Can epigenetic inheritance be reset during gametogenesis?

Epigenetic effects of in vitro maturation of egg cells on DNA methylation profiles of developmentally relevant genes in the model organism Bos taurus

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1 Epigenetic effects of the in vitro maturation of egg cells on DNA methylation profiles of developmentally relevant genes in the model organism Bos taurus Dissertation for obtaining the scientific doctorate from the Bavarian Julius Maximilians University of Würzburg presented by Tamara Schneider née Hansmann, born in Munich Würzburg, March 2014

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3 Submitted on: Members of the doctoral committee: Chairman: Reviewer: Univ.-Prof. Dr. med. Thomas Haaf Reviewer: Univ.-Prof. Dr. Georg Krohne Day of the doctoral colloquium: Doctoral certificate handed out on:

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5 The present work was carried out from November 2008 to April 2012 at the institutes for human genetics of the Universities of Mainz and Würzburg under the supervision of Univ.- Prof. Dr. med. Thomas Haaf made. Affidavit: I hereby declare that I wrote this doctoral thesis independently and without the help of a commercial doctoral consultancy and exclusively using the sources and resources given. The passages taken verbatim or correspondingly from the works referred to are marked as such. Furthermore, I affirm in lieu of an oath that I did not get the opportunity to undertake the doctoral project commercially and, in particular, did not involve a person or organization that is looking for a supervisor for the preparation of dissertations for a fee. I hereby declare that the rules of the University of Würzburg on good scientific practice have been complied with. The dissertation has not yet been submitted in the same or similar form to another university or in a different examination subject with the aim of acquiring an academic degree. On the Bavarian Julius Maximilians University of Würzburg I was awarded the academic degree of diploma in biology. I have neither acquired nor attempted to acquire any further academic degrees. Würzburg, March 2014 Tamara Schneider

6 ACKNOWLEDGMENTS First of all, I would like to thank Prof. Dr. Thomas Haaf for awarding the interesting dissertation topic and the doctoral candidate position, as well as for the supervision and assistance in the past five years. I also thank Prof. Dr. Georg Krohne for taking over the second report. I would also like to thank my colleagues and fellow doctoral students in Würzburg and Mainz for the pleasant and friendly atmosphere at the institute. I would especially like to thank Dr. Eberhard Schneider, Felix Mattern and Katharina Eirich for proofreading my dissertation and their suggestions for improvement, as well as Dr. Nady El Hajj for many scientific discussions, advice and helpful words in difficult laboratory times. I am very close to Prof. Heiner Niemann, Dr. Julia Heinzmann and Dr. Mike Diederich of the Friedrich Löffler Institute in Mariensee for the intensive and successful cooperation. Many thanks to my mother-in-law Romy Schneider for countless hours of childcare, in which I could devote myself to creating the work. I would especially like to thank my parents and grandparents for all these years of confidence and their faith in me, their financial support during the academic years, for the always correct and encouraging words and motivation. Finally, I would like to thank my husband Christian Schneider for his patience and understanding during the time of writing, for cooking and keeping our backs free, and especially for his love and our daughter Mila.

7 TABLE OF CONTENTS TABLE OF CONTENTS SUMMARY ... 57

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9 CONTENTS 4.2. Influence of IVM on the DNA methylation of imprinted genes Comparison of in vitro and in vivo matured oocytes Influence of IVM media msof and TCM on bovine oocytes Effects of the culture medium in embryo and follicle culture systems Low risk of epimutation in IVM? Influence of oocyte age and hormonal stimulation of follicle growth on the epigenetics of prepuberal and adult cows Influence of age on promoter methylation of oocyte-specific genes Influence of ovarian stimulation on promoter methylation of oocyte-specific genes Epigenetic risks of ovarian stimulation and its implications for human assisted reproduction Maturity for the development competence of egg cells Efficiency of the limiting dilution method for the study of individual cells Conclusion and outlook REFERENCES Bibliography Electronic sources DIRECTORIES List of abbreviations List of figures List of tables List of tables CURRICULUM VITAE PUBLICATIONS AND CONGRESS ARTICLES Publications Conference contributions

