Academic literature on the topic 'Epigenetic reprograming'

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.

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.

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.<br>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.

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 reproduction<br>DNA 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

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.

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.