La méiose est une étape de la différenciation germinale qui permet la formation des gamètes. Elle est composée de deux divisions successives. La ségrégation des chromosomes homologues à la première division nécessite des connexions entre homologues, mises en place par des événements de crossing-over (CO). Les CO augmentent également la diversité génétique, et leur fréquence et leur distribution sont étroitement régulées. Ils sont générés par un mécanisme de formation et réparation de cassures double brins de l'ADN (CDBs), catalysées par la protéine SPO11 et préférentiellement localisées dans des régions de 1-2 kb appelées points chauds de recombinaison méiotique. Une question majeure est de comprendre comment sont régulés ces CO, ce qui détermine leur fréquence et leur distribution, car toute altération de cette régulation peut conduire à des anomalies chromosomiques graves.Dans ce travail de thèse, pour la première fois chez les mammifères, nous avons montré que des modifications de la chromatine sont associées à l'initiation de la recombinaison méiotique (formation des CDBs par SPO11). Ces résultats ont été obtenus par des analyses d'immunoprécipitation de chromatine (ChIP) sur des spermatocytes purifiés ou non, isolés de différentes lignées de souris. Une des modifications associées à l'activité de deux points chauds testés est la triméthylation de la lysine 4 de l'histone H3 (H3K4Me3). Une analyse fonctionnelle et temporelle de cette modification a permis de montrer qu'elle ne dépend pas de SPO11 et apparaît avant la formation de CDBs. Nous avons montré ici que c'est la protéine PRDM9, récemment identifiée comme un déterminant majeur des points chauds de recombinaison chez les mammifères et possédant une activité méthyltransférase, qui appose H3K4Me3. Nous proposons un modèle où H3K4Me3 et d'autres caractéristiques inconnues constitueraient un substrat pour la machinerie d'initiation et recruteraient SPO11 en des points précis du génome, qui deviendront des points chauds. / Meiosis is a specialized cell division to produce haploid gametes from a diploid cell. It segregates parental genomes by two successive divisions. The faithful segregation of homologous chromosomes is achieved during the first unique division via formation of crossovers (COs). COs establish physical connections between homologs by the reciprocal exchange of genetic material and require the formation and subsequent repair of SPO11-dependent DNA double-strand breaks (DSBs). Studies in many organisms revealed that COs are distributed in highly localized regions (1-2Kb) of genomes called recombination hotspots. The mechanisms of COs regulation are elusive and a main question in the field is to understand how the frequency and distribution of CO are regulated, because either absence or defects of recombination can lead to aneuploidy or reduced fertility. In the present study, for the very first time in mammals, we investigate whether recombination hotspots are associated with any chromatin modifications. We performed chromatin immunoprecipitation (ChIP) on spermatocytes isolated from different mice strains harbouring either active or inactive hotspots. Comparison of hot and cold spots revealed that a specific histone modification i.e. trimethylation of the lysine 4 of histone H3 (H3K4Me3) is enriched at two tested hotspots in mice. Temporal and functional analysis show that H3K4Me3 is not dependent on SPO11 and appears before DSBs formation. Furthermore, we demonstrate here that H3K4Me3 is methylated via the histone methyltransferase activity of PRDM9, recently identified as a major determinant of recombination hotspots in mammals. We propose a model that H3K4Me3 and other unknown chromatin features may specify recruitment of SPO11 initiation machinery to initiate meiotic recombination at the hotspots.