SUMMARY SUMMARY Assisted Reproductive Techniques (ARTs) for the treatment of infertility have been associated with an increased incidence of epigenetic aberrations during gametogenesis and early embryonic development, particularly through impairment of imprinted genes. The in vitro maturation (IVM) of egg cells is an ART that is already routinely used for the reproduction of economically valuable breeding animals such as domestic cattle (Bos taurus). IVM oocytes, however, show a reduced developmental competence to the blastocyst stage, which is possibly due to an impaired epigenetic regulation. Of all known epigenetic mechanisms, DNA methylation is the most studied DNA modification. In this work, oocytes of domestic cattle were analyzed to clarify the question of the effects of IVM on the DNA methylation of both imprinted and non-imprinted genes. This animal species has a similar pre-implantation development and gestation period as humans and is therefore increasingly used as a model for studying human germ cell and embryonic development. In contrast to humans and mice, there is so far little information about bovine genes. The first aim of the research presented here was therefore the identification and characterization of the bovine differentially methylated regions (DMRs) of the three imprinted gene locations of IGF2 / H19, SNRPN and PEG3, which are associated with imprinting defects in humans and / or in the mouse model. The first description of several intergenic DMRs using bisulfite sequencing and pyrosequencing proves the existence and evolutionary conservation of the IGF2 / H19 imprinting control region (ICR) in cattle. The imprinted state of the IGF2 / H19-ICR as well as the bovine genes SNRPN and PEG3 was confirmed by the detection of differential methylation in placental and somatic tissues as well as in sperm and parthenogenetic embryos. The methylation profiles observed were typical of genomic imprinting. The direct bisulfite sequencing after previous limiting dilution (LD) allows the analysis of methylation patterns of individual alleles (DNA molecules) of a few or even just a single cell (El Hajj et al., 2011). In a first LD experiment on bovine oocytes, the three previously characterized and imprinted genes were examined with regard to possible epigenetic changes that could be caused by various IVM conditions and media (TCM and msof). The overall rate of methylation errors of individual CpG sites as well as that of whole alleles (imprinting errors) did not differ significantly between the two IVM groups and the in vivo group. This result indicates that the current IVM protocols have little or no impact on these crucial epigenetic markings. IVM oocytes from prepuberal calves show a reduced developmental competence compared to IVM oocytes from adult animals. For this reason, the promoter methylation of three developmentally relevant, non-imprinted genes (SLC2A1, PRDX1, ZAR1) after ovarian stimulation with FSH and / or IGF1 was investigated in a second LD test. Both immature and in vitro-matured oocytes of prepuberal and adult cows showed a clear, unimpaired hypomethylation of the three gene promoters without any differences between the different age types of the donor animals or 8

11 SUMMARY their treatment. Neither age, hormonal stimulation nor IVM seem to have an influence on the methylation status of these three genes. In summary, the reduced development potential of IVM egg cells from adult and prepuberal cows was not reflected in abnormal methylation patterns of the imprinted and non-imprinted genes examined. This suggests a general stability of the established DNA methylation profiles in oocytes. For this reason, epigenetic mechanisms other than DNA methylation, such as ncrnas or histone modifications, must contribute to the reduction of the developmental competence of prepuberal and IVM oocytes. These changes presumably hinder the cytoplasmic maturation of the egg cell, which in turn leads to a later impairment of the development of the zygote and the embryo. 9

