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Les éléments transposables (ET) et leur reliques sont des composants majeurs des génomes eucaryotes. Ils sont potentiellement hautement mutagéniques car leur prolifération peut engendrer des réarrangements chromosomiques, des interruptions de gènes ou affecter l’expression génique par interférence transcriptionnelle. Néanmoins, peu d’ET sont généralement mobiles dans les génomes grâce à l’action de mécanismes qui restreignent leur activité comme la méthylation de l’ADN chez les mammifères et les plantes. De fait, chez Arabidopsis thaliana, une perte sévère de méthylation de l’ADN causée par une mutation dans le gène codant la protéine remodeleuse de chromatine DDM1 (DECREASE IN DNA METHYLATION 1) engendre l’accumulation massive de transcrits correspondants à des séquences d’ET. En revanche, peu d’ET semblent être mobilisés suite à cette réactivation transcriptionnelle. Nous proposons ici de déterminer (1) l’étendue de la mobilisation des ET suite à la perte de méthylation de l’ADN, (2) la distribution des nouvelles insertions d’ET le long du génome d’Arabidopsis et (3) les conséquences des nouvelles insertions d’ET sur l’expression des gènes situés à proximité. Dans ce but, nous avons séquencé le génome d’une cinquantaine d’epiRIL (epigenetic Recombinant Inbred Lines) dérivées d’un croisement entre une plante sauvage et un mutant ddm1. Suite au croisement retour de la F1 avec une plante sauvage et sélection des individus F2 homozygotes pour l’allèle sauvage DDM1, les epiRIL ont été propagées au travers de 6 autofécondations successives. Les epiRIL permettent donc l’étude détaillée des évènements de transpositions juste après qu’ils aient eu lieu. Pour identifier les évènements de transpositions dans ces lignées nous avons mis au point TE-tracker, un programme basé sur les données issues du séquençage Illumina de banques maite-pair. Par cette approche, nous avons montré que les ET mobiles dans ddm1 et les epiRIL appartiennent à seulement une quinzaine environ des >300 familles identifiées dans le génome d’Arabidopsis. Qui plus est, on observe des variations importantes de fréquences et dynamiques de transpositions entre les différentes familles d’ET ce qui suggère l’existence de mécanismes additionnels contrôlant la transposition. Les analyses moléculaires réalisées sur un sous-ensemble des ET mobilisés appartenant à différentes familles ont notamment montré que ces différences sont dues en grande partie aux différentes modalités d’établissement du contrôle épigénétique sur les ET nouvellement insérés. D’autre part, nos analyses indiquent que la distribution des nouvelles insertions d’ET diffère grandement de celle des copies résidentes. Ce résultat suggère donc que la suraccumulation des séquences d’ET dans les régions péricentromériques du génome d’Arabidopsis n’est pas due à un ciblage spécifique des insertions dans ces régions, mais est plutôt la conséquence de leur élimination des bras chromosomiques. Enfin, nous avons cherché à déterminer dans quelle mesure la méthylation de l’ADN associée aux séquences répétées a un impact sur l’expression des gènes situés à proximité en étudiant des mutants affectés dans les différentes voies de la méthylation de l’ADN. Par des analyses phénotypiques et moléculaires nous avons montré que, même si la plupart des gènes d’Arabidopsis n’est pas affectée par l’état de méthylation des séquences répétées situées à proximité, deux voies de la méthylation de l’ADN agissent ensemble pour maintenir l’expression normale d’un petit nombre de gènes ayant des effets pléiotropes situés proximité de séquences répétées. La méthylation de l’ADN agit donc comme un double système de contrôle pour assurer l’expression normale d’un petit nombre de gènes clefs localisés a proximité de séquences répétées. / Transposable elements (TEs) and their relics are major components of eukaryotic genomes. TEs are potentially highly mutagenic as their proliferation can cause chromosomal rearrangements, disrupt genes or affect gene expression through transcriptional