La différenciation et le développement requièrent une régulation fine de l’expression desgènes, médiée en partie par les modifications épigénétiques. Parmi les modificationsd’histones, la chromatine bivalente, signature chromatinienne atypique associant lesmarques permissive H3K4me2/3 et répressive H3K27me3, est de par sa plasticité, pressentiepour jouer un rôle décisionnel dans l’acquisition d’une identité cellulaire. Pour étudier le rôlede la chromatine bivalente au cours du développement, nous avons choisi d’utiliserl’empreinte parentale. Ce cadre développemental bien caractérisé, conduit à l’expression decertains gènes à partir d’un seul des deux allèles selon son origine parentale. La méthylationdifférentielle de l’ADN d’une région clé, appelée ICR (Imprinting Control Region), bienqu’absolument requise pour l’expression mono-allélique de ces gènes, n’est pas suffisantepour rendre compte de la complexité du profil d’expression de ces gènes suggérantl’implication d’autres mécanismes. Sur 15 ICR méthylés sur l’allèle maternel, nous avonsprécisément mis en évidence que la chromatine bivalente est présente par défaut sur l’allèlenon-méthylé lorsque celui-ci est transcriptionnellement inactif, quel que soit le stadedéveloppemental ou le tissu étudié, participant ainsi à la régulation fine de l’expressiontissu-spécifique à partir de ces régions. Dans leur ensemble, nos données révèlent que lachromatine bivalente joue un rôle moins dynamique que pressentie. Ainsi, au niveau del’empreinte parentale, sa fonction principale serait de protéger l’allèle non-méthylé des ICRcontre l’acquisition de méthylation tout en aidant à le maintenir réprimé dans certainstissus. Nous proposons que la chromatine bivalente joue un rôle similaire sur l’ensemble desîlots CpG du génome, contribuant ainsi à la protection de l’identité cellulaire. Afin decompléter cette première étude, j’ai étudié la régulation de l’expression d’un candidat de larégulation de la dynamique de la chromatine bivalente, l’histone déméthylase pourH3K27me3, JMJD3. Les résultats obtenus suggèrent que l’induction d’expression observéeau cours de la différenciation neurale s’appuie sur une dynamique de la structuretridimensionnelle de la chromatine qui pourrait elle-même être régulée par la transcriptiond’un eARN (enhancer ARN) et l’hydroxyméthylation. Ce modèle souligne un mode derégulation complexe de ce nouvel acteur épigénétique, impliquant des régionsintragéniques, et pourrait notamment permettre de comprendre les mécanismes impliquésdans sa dérégulation dans les cancers. / Fine-tuned regulation of gene expression is required for cell fate determination anddevelopment. Epigenetics modifications are well documented to be instrumental in thisprocess. Among them, bivalent chromatin, an unusual chromatin signature, which associatesthe permissive mark H3K4me2/3 and the repressive mark H3K27me3, is believed to arbitrategene expression during cell commitment. To study its precise role in development, we haveundertaken to study bivalency in the context of genomic imprinting. This well-defineddevelopmental frame is a process restricting expression of some genes to one parental alleleonly. The constitutive differential DNA methylation at the key region called ICR (ImprintingControl Region), is absolutely required but not sufficient to explain the complexity of themono-allelic expression pattern of imprinted genes, indicating that other mechanisms couldbe involved. Specifically, on 15 maternally methylated ICR, we showed that bivalentchromatin is acquired by default on the unmethylated allele of ICR when it istranscriptionally inactive whatever the developmental stage or the tissue studied and thuscontribute to tissue-specific expression from these regions. Altogether, our results revealthat chromatin bivalency is much less dynamic than proposed. In the context of genomicimprinting, it seems to plays more a safeguard function at ICR by protecting theunmethylated allele against DNA methylation acquisition while keeping it silent in a subsetof tissues. To complete this study, I studied the regulation of JMJD3, a histone demethylasefor H3K27me3, candidate to regulate bivalency dynamic. Our results suggest that theinduction of Jmjd3 expression observed during neural differentiation rely on the dynamic ofthe tridimensional architecture at the locus which could be regulated by the transcription ofan eRNA (enhancer RNA) and by hydroxymethylation. This model highlight a complex way ofregulation for this new epigenetics actor, involving intragenic regions and could help tounderstand how Jmjd3 expression is deregulated in a pathological context such as in cancer.