12 SUMMARY SUMMARY Infertility treatments by assisted reproductive technologies (ARTs) are associated with an increased incidence of epigenetic aberrations during gametogenesis and early embryogenesis, specifically in imprinted genes. In vitro maturation (IVM) of oocytes is an ART which is routinely applied for reproduction of agriculturally and economically important species like cattle (Bos taurus). However, IVM oocytes exhibit a reduced developmental competence to the blastocyst stage which may be caused by an impaired epigenetic regulation. Of all known epigenetic mechanisms DNA methylation is the most studied DNA modification. In this thesis, bovine oocytes have been analyzed in order to investigate the impact of IVM on the DNA methylation of imprinted and non-imprinted genes. Because this species exhibits a similar pre-implantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. In contrast to humans and mice, only little information on bovine imprinted genes is available. Thus, the first attempt of the research presented here was to identify and characterize the bovine differentially methylated regions (DMRs) of the three imprinted loci, namely IGF2 / H19, SNRPN and PEG3 which are each associated with imprinting defects in humans and / or the mouse model. The first description of several intergenic DMRs by bisulfite sequencing and pyrosequencing proved the existence of an intergenic IGF2 / H19 imprinting control region (ICR) in the bovine. The imprinted status of the IGF2 / H19-ICR as well as the bovine genes SNRPN and PEG3 was confirmed by differential methylation consistent with genomic imprinting in placental and somatic bovine tissues, in sperm and parthenogenetic embryos. Limiting Dilution (LD) Bisulfite Sequencing (El Hajj et al., 2011) followed by direct bisulfite sequencing allows the analysis of methylation profiles of individual alleles (DNA molecules) from only a few or even single cells. In a first approach using LD, the three characterized imprinted regions were analyzed to determine putative epigenetic alterations in bovine oocytes cultured with different types of IVM conditions and media (TCM and msof). The total rate of individual CpG and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. The developmental capacity of IVM oocytes from prepubertal calves is reduced compared with their IVM oocyte counterparts from adult animals. Therefore, in a second LD approach, the promoter methylation of three developmentally important, non-imprinted genes (SLC2A1, PRDX1, ZAR1) has been studied in IVM oocytes from prepubertal cattle after ovarial stimulation with FSH and / or IGF1. Both immature and in vitro matured prepubertal and adult oocytes showed unimpaired hypomethylation of the three gene promoters without differences between the different ages of donors and treatments. Thus, neither age nor hormonal treatment or IVM seem to influence the methylation status of these three genes. In conclusion, the reduced developmental capacity of IVM oocytes from adult and prepubertal cattle were not associated with aberrant methylation patterns of the investigated imprinted and non-imprinted genes suggesting a general stability of established DNA methylation marks in oocytes. Therefore, epigenetic mechanisms other than DNAmethylation such as ncrnas or histone modifications might confer to the reduced developmental competence of prepubertal and IVM oocytes. These factors are supposed to interfere with cytoplasmic maturation of the oocyte leading to an impaired development of the zygote and embryo rather than to influence nuclear maturation of the oocyte. 10

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14 INTRODUCTION 1. INTRODUCTION 1.1. Epigenetics Epigenetics (Greek: epi = over, above, on) deals with all those biochemical changes that can lead to a modulation of gene expression regardless of the DNA sequence. The epigenome summarizes all epigenetic mechanisms of the genome. Epigenetic changes are covalent modifications of the DNA or the histone proteins (Smallwood and Kelsey, 2012), which form an additional layer of information on the DNA and thereby regulate the physical accessibility of the DNA for transcriptional or repressive processes in the cell nucleus (Tomizawa et al., 2012 ). While the identical genetic information of the diploid genome is present in every body cell, the various epigenetic mechanisms of plastic control serve both spatial (cell or tissue-specific), temporal (development-specific) and parent-specific expression of the genetic information (El Hajj and Haaf, 2013) . Therefore, they play an important role in specific gene expression, especially during embryonic development and cell differentiation (Jaenisch and Bird, 2003). In differentiated body cells, only about 5-10% of the genes are activated to carry out the cell and tissue-specific tasks, while the majority of the genes are epigenetically shut down (El Hajj and Haaf, 2013). Epigenetic modifications also represent reactions to environmental influences, which allow conclusions to be drawn about past external influences (Dolinoy et al., 2007). Existing epigentic adaptations can in turn have a positive or negative influence on the later response to new environmental changes. Thus, the epigenome not only determines the cell and tissue-specific functions of the genome, but also acts as a cellular memory for its development history and reactions to external environmental influences (Tomizawa et al., 2012). Epigenetic mechanisms are mostly biochemical modifications of the DNA that lead to a more or less dense chromatin packing. These are mainly histone modifications through phosphorylation, methylation or acetylation as well as DNA methylation. The binding of transcription factors, micrornas or complexes of regulatory active proteins to the DNA is also counted as part of epigenetics. Finally, there are also cis- or trans-regulatory chromatin interactions that work together in a special way to control gene expression, in the broadest sense 12