interference. However, few TEs are usually mobile within genomes at any one time thanks to potent mechanisms that restrain their activity, such as DNA methylation in mammals and plants. Thus, in the flowering plant Arabidopsis, severe loss of DNA methylation caused by mutations in the chromatin remodeler gene DDM1 triggers massive accumulation of transcripts corresponding to TEs. Yet, comparatively few TEs appear to be mobilized as a result. Here, we set out to determine (1) the extent to which DNA methylation prevents TE mobilization, (2) where do TEs insert following their reactivation and (3) what are the consequences of new TE insertions on the expression of neighboring genes. To this ends, we have sequenced the genome of over 50 epigenetic Recombinant Inbred Lines (epiRILs) that were derived from a cross between a wild type and an isogenic ddm1 mutant line. After backcrossing of the F1 and selection of the progeny homozygous for wild-type DDM1, the epiRILs were propagated through six rounds of selfing. The epiRILs therefore permit a detailed assessment of transposition events soon after they have occurred. In order to identify TE mobilization in the epiRILs we developed TE-tracker, a pipeline based on Illumina sequencing of mate pairs libraries. Using this approach, we could show that although both retroelements and DNA transposons are mobilized in ddm1 and the epiRILs, mobile TEs belong to only a dozen or so of the >300 TE families identified in the Arabidopsis genome. Furthermore the rate and dynamics of transposition vary dramatically between TE families, suggesting the existence of additional mechanisms controlling transposition. Molecular analysis performed on a subset of those mobile TEs belonging to distinct families show that these differences are greatly due to different modality of establishment of epigenetic control over newly inserted TEs. In addition, our analysis indicates that the distribution of new TE insertions differs dramatically from the one of resident copies. These findings provide compelling evidence that over accumulation of TE sequences in the pericentromeric regions of the Arabidopsis genome is not due to specific targeting of TEs, but rather to their elimination from chromosome arms. Finally, we assessed the extent to which repeat-associated DNA methylation impacts the expression of neighboring genes by studying mutants affected in different methylation pathways. Phenotypic and molecular analyses reveal that, even though most Arabidopsis genes are not detectably sensitive to the methylation status of neighboring repeats, two DNA methylation pathways act together to maintain the normal expression of only a very small number of genes near repeats and that these genes tend to have pleiotropic effects. Then, DNA methylation acts as a double-lock system to ensure the normal expression of a small number of key genes located near repeats.

O aborto espontâneo recorrente (AER) caracteriza-se pela ocorrência de três ou mais abortos consecutivos espontâneos até a 20ª semana de gestação. É uma condição patológica multifatorial, em que alterações morfológicas uterinas, distúrbios endócrinos, alterações no cariótipo, polimorfismos genéticos relacionados aos genes envolvidos no metabolismo da homocisteína, hemostasia, infecções, autoanticorpos e o processo inflamatório podem contribuir para a ocorrência de AER. O estado fisiológico do endométrio é essencial para a implantação do embrião no útero durante a gestação. Na interface materno-fetal, há uma modulação de citocinas, necessária para o estabelecimento da angiogênese e desenvolvimento da placenta. Um desequilíbrio entre as citocinas pode diminuir a tolerância ao feto e ocasionar rejeição fetal. A concentração de citocinas pode ser modificada por conta de uma diminuição na expressão de alguns genes, e esta pode ser regulada pelo seu estado de metilação sítio-específica. A metilação do DNA é um mecanismo epigenético de regulação gênica, e que corresponde à incorporação de grupos metila em ilhas CpG localizadas próximas às regiões promotoras