica Sam68 si sposta dal nucleo al citoplasma per poi tornare lentamente nel nucleo. Al fine di comprendere la funzione di Sam68, abbiamo ridotto la sua espressione in vivo microiniettando un RNA a doppio filamento negli zigoti allo stadio di pronuclei. Gli embrioni sono stati mantenuti in coltura fino a 96h dalla fecondazione, quando la maggior parte degli embrioni di controllo ha raggiunto lo stadio di blastocisti ed abbiamo osservato come la deplezione di Sam68 inficia fortemente lo sviluppo preimpianto, abbassando la percentuale di formazione delle balstocisti (40% contro 80% nei controlli). Esperimenti di immunofluorescenza hanno mostrato che Sam68 associa con i fattori di inizio della traduzione (complesso eIF4F) nello zigote, indicando che questa RNA-binding protein possa essere implicata nell’utilizzo citoplasmatico di specifici mRNA materni.Early embryogenesis is a remarkable example of how a stem cell arises from terminally differentiated cells. The gametes are non-dividing haploid cells that upon fertilization initiate the functional conversion of their genomes into a single embryonic one. Particularly, the zygote has to resume the mitotic cell cycle, to remodel the parental chromatin, to activate transcription, and to initiate the embryonic developmental program, which requires acquisition of totipotency of the early blastomeres. Evidence gathered in several organisms demonstrates that translational activation of dormant maternal mRNAs is crucial to set in motion the molecular mechanisms of development. To investigate such mechanisms, we have focused our attention on two aspects: a) the identification of maternal mRNAs recruited for translation at the onset of embryogenesis; b) the role played by the RNA-binding protein Sam68 in this process. a) The oocyte is transcriptionally inactive throughout the meiotic divisions and the first embryonic cell cycle. In order to identify the maternal mRNAs involved in early embryogenesis we isolated the polysomes from two different developmental stages: the ovulated oocytes arrested in metaphase II (MII oocytes) and zygotes at the pronuclear stage (PN embryos). Purified mRNAs from the polysomal fractions were analysed by microarrays. Comparative analyses were performed using both DAVID database screen and manual analysis based on the NCBI database. These analyses allowed us to group RNAs in specific functional categories and pointed out some interesting differences among the two stages examined. In MII oocytes the polysomes are enriched with mRNAs that encode for proteins involved in metabolism and ribosome biogenesis, suggesting that the ovulated oocytes are mainly involved in maintaining their energy status awaiting for fertilization. On the other hand, after fertilization the polysomes become enriched with RNAs coding for proteins involved in cell cycle, chromatin remodelling and transcription. Particularly the chromatin remodelling group includes some interesting genes as Epc1 and 2, Nalp9b, Suv39h1 and Metll8 which could play relevant role in early embryogenesis and totipotency acquisition. b) Sam68 belongs to the STAR family of RNA-binding proteins, which play evolutionarily conserved functions in development. We have investigated the expression and function of this protein during early embryogenesis. Sam68 localizes in the germinal vesicle of GV oocytes, and after the nuclear breakdown, its levels in the oocyte decrease. Residual Sam68 remains in the cytoplasm during the first meiotic division, until the cytostatic arrest. At fertilization, Sam68 localizes in the cytoplasm until the complete formation of pronuclei. During each mitotic division, Sam68 shuttles from the nucleus to the cytoplasm, and then slowly re-enters the nucleus. To understand the function of Sam68, we knocked-down its expression in vivo by microinjecting a double stranded RNA in zygotes at pronuclear stage. Embryos were cultured up to 96 hours after fertilization, when most of the control embryos had reached the blastocyst stage. We observed that depletion of Sam68 strongly affected pre-implantation development, lowering the rate of blastocyst formation (40% versus 80% in control embryos). Immunofluorescence experiments have shown that Sam68 associates with translation initiation factors (eIF4F complex ) in the zygote, indicating that this RNA-binding protein could be involved in the cytoplasmic utilization of specific maternal mRNAs.
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Chen, Zhaoyi. „Modeling Defective Epigenetic Inheritance in Vascular Aging Using Hutchinson-Gilford Progeria Syndrome Vascular Smooth Muscle Cells“. Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41096.