15 INTRODUCTION to epigenetic processes (Meehan, 2003). All these epigenetic changes are reversible, but, as in the case of DNA methylation (see chapter), can be passed on stably to daughter cells and from across generations (Hitchins et al., 2011). Therefore, inherited epigenetic changes can have phenotypic effects even though there has been no change in the DNA sequence. A faulty epigenetic modification that has effects that deviate from the normal state is referred to as epimutation, analogous to the term mutation in genetics. Since epimutations are reversible, they do not represent mutations in the sense of Mendel's genetics. The inheritance of the epigenetically based imprinting diseases known so far does not follow Mendel's theory of segregation, but depends on the gender of the parent from whom the epimutation is inherited. In these cases, one speaks of parent-specific or parental inheritance, which is discussed in more detail in Chap. DNA methylation DNA methylation is probably the best studied epigenetic modification (Reik et al., 2001). It is a comparatively stable change in the DNA that is accompanied by a reorganization of the chromatin structure and thereby mediates permanent repression of transcription. The methylated DNA forms complexes with various proteins that have methylcytosine binding domains. This complexation causes a locally denser packaging of the chromatin (condensation), which in turn leads to an inaccessibility for transcriptional factors and proteins (Jaenisch and Bird, 2003; Meehan, 2003). For this reason, low DNA methylation in the promoter region of a gene is associated with active transcription of the same (Neumann and Barlow, 1996), while promoter methylation is associated with repression of transcription (Bird and Wolffe, 1999). By means of DNA methylation, cells can on the one hand repress the intrinsic expression of genes, but on the other hand also render mobile extrinsic DNA elements harmless, such as viral DNA, retrotransponsons or invasive plasmids (Yoder et al., 1997b; Bestor, 2000; Jaenisch and Bird , 2003). Special functions of DNA methylation include X chromosome inactivation, genomic imprinting and the shutdown of genes during the differentiation of cells. The methylation takes place at the carbon atom 5 of the pyrimidine ring of the cytosine (5mC), which in the 3 -direction of the 13th

16 INTRODUCTION The base sequence is usually followed by a guanine (cytosine phosphatidyl guanine or CpG dinucleotide). The methylation is mirror-symmetrical on both strands of the DNA double strand. The asymmetric hemimethylation of the DNA double strand existing after the DNA replication is completed by the enzymatic transfer of a methyl group to the cytosine of the newly synthesized subsequent strand. The transfer is catalyzed by the methyltransferase DNMT1, which uses the substrate S-adenoyslmethionine (SAM) as the methyl donor. The de novo methyltransferases of the group of three (DNMT3A, DNMT3B and DNMT3L), on the other hand, are not responsible for maintaining methylation after mitosis, but are responsible for re-establishing it on unmethylated DNA (see Chapter; Meehan, 2003; Edwards and Ferguson-Smith , 2007). The demethylation of 5-methylcytosines usually takes place passively through the successive loss of the methyl group in the course of several rounds of DNA replication, more rarely through active enzymatic modification and elimination of the methyl group. The mechanism of active demethylation of cytosines has long been the subject of controversial discussion (Wu and Zhang, 2010), but was only recently identified: TET proteins catalyze the hydroxylation of 5-methylcytosines to 5-hydroxymethylcytosine (5hmC), which in turn serves as a substrate for the Thymine-DNA-glycosylase-mediated base excision repair (BER) to unmethylated cytosine is used (summarized by Kohli and Zhang, 2013). About 80% of all CpG dinucleotides in the genome are methylated (Antequera, 2003). These are almost exclusively in non-coding regions of the genome in order, for example, to suppress the activity of parasitic retrotransposons (Yoder et al., 1997b). Due to the spontaneous deamination of methylated cytosines in the genome, CpGs occur statistically significantly less than all other 15 dinucleotides. At 1%, their proportion in the human genome is only around 20% of the expected frequency of around 5% (Sved and Bird, 1990). In the promoter area of ​​60-70% of all genes, however, there are 0.2 to 2 kb long sections with an above-average high CpG density (> 60% GC content compared to approx. 40% GC in the rest of the genome). These sequences are called CpG islands and have many binding sites for transcription factors. The promoter CpG islands are normally unmethylated, which ensures transcription (summarized in Antequera, 2003; Weber et al., 2007). The methylation of the CpG islands in the development of diseases or in their course is associated with the post-translational modification of histone proteins, which leads to a locally condensed, inactive chromatin structure and thus to the switching off of the cis-regulated genes. 14th