Les plantes sont des organismes sessiles qui présentent plusieurs caractéristiques leur permettant de s'adapter rapidement aux variations de conditions environnementales. En particulier la lumière représente une source d’information essentielle utilisée tout au long du cycle de vie pour ajuster leur développement. Cette thèse avait pour objet l’étude de l’impact des mécanismes chromatiniens dans la régulation de l’expression des gènes pouvant influencer l’adaptabilité des plantes aux variations de signaux lumineux, à travers deux types de réponses caractérisées par des échelles de temps différentes chez la plante modèle Arabidopsis thaliana. La première étude portait sur des processus chromatiniens dynamiques participant à la régulation de l’expression génique, et utilisait comme modèle le dé‐étiolement. La triméthylation de la lysine 4 de l’histone H3 (H3K4me3), une modification posttraductionnelle généralement associée à un état transcriptionnel actif a été plus particulièrement étudiée. Afin de mieux connaître cette voie, le gène SWD2‐Like b (S2Lb) a été caractérisé. Il s’agit d’un nouveau partenaire de complexes COMPASS‐like et un déterminant important du niveau global de H3K4me3. L’analyse de plantes dans lesquelles ce gène est inactivé a montré qu’un défaut d’accumulation de H3K4me3 corrélait avec une induction plus faible de gènes de réponse à la lumière au cours du dé‐étiolement. Ces résultats et les nouveaux outils obtenus constituent une base solide pour étudier l’influence de cette marque et des facteurs associés sur la modulation fine de l’expression génique en relation avec d’autres marques chromatiniennes. La seconde étude cherchait à déterminer l’impact des variations épigénétiques sur la capacité des plantes à induire un syndrome d’évitement de l’ombre, une réponse adaptative à des conditions de lumière défavorables produites par des compétiteurs. Un phénotypage à grande échelle dans deux conditions de lumière induisant des réponses opposées a été réalisé sur une population de lignées recombinantes inbred (epiRIL), dans laquelle les variations épigénétiques (méthylation de l’ADN) sont maximisées mais les variations de séquence nucléotidique sont minimes. Une plus grande variation phénotypique ainsi qu’une plus grande amplitude dans la capacité de réponse à l’ombre ont été observées dans la population epiRIL. De plus, une cartographie QTL a permis d’identifier une région au début du chromosome 3 spécifiquement associée à la réponse d’évitement de l’ombre. Bien qu’une caractérisation plus fine soit nécessaire, le locus impliqué pourrait correspondre à une première description de QTL « épigénétique » influençant la plasticité phénotypique des plantes en réponse à une variation des conditions de l’environnement. / Plants are sessile organisms that successfully face variations of the environment by taking advantage of their ability to adapt their physiology and morphology. In particular, light perception constitutes an essential source of information used throughout their life cycle to fine‐tune development. The work presented was aimed at studying the role of chromatin‐associated mechanisms on adaptive responses to light cues at two different timescales in the model plant species Arabidopsis thaliana. In a first part, the role of chromatin dynamics in the regulation of gene expression was assessed during de‐etiolation, a developmental transition of seedlings that is triggered upon the first perception of light. It focused mainly on the trimethylation of histone H3 at lysine 4 (H3K4me3), a post‐translational modification associated with transcriptionally active states. To gain new insights into this pathway, the SWD2‐Like b (S2Lb) gene was characterized and shown to represent a new partner of plant COMPASS‐like complexes and a major determinant of H3K4me3 in A.thaliana. Loss‐of‐function plant lines for the S2Lb gene revealed that a default in H3K4me3 enrichment correlates with impaired inducibility of several light‐responsive genes during de‐etiolation. The findings described here set the bases to investigating how this mark and the associated factors influence the modulation of gene expression in relation with other chromatin marks. The second part of this thesis was aimed at assessing the impact of epigenetic variation on the capacity of plants to undergo the shade‐avoidance response (SAR), an adaptive developmental response to unfavorable light conditions produced by competitors. A population of epigenetic Recombinant Inbred Lines (epiRILs), in which epigenetic variation (DNA cytosine methylation) is maximized and nucleotidic sequence variation is minimized, was used for a large‐scale phenotyping under two light conditions triggering opposite responses. The epiRIL population exhibited larger amplitude of phenotypic variation than wild‐type parents in each condition as well as a wider range of response to shade. A region at the beginning of chromosome 3 was identified by QTL mapping to specifically associate to the SAR. Though it remains to be characterized, the locus involved may represent a first “epigenetic QTL” influencing phenotypic plasticity in response to environmental changes.