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Cardiovascular disease (CVD) is the leading cause of death due to its prevalence in tandem with the propensity of atherosclerosis to worsen and cause myocardial infarction and stroke. The greatest risk factor for CVD development is age. The multifactorial etiology of atherosclerosis has made CVD difficult to model and consequently little is known about CVD onset and progression. Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe human premature aging disorder caused by a mutation in Lamin A that leads to the accumulation of an aberrant Lamin A protein termed progerin. Patients who harbour this mutation develop atherosclerosis and die from myocardial infarction or stroke at an average age of 13 years old. Autopsies reveal deterioration of vascular smooth muscle cells (VSMCs) in HGPS patients, underlining a strong connection between VSMC loss and predisposition to CVD development. The major aim of this thesis was to model normative vascular aging and disease using HGPS induced pluripotent stem cell (iPSC)-derived VSMCs and monitor the onset of defective epigenetic inheritance in vitro. My results indicate reprogramming of patient fibroblasts to restores a normal nuclear phenotype. Patient derived iPSC lines generated from fibroblasts are nearly indistinguishable from healthy controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles. However, differentiation of HGPS iPSCs to generate HGPS VSMCs recapitulates many aspects of normative vascular aging exemplified by increased ROS, DNA damage and transcriptomic aberrations. Furthermore, using a multi-omic approach including RNA-sequencing, and accelerated native isolation of protein on nascent DNA, HGPS VSMCs demonstrate loss of histone acetylation due to defective MOF abundance that contributed to impaired engagement with DNA damage repair pathway. This dissertation provides insights on the mechanisms that drive the epigenetic and transcriptomic changes in HGPS vasculature, illuminating druggable pathways that may also drive CVD in the general population.
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Collier, Amanda. „Characterising the reprogramming dynamics between human pluripotent states“. Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/287952.
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Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.
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FARIA, PEREIRA MARLENE CRISTINA. „EPIGENETIC AND FUNCTIONAL ASSESSMENT OF ENHANCEROPATHIES ACROSS HUMAN MODELS: FOCUS ON GABRIELE-DE VRIES SYNDROME“. Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/945230.
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Yin Yang 1 (YY1) is a ubiquitous zinc finger transcription factor (TF) that occupies active enhancers and promoters contributing to physical interactions between these regions via DNA looping. Increasing evidence shows that disruption of non-coding regions such as enhancers is prevalent across different neurodevelopmental disorders (NDDs) with intellectual disability (ID) features. Indeed, YY1 haploinsufficiency causes a NDD with ID, named Gabriele-de Vries syndrome (GADEVS). Although it is known that YY1 controls the expression of a dazzling list of genes and influences various cellular processes in numerous cell types, the impact of this TF in the neurodevelopment of the human cortex is yet to be unraveled. By taking advantage of disease-modeling as a tool to investigate the pathogenesis of GADEVS across different time points and tissues we gathered new insights about how YY1 haploinsufficiency exerts such a dramatic phenotype in individuals carrying mutations. We reprogrammed patient-derived and healthy somatic cells into induced-pluripotent stem cells (iPSCs) and observed, already at the pluripotent stage, a major transcriptional dysregulation. Moreover, since YY1-mutated patients exhibit ID features, we differentiated our cohort of iPSCs into cortical neurons as well organoids and were able to capture stage-specific striking features, not only at the transcriptomic level, but also structural and compartmentalization impairments. Of note, YY1-mutated neurons displayed synaptic disparities, sufficient to induce astrogliosis-like features in surrounding astrocytes, both shown to be critical for proper brain function and plasticity forms in the CNS. Instead, in cortical organoids we recapitulated features of abnormal ventricle formation, pathological hallmarks observed in GADEVS patients and mice models followed by ID and developmental delay. This study showed, for the first time, the molecular signatures that possibly lead to cognitive defects in human patients and provide the first solid foundation for the development of therapeutic strategies and drug screening in the future.
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Leong, Yeh Chwan. „Reprogramming to cancer induced pluripotent stem cells elucidates the contribution of genetic and epigenetic alterations to breast carcinogenesis“. Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/53330/.
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The induced pluripotent stem cells (iPSCs) technology has revolutionized disease modelling by enabling the generation of patient-specific pluripotent stem cells for the study of complex disorders such as cancer. Somatic cell reprogramming through iPSCs induces global epigenetic reconfiguration of the chromatin which converts cancer cells to an embryonic stem cell-like state with potential reversion of tumorigenicity. Therefore, reprogramming can be used to answer the question as to whether epigenetic alterations alone can be sufficient to induce carcinogenesis, independent of genetic defects. In addition, it can used to dissect the relative contribution of genetics and epigenetics and epigenetics to tumorigenicity. In this study, the triple negative breast cancer (TNBC) cell line BT-549 and oestrogen receptor positive (ER+) cell line MCF7 were successfully reprogrammed by using the non-integrative episomal vectors expressing OCT4, SOX2, L-MYC, KLF4, LIN28, EBNA1, shRNA against TP53, and microRNA-302/367 cluster together with treatment of sodium butyrate. Pluripotency of cancer-derived iPSCs was confirmed by RT-PCR, RT-qPCR and immunofluorescence staining for expression of pluripotency markers. Differentiation potential of iPSCs was also assessed by using in vitro differentiation either spontaneous or directed to the mammary lineage. Functional assays indicated potential loss of tumorigenicity in re-differentiated cells derived from cancer iPSCs. The same approach was applied to study an immortalised, non-malignant mammary epithelial cell line MCF10A and two of its derived isogenic lines harbouring the two most frequent mutations in breast cancer, PIK3CAH1047R (+/-) and TP53(-/-), created by using CRISPR-Cas9 gene editing. Reprogramming induced a tumorigenic phenotype in iPSCs (PIK3CAH1047R (+/-) isogenic line only) and re-differentiated progenies (in both wild type MCF10A and PIK3CAH1047R (+/-) cell lines), suggesting the contribution of PIK3CA mutation in enhancing malignant transformation. Results in this study suggested that epigenetics alone and/or its interaction with genetic defects (e.g. PIK3CA mutation) has significant impact on breast cancer carcinogenesis. The dissection of the molecular mechanisms underlying the loss and gain of tumorigenicity using the iPSC models generated in this study could provide general understandings on breast carcinogenesis, which in turn could have important clinical implications.