17 INTRODUCTION Genomic imprinting Genomic imprinting is an epigenetic modification of DNA for the regulation of genes, which is essential for the development of the fetus and the placenta (Miozzo and Simoni, 2002; Reik et al., 2003). It occurs almost exclusively in higher mammals (Eutheria), but also in flowering plants and some insect species (Sha, 2008). A prominent mechanism of genomic imprinting is the DNA methylation of these genes, which results in a specific shutdown of one of the two parental alleles (Reik and Walter, 2001; Kelsey, 2007; Ideraabdullah et al., 2008; Tomizawa and Sasaki, 2012). The monoallelic suppression is strictly dependent on the parental origin, so that part of the imprinted genes is always imprinted on the maternal chromosome, the other part is always imprinted on the paternal chromosome (Kelsey, 2007). The preferential or mostly sole expression always takes place from the non-imprinted allele. Due to the allele-specific methylation, the imprinted regulatory loci are referred to as differentially methylated regions (DMRs). A distinction is made between two groups of DMRs. In the case of primary DMRs, DNA methylation is already established in the germ cells, which is why they are also known as germline DMRs (gdmrs). Often the primary DMRs are also imprinting control regions (ICRs; see Chapter). The secondary or somatic DMRs, on the other hand, receive differential methylation only after fertilization and are controlled by neighboring ICRs (Tomizawa and Sasaki, 2012). Genomic imprinting is a reversible modification that has to be re-established in the germ cells of each generation. The gender-specific marking already takes place in the primordial germ cells during the early embryonic development of an individual. The parental imprints are completely deleted and re-established depending on the gender of the embryo. The egg-specific and sperm-specific imprinting patterns are retained in the next generation even after the mature germ cells have been fertilized (Kelsey, 2007; Ideraabdullah et al., 2008) and escape fetal reprogramming (see section). So far, 100 to 200 imprinted genes have been identified in mammals (Jirtle, 2012; Williamson et al., 2012). 15th

18 INTRODUCTION The function of imprinted genes Imprinted genes play an important role in fetal and placental development (Miozzo and Simoni, 2002). Due to their monoallelic expression, the imprinted genes are usually dose-sensitive, since changes in the expression level have functional effects. In particular, dysregulations affect prenatal growth control, the development of special organs and cell lines, normal brain function and postnatal energy homeostasis (Bartolomei and Ferguson-Smith, 2011). Due to their growth-regulating role, some imprinted genes also have an oncogenic or tumor-suppressing function, so that a change in the gene dose due to the loss of the imprint is associated with the development of neoplasms (summarized in Uribe-Lewis et al., 2011). The first evidence of genomic imprinting in mammals is provided by the lack of parthenogenesis, which is widespread in the animal and plant world. This form of single-sex reproduction, also known as virgin generation, allows the emergence of a genetically identical offspring from an unfertilized egg cell, which starts embryogenesis through hormonal activation without the involvement of a sexual partner. Corresponding to the lack of natural parthenogenesis in mammals, the artificial creation of parthenogenetic mouse embryos also led to an early lethality of the embryos (Kaufman et al., 1977; Obata and Kono, 2002). The fact that a uniparental diploid genome is incompatible with life is also shown by diandric pregnancies, which have two male genomes due to the fertilization of an egg cell without a nucleus with one or two sperm cells. A hydatiform mole develops from the diandric zygote, but only contains placental structures and no fetal or embryonic tissue. More specific evidence of genomic imprinting was provided by experiments with mice in which pronuclear transplants were performed on fertilized egg cells (Barton et al., 1984; McGrath and Solter, 1984; Surani et al., 1984). For this purpose, the egg cells were enucleated and equipped with either two different (biparental) female pronuclei or two male pronuclei. Despite the presence of a diploid set of chromosomes, neither the digynic nor the diandric embryos developed beyond an early post-implantation stage (McGrath and Solter, 1984). In addition to the early mortality, characteristic morphological differences between diandric and digynic embryos and their trophoblasts were also observed