Au cours de la gestation précoce, la régulation de la physiologie endométriale est cruciale au bon déroulement de l’implantation. Chez les mammifères, une famille de facteurs de transcription est fortement impliquée dans la régulation de la physiologie endométriale, les facteurs STAT. Chez la vache, des analyses haut-débit ont révélé que l’expression endométriale de STAT1 est régulée au cours de la période préimplantatoire. Le but de cette thèse est donc d’apporter de nouvelles données sur l’expression et la régulation endométriales de STAT1 mais également sur ses fonctions biologiques au cours de la gestation précoce chez la vache.Grâce à différents modèles physiologiques et expérimentaux, l’impact de la progestérone, de l’IFNT (signal majeur de reconnaissance maternelle de la gestation chez les ruminants) et de la gestation sur l’expression et la régulation de STAT1 (y compris sa phosphorylation) a été analysé dans l’endomètre bovin et sur des cultures primaires de cellules endométriales. Ainsi, l’expression de STAT1 (transcrit et protéine) ainsi que sa phosphorylation sont augmentés en présence du conceptus et de l’IFNT, indépendamment du taux circulant de progestérone à l’implantation chez la vache. Pour avoir une meilleure connaissance des rôles de STAT1, l’identification de ses gènes cibles a été entreprise : d’abord avec une approche gènes candidats (avec la famille des gènes SOCS), puis par une approche exploratoire.Les facteurs SOCS sont connus pour être des régulateurs négatifs de la voie de signalisation des cytokines. L’utilisation des différents modèles physiologiques et expérimentaux évoqués plus haut a permis l’analyse de l’expression et de la régulation des huit membres de la famille des gènes SOCS au cours de la gestation précoce chez la vache. L’application d’un protocole d’immunoprécipitation de la chromatine sur des cultures primaires de cellules stromales bovines montre le recrutement rapide de STAT1 par l’IFNT sur les promoteurs des gènes SOCS IFNT-dépendants. D’autre part, l’identification systématique des gènes cibles de STAT1 a été entreprise via l’élaboration d’un protocole d’immunoprécipitation de la chromatine suivit de séquençage haut-débit, appliqué à des échantillons d’endomètre bovin. L’ensemble de ces travaux suggèrent l’implication de STAT1 dans la signalisation endométriale de l’IFNT, dans la régulation du système immunitaire maternel et également dans le contôle des phénomènes d’apposition et d’adhérence, fonctions cruciales à l’implantation chez la vache. / During the early stage of the pregnancy, the regulation of the endometrial physiology is crucial to the right establishment of the implantation. In mammals, a transcription factor family is highly involved in the regulation of endometrial physiology, the STAT family. In cattle, high-throughput analyses light up the regulation of endometrial STAT1 expression during the pre-implantation period. Thus, the aim of this work is to bring new insights about endometrial STAT1 expression and regulation but also on its biological functions during the early pregnancy in cattle. Using physiological and experimental models, the impact of progesterone, IFNT (major signal of the maternal recognition of pregnancy in ruminants) and pregnancy on the expression and the regulation of STAT1 transcript and protein (including its phosphorylation status) have been analyzed in the bovine endometrium and endometrial cells. Thus, STAT1 (transcript, protein and phosphorylation) is up-regulated by the presence of the conceptus and by IFNT but independent of progesterone level at implantation in cattle. To better understand endometrial STAT1 functions, the identification of STAT1 target genes has been initiated: first, on a candidate genes family, SOCS genes, and secondly, with an explorative approach.The proteins SOCS are known to be negative regulator of cytokine signalling pathway. Using physiological and experimental models previously quoted, the eight members of SOCS genes expression and regulation were analyzed during the early pregnancy in cattle. Chromatin immunoprecipitation protocol applied on stromal cells show the recruitment of STAT1 on SOCS promoters by a rapid treatment of IFNT. Moreover, the exhaustive identification of STAT1 target gene has been initiated, using a chromatin immunoprecipitation followed by high-throughput sequencing on bovine endometrium samples. Collectively, this data suggests the involvement of STAT1 in IFNT signalling pathway but also in the regulation of maternal immune system and the apposition/adhesion process, all that being crucial for the implantation in cattle.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 149-167).