de genes humanos, e isso pode ser importante na avaliação do risco de complicações gestacionais. Além disso, o estado nutricional de vitaminas foi relacionado a alterações no padrão de metilação de alguns genes. Os objetivos deste trabalho foram avaliar as concentrações dos mediadores inflamatórios em mulheres com aborto e em grupo controle, verificar correlações entre as concentrações de vitaminas, homocisteína total e taxa de metilação do DNA, verificar correlações entre concentração de citocinas e taxa de metilação do DNA e determinar odds ratio (IC 95%) de ter aborto em modelos multivariados. Foram incluídas 253 mulheres com história de aborto recorrente e 264 mulheres saudáveis (controle). O DNA foi extraído de leucócitos de sangue periférico para o estudo de metilação. Foram separadas alíquotas de soro e plasma para dosagem de vitaminas, metabólitos e citocinas. Não foram encontradas diferenças nas taxas de metilação do DNA entre os grupos aborto e controle. A citocina TNFα está aumentada nos grupos de aborto em comparação ao controle. A taxa de metilação do DNA no gene IFNG foi correlacionada inversamente às concentrações de folato sérico e citocina IFNγ no grupo controle. E as concentrações de IL10 foram inversamente correlacionadas à taxa de metilação do DNA nos grupos de aborto secundário e controle. Neste trabalho, verificou-se que as vitaminas e as citocinas influenciam na taxa de metilação do DNA do gene IFNG e a citocina pró-inflamatória TNFα apresenta-se aumentada em mulheres com história de aborto. / Recurrent spontaneous abortion (RSA) is characterized by the occurrence of three or more consecutive spontaneous abortions until the 20th week of gestation. It is a multifactorial pathological condition in which morphological uterine, endocrine disorders, changes in the karyotype genetic polymorphisms related to genes involved in homocysteine metabolism, infection, autoimmunity and inflammatory processes may contribute to the occurrence of RSA. The physiological state of the endometrium is essential for embryo implantation in the uterus during pregnancy. In maternal-fetal interface, there is a modulation of cytokines necessary for the establishment and development of placental angiogenesis. An imbalance between cytokines can decrease tolerance to fetus and cause fetal rejection. Concentration of cytokines may be modified due to a decrease in the expression of genes related to some of these cytokines that can be regulated by DNA methylation, which is an epigenetic mechanism of gene regulation and which corresponds to the incorporation of groups Methyl CpG islands located near the promoter regions of human genes, and this may be important in assessing the risk of pregnancy complications. In addition, the nutritional status of vitamins was associated with changes in the methylation pattern of certain genes. The aims of this study were to determine the concentrations of inflammatory mediators in women with abortion and the control group, examine correlations between concentrations of vitamins, total homocysteine and DNA methylation rate, examine correlations between cytokine concentration and DNA methylation and determine odds ratio (95% CI) of having abortion in multivariate models. We included 253 women with a history of recurrent miscarriage and 264 healthy women (control). DNA was extracted from peripheral blood leukocytes for the study of methylation. Serum and plasma aliquots were used for determination of vitamins, metabolites and cytokines. There were no differences in rates of DNA methylation between control and abortion groups. The cytokine TNFα is increased in abortion groups compared to the control. DNA methylation rate in gene IFNG was inversely correlated with serum folate and serum cytokine IFNγ in the control group. Also IL10 concentrations were inversely correlated to DNA methylation rate in groups of miscarriage and secondary control. In this work, it was found that vitamins and cytokines influence DNA methylation rate in the promoter region and are different in the study and control groups.