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Lucas, Emma S. „Defining global DNA methylation differences betwen embryonic stem cells and fibroblasts for exploitation in Epigenetic reprogramming in vitro“. Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519395.
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Saadeh, Heba. „The role of DNA sequence signals in the epigenetic reprogramming of CpG islands during oogenesis and early embryogenesis“. Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-dna-sequence-signals-in-the-epigenetic-reprogramming-of-cpg-islands-during-oogenesis-and-early-embryogenesis(76174739-3129-4655-b3f7-a72a9ea9a11a).html.
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The reprogramming of epigenetic marks is a genome-wide process and yet CpG islands escape the overall trend. In addition, not all CpG islands behave in the same way. While the majority of CpG islands resist the de novo DNA methylation establishment in the germ lines, ~1600 CpG islands acquire methylation during oogenesis. The majority of these oocyte-methylated CpG islands remain un-methylated during spermatogenesis, that is, they are maternal germ line differentially methylated regions (maternal gDMRs). All but 25 (permanent) maternal gDMRs lose their methylation post fertilisation and pre-implantation. The DNA sequences of CpG islands was investigated - in the context of transcription in the oocyte - to identify targeting signals for either establishing and/or maintaining, or altogether escaping DNA methylation during oogenesis and in the pre-implantation embryo. The methylation of CpG islands in the oocyte was found to be significantly associated with transcription through CpG islands, as previously observed. However, the sequences of oocyte-methylated CpG islands do not contain a characteristic DNA sequence motif, and neither do the upstream promoters from which transcription through oocyte-methylated CpG islands originates. An analogous de novo motif search successfully identifies TGCCGC, the recognition site of the Zfp57/Kap1 imprint maintenance complex, as a DNA sequence motif that is characteristic for PPM-DMRs. Analysis of the incidence of TGCCGC indicates that not just its presence but also multiple occurrences within a sequence may be required for imprint maintenance. Furthermore, the lack of additional characteristic motifs suggests the absence of additional DNA-binding factors that specifically interact with PPM-DMRs. A period of 8-10bp in the spacing of CpGs in PPM-DMRs was previously observed and proposed as a targeting signal for de novo methylation in the germ line. This observation was reproduced, and the property of the average was found to be representative to only less than the half of the PPM-DMRs. Moreover, the pairs of CpGs 8-10bp apart are in fact depleted in oocyte-methylated CpG islands, consistent with the consequence of accidental deamination over time. The absence of a DNA sequence motif or CpG spacing characteristic of oocyte-methylated CpG islands (including PPM-DMRs) supports a sequence-independent model of de novo methylation establishment during oogenesis. However, the CpG islands that escape this mechanism contain a characteristic CGrich motif that is highly similar to the recognition site of E2F1/2, DNA-binding protein involved in chromatin remodelling that is expressed in oocytes. Logistic-linear regression analyses indicate that the presence of the motif independently conveys significant protection from methylation, regardless of, for example, whether the CpG island is an active promoter. Therefore, the following is proposed: E2F1/2 are part of a mechanism for the active protection of specific CpG islands from de novo methylation in the oocyte.
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Abdallah, Hussein(Hussein M. ). „The core mammalian pluripotency network in induced pluripotent stem cell (iPSC) formation : models for genetic and epigenetic reprogramming“. Thesis, Massachusetts Institute of Technology, 2018. https://hdl.handle.net/1721.1/122910.
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This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2018
Cataloged from student-submitted PDF version of thesis. "February 2018."
Includes bibliographical references (pages 23-37).