Erythropoiesis, the process of producing mature erythrocytes from the haematopoietic stem cells (HSCs) that reside in the bone marrow, is tightly regulated at both the cell and molecular level by a well defined set of extracellular cytokine signals and intracellular transcription factors. Diseases affecting erythropoiesis are among the most commonly inherited conditions and result from disturbances to the cellular and molecular events that normally regulate this process. Erythroid Kruppel-like factor (EKLF/KLF1) is a transcription factor that is essential for erythropoiesis. EKLF is the founding member of the Kruppel-like factor family of transcription factors that bind to GC rich CACC-Box elements within gene promoters and activate transcription. The β-like globin genes are critical targets of EKLF through its binding at sites within the proximal promoters and the upstream locus control region (LCR) enhancer. Mice lacking EKLF die prior to birth by E16 with a phenotype that closely resembles the human disease thalassaemia. Thalassaemia is due to mutations in the α or β-globin genes, leading to globin chain imbalance, red cell destruction and ineffective erythropoiesis. However, restoration of expression of γ-globin (a β-like gene) failed to prevent embryonic lethality in EKLF knockout mice and suggested that additional target genes were critical to erythropoiesis. This thesis describes investigation into the transcriptional network of EKLF and an in depth analysis of previously uncharacterised phenotypes present in the EKLF knockout mouse. I have identified a suite of target genes for EKLF that include critical components of the cells cycle. I have also tested the hypothesis that EKLF is able to function in vivo as a tumour suppressor gene. Additionally, I report a role for EKLF in the determination of cell fate within the haematopoietic system and describe the development of a new approach to globally understanding erythroid transcription factor function. A previously performed microarray transcriptional profiling study provided a set of potential target genes for EKLF. I have expanded on this study by identifying that the cell cycle genes p18INK4c, and E2f2 are direct transcriptional targets of EKLF, where binding of EKLF occurs at the promoter and a novel intronic enhancer region, respectively. I have also described a previously undiscovered cell cycle phenotype of aberrant entry into S-phase in EKLF -/- erythroid cells that is directly related to abrogated expression of E2f2. The Kruppel-like factor family of genes have been implicated as players in the tumour process. By constructing a model for the loss of EKLF within HSCs in vivo, I have tested whether EKLF is functional as a tumour suppressor. The loss of EKLF in vivo was found to be insufficient to generate erythroleukaemia, however did result in erythroid hyperplasia, extramedullary haematopoieis and a mild macrocytic anaemia. In addition to regulation of erythropoiesis, EKLF performs a critical role in the lineage choice for a megakaryocyte-erythroid progenitor (MEP) between the megakaryocytic and erythroid lineages. This thesis describes that in the absence of EKLF, MEPs fail to commit properly to either lineage and proceed along a promiscuous pathway sharing the hallmarks of both megakaryocytes and erythroid cells. A detailed molecular mechanism for this phenotype remains undetermined, but is likely to involve interactions with the megakaryocyte transcription factor Fli1 and other members of the Kruppel-like factor family, such as BKLF (KLF3). While the transcriptional mechanisms that drive erythropoiesis have been slowly discovered, the development of chromatin immunoprecipitation (ChIP) assays and next generation DNA sequencing technology has presented the potential to rapidly enhance the progression of these studies. In this thesis I describe the development of ChIP-seq using Applied Biosystems SOLiD technology, an approach to rapidly identify binding sites for erythroid transcription factors in an unbiased genome wide approach. The work described in this thesis has expanded the transcriptional network of EKLF to include critical components of the cell cycle and has suggested many additional target genes from ChIP-seq requiring validation. As one of the major transcription factor players during erythropoiesis, EKLF performs many critical functions that include the regulation of the cell cycle, lineage selection and erythroid development. I suggest that current and future studies of EKLF function will influence our understanding of erythropoiesis and refine our understanding of human conditions such as thalassaemia and erythroleukaemia.