Le cancer du sein est le cancer le plus fréquent et la première cause de mortalité par cancer chez la femme dans le monde [1]. De nombreux facteurs participent au développement de cette maladie et les gènes BRCA1 et BRCA2 sont particulièrement impliqués. En effet, des mutations dans ces deux oncosuppresseurs sont responsables de 5 à 10% des cancers du sein héréditaires [2]. De plus, une baisse de leur expression est retrouvée dans un grand nombre de cancers du sein sporadiques [3]. Les mutations héréditaires des gènes BRCA1 et BRCA2 sont également à l’origine de cancers de l’ovaire [4]. Ce cancer est beaucoup moins fréquent que le cancer du sein, mais il est associé à un mauvais pronostic. En plus de ces facteurs génétiques, des facteurs hormonaux semblent également intervenir dans les processus de carcinogenèse mammaire et ovarienne, mais aussi des facteurs environnementaux et plus particulièrement l’alimentation. En effet, la consommation de soja, fréquente dans certaines régions de l’Asie serait responsable d’une diminution du risque de développer un cancer du sein dans les pays Asiatiques par rapport aux pays Occidentaux. Ce sont les phyto-oestrogènes contenus dans le soja qui agiraient, grâce à leur similarité de structure avec le 17-β-oestradiol de la femme [5]. Les phyto-oestrogènes du soja pourraient également agir sur le développement du cancer de l’ovaire puisque celui-ci est un cancer oestrogéno-dépendant, comme le cancer du sein. L’équipe Nutrition et Cancer du Département d’Oncogénétique du Centre Jean Perrin étudie les effets potentiellement préventifs des phyto-oestrogènes du soja dans le processus de cancérogenèse. Une première étude, menée au sein de l’équipe, a montré que l’expression des gènes BRCA1 et BRCA2 dans la glande mammaire pouvait être modulée par la consommation de soja chez des rates ovariectomisées [6]. Aussi, des études transcriptomiques, ont montré que les conséquences de l’inactivation des oncosuppresseurs BRCA1 et BRCA2 par l’utilisation d’un petit ARN interférent dans les cellules mammaires pouvaient être contrées par un traitement avec les phyto-oestrogènes du soja [7, 8]. Suite à l’émergence de travaux montrant des effets des phyto-oestrogènes du soja sur la méthylation de l’ADN, et la présence de méthylation dans le promoteur des gènes BRCA1 et BRCA2 dans les cancers sporadiques du sein, nous avons voulu voir si les phyto-oestrogènes du soja pourraient agir directement sur la méthylation de ces deux oncosuppresseurs, que nous avons au préalable mis en évidence dans les cancers du sein et de l’ovaire. / Breast cancer is the most common cancer and the leading cause of cancer death among women worldwide [1]. Many factors contribute to the development of this disease and the BRCA1 and BRCA2 genes are particularly involved. Indeed, mutations in these two oncosuppressors are responsible for 5 to 10% of hereditary breast cancers [2]. In addition, a decrease in their expression is found in a large number of sporadic breast cancers [3]. Hereditary mutations of the BRCA1 and BRCA2 genes are also at the origin of ovarian cancers [4]. This cancer is much less common than breast cancer, but it is associated with a poor prognosis. In addition to these genetic factors, hormonal factors also seem to be involved in the processes of breast and ovarian carcinogenesis, but also environmental factors and more particularly food. Soybean consumption, which is common in some parts of Asia, is thought to reduce the risk of developing breast cancer in Asian countries compared to Western countries. It is the phytoestrogens contained in soy that act, thanks to their similarity of structure with the 17-β-estradiol of the woman [5]. Soy phytoestrogens may also affect the development of ovarian cancer since it is an estrogen-dependent cancer, such as breast cancer. The Nutrition and Cancer team of the Department of Oncogenetics at the Jean Perrin Center is studying the potentially preventative effects of soy phytoestrogens in the carcinogenesis process. A first study, conducted within the team, showed that the expression of BRCA1 and BRCA2 genes in the mammary gland could be modulated by the consumption of soy in ovariectomized rats [6]. Also, transcriptomic studies have shown that the consequences of the inactivation of BRCA1 and BRCA2 oncosuppressors by the use of a small interfering RNA in mammary cells could be countered by treatment with soy phytoestrogens [7, 8]. Following the emergence of studies showing the effects of soy phytoestrogens on DNA methylation, and the presence of methylation in the BRCA1 and BRCA2 gene promoter in sporadic breast cancers, we wanted to see if the soy phytoestrogens could directly affect the methylation of these two oncosuppressors, which we have previously identified in breast and ovarian cancers.