In 2006, history was made in a seminal experiment that converted mouse fibroblasts to a pluripotent phenotype coined the 'induced pluripotent stem cell' (iPSC) state. Unhindered by ethical or immunogenic constraints, iPSCs potentially hold the keys to tremendous applications in therapeutic and regenerative medicine. Furthermore, on-demand iPSC generation has the capacity to revolutionize basic research in disease modeling and drug discovery. These promises notwithstanding, the economics of iPSC formation--which remains a slow, inefficient, expensive, and laborious process--still stand in the way of fully making use of this extraordinary technology. In this thesis, I present mathematical models aimed at understanding the theoretical reprogrammability of the core pluripotency gene regulatory network being awakened in iPSC reprogramming. Using these modeling insights, I discuss the merits of current reprogramming strategies, which can be viewed as open-loop perturbations in control theoretic terms. I then discuss an alternative paradigm of closed-loop reprogramming, which is theoretically shown to be far superior when it comes to the reprogrammability of the pluripotency network. Finally, I propose a reprogramming model that incorporates the eæect of DNA demethylation on the activation of the network, with attention given to the relationship between this epigenetic transformation and the cell proliferation barrier that somatic cells seemingly face on the road to pluripotency.
by Hussein Abdallah.
M. Eng.
M.Eng. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science
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Dubé, Delphine. „Différence dans la capacité de fibroblastes à être reprogrammés par le cytoplasme de l'ovocyte : étude d'une situation différentielle chez le bovin“. Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS252.
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La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneusesReprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells
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Yamashiro, Chika. „Generation of human oogonia from induced pluripotent stem cells in vitro“. Kyoto University, 2019. http://hdl.handle.net/2433/242826.
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Shimamoto, Ren. „Generation and Characterization of Induced Pluripotent Stem Cells from Aid-deficient Mice“. Kyoto University, 2014. http://hdl.handle.net/2433/189672.
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Shimamoto R, Amano N, Ichisaka T, Watanabe A, Yamanaka S, et al. (2014) Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice. PLoS ONE 9(4): e94735. doi:10.1371/journal.pone.0094735Kyoto University (京都大学)
0048
新制・課程博士
博士(医科学)
甲第18515号
医科博第56号
新制||医科||4(附属図書館)
31401
京都大学大学院医学研究科医科学専攻
(主査)教授 斎藤 通紀, 教授 平家 俊男, 教授 山田 泰広
学位規則第4条第1項該当
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Payne, Kyle K. „Immunotherapy of Cancer: Reprogramming Tumor/Immune Cellular Crosstalk to Improve Anti-Tumor Efficacy“. VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3939.
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Immunotherapy of cancer has been shown to be promising in prolonging patient survival. However, complete elimination of cancer and life-long relapse-free survival remain to be major challenge for anti-cancer therapeutics. We have previously reported that ex vivo reprogramming of tumor-sensitized immune cells by bryostatin 1/ionomycin (B/I) and the gamma-chain (γ-c) cytokines IL-2, IL-7, and IL-15 resulted in the generation of memory T cells as well as CD25+ NKT cells and CD25+ NK cells. Adoptive cellular therapy (ACT) utilizing these reprogrammed immune cells protected FVBN202 mice from tumor challenge, and overcame the suppressive functions of myeloid-derived suppressor cells (MDSCs). We then demonstrated that the presence of CD25+ NKT cells was required for anti-tumor efficacy of T cells as well as their resistance to MDSCs. Similar results were obtained by reprogramming of peripheral blood mononuclear cells (PBMC) from patients with early stage breast cancer, demonstrating that an increased frequency of CD25+ NKT cells in reprogrammed immune cells was associated with modulation of MDSCs to CD11b-HLA-DR+ immune stimulatory cells. Here, we tested the efficacy of immunotherapy in a therapeutic setting against established primary breast cancer (Chapter One), experimental metastatic breast cancer (Chapter Three) as well as against minimal residual disease (MRD) in patients with multiple myeloma (Chapter Two). We evaluated the ability of reprogrammed immune cells, including CD25+ NKT cells, to convert MDSCs to myeloid immune stimulatory cells, in vivo; this resulted in the identification and characterization of a novel antigen presenting cell (APC). These novel immune stimulatory cells differed from conventional APCs, including dendritic cells (DCs) and macrophages. We have also demonstrated that enhancing immunogenicity of mammary tumors by treatment with Decitabine (Dec) along with overcoming MDSCs by utilizing reprogrammed T cells and NKT cells in ACT prolongs survival of animals, but fails to eliminate the tumor. However, targeting cancer during a setting of MDR, when tumor cells are dormant, results in objective responses as evidenced in our multiple myeloma studies. This suggests that targeting breast cancer with immunotherapy following conventional therapies, in a setting of residual disease when tumor cells are dormant, may be effective in eliminating such residual cells or maintaining dormancy and extending time-to-relapse for breast cancer patients.
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Tiwari, Vijay Kumar. „The Epigenetics of Gene Transcription and Higher Order Chromatin Conformation“. Doctoral thesis, Uppsala universitet, Zoologisk utvecklingsbiologi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6302.
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It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.