<p>An overall coordination between the expressions of genes is required for the proper development of an individual. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known as genomic imprinting. </p><p>The imprinted <i>H19</i> and <i>Igf2</i> genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of <i>H19</i>, known as the imprinting control region (ICR). This region shows differential methylation, with hypermethylation specifically on the paternal allele. On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. </p><p>The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF with the region. This association was investigated in both an <i>in vitro</i> episomal system and in an <i>in vivo</i> mouse model system by mutating the CTCF target sites at the <i>H19</i> ICR. The importance of CTCF for the insulator property of the region was confirmed in both instances. In the mouse model, the disruption of the binding was also observed to affect the methylation profile of the ICR, which ultimately resulted in the de-repression of the maternal <i>Igf2</i> allele.</p><p>The relevance of multiple CTCF target sites in higher vertebrates for the proper insulator function was investigated using another knock-in mouse model with mutation at a single CTCF target site in the <i>H19</i> ICR. The investigation confirmed the cooperation between the target sites for the establishment of a functional insulator on the maternal allele. Target sites in the ICR were also analysed for their differential binding affinity for the CTCF protein.</p><p>The utilisation of the CTCF target sites was examined in different human tumours and cell lines. Methylation analysis conveyed a lack of correlation between the loss of insulator function and methylation status of the ICR with the loss of imprinting (LOI) of <i>IGF2</i>. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the <i>H19</i> ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of <i>IGF2</i> imprinting observed in some instances.</p><p>In conclusion, this thesis confirms the importance of CTCF in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of <i>H19</i> and <i>Igf2</i>. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of <i>IGF2 </i>imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.</p>

<p>Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the <i>Kcnq1, Igf2</i> and <i>H19</i> genes, located at the distal end of mouse chromosome 7.</p><p>The <i>H19</i> ICR, located in the 5' flank of the <i>H19</i> gene represses paternal <i>H19</i> and maternal <i>Igf2</i> expression, respectively, but has no effect on <i>Kcnq1</i> expression, which is controlled by another ICR located at the intron 10 of the <i>Kcnq1</i> gene. This thesis demonstrates that the maternal <i>H19</i> ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the <i>H19</i> ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the <i>H19</i> ICR and CTCF. The <i>Kcnq1</i> ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the <i>Kcnq1</i> ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions.</p><p>In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.</p>

<p>A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting.</p><p>The imprinted state of the <i>H19</i> and <i>Igf2</i> genes is controlled by a short stretch of sequences upstream of <i>H19</i> known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited <i>Igf2</i> allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the <i>H19</i> ICR, which plays an important role in maintaining the imprinting status of <i>H19</i> and <i>Igf2</i>, was shown to lose the insulator property upon CpG methylation.</p><p>Another ICR in the <i>Kcnq1</i> locus regulates long-range repression of <i>p57Kip2</i> and <i>Kcnq1</i> on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing <i>H19</i> and <i>Igf2</i>, on the distal end of mouse chromosome 7. Similarly to the <i>H19</i> ICR, the <i>Kcnq1</i> ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions.</p><p>The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the <i>H19</i> ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF <i>in vivo</i> occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. </p><p>In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.</p>