Dong Shumin. / "February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 187-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Gastric cancer is the second leading cause of cancer death worldwide and in China. The mechanism of gastric carcinogenesis is not fully understood. Epigenetic studies indicated that inactivation of tumor suppressor genes by DNA hypermethylation plays a crucial role in the progression of gastric cancer. Epigenetic inactivation of secreted frizzled-related protein (SFRP 1) by methylation plays a pivotal role on the development of various cancers. However, the role of SFRP family genes in gastric cancer remains largely unknown. We aimed to characterize the epigenetic abnormalities and discover novel biomarkers for early detection of gastric cancer. We investigated the epigenetic alterations in gastric adenocarcinoma by microarray based analysis and gene promoter hypermethylation. Based of the microarray data, we determined the functional significance and frequency of SFRP family genes hypermethylation in human gastric cancer. We screened the mRNA expression and methylation status of the SFRP family members in human gastric cancer cell lines and primary gastric cancer samples. Demethylation study of SFRP family genes were done by treating gastric cancer cell lines with 5'Aza. The biological effects of SFRP were analyzed by flow cytometry, cell viability assay and tumor growth in nude mice. SFRP1, 2, 4 and 5 were undetectable in 100% (7/7), 100% (7/7), 42.8% (3/7) and 85.7% (6/7) of gastric cancer cell lines, respectively. However, only SFRP2 showed significant down-regulation in gastric cancer compared with adjacent non-cancer samples (P<0.01). Treatment with demethylation agent, 5'-Aza, restored the expression of SFRP2 in all 7 cancer cell lines. Promoter hypermethylation of SFRP2 was detected in 73.3% of primary gastric cancer samples and 20% of adjacent non-cancer tissue (P<0.01). Bisulfite sequencing confirmed the density of promoter methylation in cell line, primary gastric cancer tissue and their adjacent non-cancer tissue. Transfection of SFRP2 induced cell apoptosis, inhibited proliferation in vitro and suppressed tumor growth in vivo. Furthermore, SFRP2 methylation was detected in 37.5% of samples showing intestinal metaplasia. Methylated SFRP2 was also detected in 66.7% of serum samples from cancer patients but not in normal controls. Epigenetic inactivation of SFRP2, but not SFRP1, SFRP4 and SFRP5 is a common and early event of carcinogenesis. Hence, detection of SFRP2 methylation in serum may have diagnostic value in gastric cancer patients. / by Cheng, Yuen Yee. / Adviser: FKL Chan. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0803. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

Chan Wing Yan Michael. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 168-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Chemical modification of the cytosine base via the addition of a methyl group to form 5-­‐methylcytosine (5-­‐mC) is a well-­‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-­‐mC is not the only DNA modification found within the vertebrate genome. 5-­‐hydroxymethylcytosine (5-­‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-­‐mC base from which it is derived, 5-­‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-­‐/-­‐) as well as and methylation hypomorphic (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-­‐hmC for both p53-­‐/-­‐ and (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.

DNA methylation is a key epigenetic mark which undergoes global reprogramming during early mammalian embryonic development, resulting in almost complete erasure of the mark after fertilisation of the zygote. Genome-wide patterns of DNA methylation are subsequently re-established in the implanting blastocyst by de novo DNA methyltransferases Dnmt3a and Dnmt3b along with their catalytically inactive co-factor Dnmt3l, while these DNA methylation patterns are maintained through cell divisions by maintenance methyltransferase Dnmt1. The exact mechanisms by which these DNA methyltransferase enzymes are targeted to specific genomic regions remain unclear, but may involve interaction with modified histones and/or the participation of co-factors. Lsh (lymphoid specific helicase), a putative chromatin remodelling helicase, has been implicated in facilitating de novo methylation, as Lsh knockout embryos and derived somatic cell lines display substantial but specific DNA methylation losses at repetitive elements and single copy genes. This study aims to define the requirement for Lsh in establishing de novo DNA methylation and gene expression patterns during the early stages of mouse embryonic development. The '2i' culture system using two small molecule kinase inhibitors was harnessed to convert lsh-/- mouse embryonic stem cells (mESCs) to a hypomethylated 'ground state' of pluripotency. Culture conditions were then altered to transition these ground state mESCs to cells representing later, more methylated stages of development ('serum' mESCs, epiblast stem cells and embryoid bodies). Implementation of this model system suggests that Lsh does not contribute to DNA methylation establishment in a pluripotent context, but rather is important for facilitating de novo DNA methylation during differentiation to culture models representing later developmental stages. These investigations also reveal that Lsh differentially regulates DNA methylation at major and minor satellite repeats depending on cellular context, and that this regulation may involve a role for Lsh in maintenance of DNA methylation.

DNA methylation is essential for mammalian development and transcriptional repression of genes and retrotransposons during embryo development and in somatic cells. The patterns of DNA methylation are established by de novo DNA methyltransferases, which are regulated by developmental signalling and require access to chromatin. Besides DNA methyltransferases, other proteins have recently been implicated in DNA methylation, such as the ATP-dependent chromatin remodeler LSH. The absence of LSH in mouse embryos leads to defects in DNA methylation and development. In relation to this, mutations in LSH have been found to cause Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome. This syndrome is characterized by centromeric instability and CpG hypomethylation of centromeric satellite repeats, and is most often caused by mutations in the catalytic domain of the DNA methyltransferase DNMT3B. LSH is essential for developmentally programmed de novo DNA methylation of large chromosomal domains including promoters of protein coding genes and repetitive sequences. Importantly, fibroblasts derived from chromatin remodeling ATPase LSH-null mouse embryos, which lack DNA methylation at transposons and specific gene promoters, are capable of re-establishing normal patterns of DNA methylation and transcriptional silencing of misregulated genes upon re-expression of LSH. The ATP hydrolysis by LSH is essential for its function in gene silencing and de novo DNA methylation. However, the molecular mechanisms of LSH-dependent gene silencing and de novo DNA methylation are yet unclear. Here we use an inducible system that enables controlled expression of LSH in Lsh-null mouse embryonic fibroblasts (MEFs) to follow chromatin dynamics, transcriptional silencing and establishment of de novo DNA methylation. This conditionally reversible Lsh knockout cellular system allowed us to study the order of events occurring immediately after LSH restoration in MEF cell lines in order to elucidate the molecular mechanism of LSH-dependent gene silencing. We have demonstrated that LSH upon its restoration localises to the promoters of LSH-dependent loci leading to a mild decrease in the occupancy of H3, which reinforces the previously shown role of LSH as a chromatin remodeler. Simultaneously, there is removal of acetyl groups from H3 tails when LSH is bound to these target regions, which might be facilitated by the interaction of HDACs with LSH. The removal of H3Ac marks is followed by deposition of H3K9me2 by G9a/GLP histone methylases at the same time point when misregulated genes are silenced. This suggests that LSH creates a suitable substrate for G9a/GLP promoting gene silencing. Surprisingly, transcriptional repression occurs without acquisition of DNA methylation at the promoters of these loci. This order of events implies that LSH plays a role as a chromatin remodeler leading to changes in chromatin structure and modifications that facilitate epigenetic gene silencing without DNA methylation in the initial period when LSH is restored in MEF cell lines. Furthermore, deposition of H3K9me2 by the G9a/GLP complex is critical for silencing of specific genes, but not for repetitive elements such as IAPs. The histone modification H3K27me3 seems to play a transitory role in the silencing of IAP retrotransposons in the absence of G9a/GLP activity. In conclusion, this work has demonstrated that changes in chromatin modifications leading to a transcriptionally repressive chromatin state can be established in somatic cells by the chromatin remodeler LSH without acquisition of DNA methylation. This suggests that the primary role of LSH is to promote changes in chromatin structure and modifications that lead to gene silencing and not DNA methylation, which most likely occurs as a consequence of transcriptional silencing.