19 INTRODUCTION the extraembryonic tissue remained underdeveloped in digynic embryos, conversely in Diandrie the embryos themselves showed insufficient development. Surani and Barton concluded that the paternal genome is essential for the normal development of extraembryonic tissues, whereas the maternal genome makes an important contribution to correct embryogenesis (Barton et al., 1984; Surani et al., 1984). These results showed for the first time that the paternal and maternal genomes are functionally not equivalent and that a biparental and bisexual origin of the gametes are essential for correct embryonic development. The evolution of genomic imprinting, as found in vertebrates only in mammals, presumably results from a conflict of interest over maternal resources between the paternal and maternal parts of the genome and is referred to as the gender conflict hypothesis (Moore and Haig, 1991). According to this, the parental genomes competitively regulate intrauterine growth by means of different sets of imprinted genes. Paternally shaped genes encourage the use of as many maternal resources as possible in order to ensure maximum fetal and postnatal growth of the offspring. In contrast, the maternally shaped genes have a restrictive effect on growth with regard to the allocation of resources for possible later pregnancies (Moore and Haig, 1991). The necessity of the evolutionary development of this competitive system results from the extensive prenatal and postnatal supply of the embryo with nutrients and space, which can only be found in the form of placenta (eutheria). The best-known example of an imprinted gene that has a regulatory effect on fetal growth is insulin-like growth factor 2 (Igf2). Loss of imprinting (LOI) results in biallelic expression of the gene and an approx. 30% increase in birth weight (Leighton et al., 1995a), while conversely the inactivation of the normally expressed paternal allele leads to a reduced birth weight by approx. 20% (Leighton et al., 1995b). This example supports the hypothesis that imprinted genes create a genetically controlled balance between supplying the fetus with nutrients from the placenta and retaining maternal resources for later pregnancies. This hypothesis makes a coevolution of genomic imprinting and the taxon Eutheria very likely (Reik et al., 2003). Since there is also a high need for maternal resources in the postnatal breastfeeding phase, it is not surprising that the concept of genomic imprinting also extends to the 17th

20 INTRODUCTION to extend the postnatal and adult periods. Although the expression of most imprinted genes is downregulated after birth, some of them also play an important role in postnatal growth, maternal care behavior, and brain development (Plagge et al., 2004; Kelsey, 2007; Wilkinson et al., 2007) . The findings suggest that the imprinted genes, which are mainly differentially expressed in the brain postnatally, serve to control nutrient resources by helping to control metabolic energy homeostasis and mother-child interaction (Bartolomei and Ferguson-Smith, 2011 ). The maternally shaped paternally expressed gene 3 (Peg3), which was examined in this work, for example, plays an important role in the care of the newborn by the mother. It is strongly expressed in the placenta and the developing embryo, as well as in the hypothalamus and the adult brain (Li et al., 1999). Mutations of the paternal allele led to growth retardation in mice without further impairment. However, mutant females later showed impaired care behavior in the sense of reduced attention and licking of the young animals as well as a lack of milk production, which led to the death of over 90% of the offspring. The lactation deficiency was attributed to a reduced release of the hormone oxytocin in neurons of the hypothalamus (Li et al., 1999; Curley et al., 2004). Male mice also showed abnormal behavior with regard to their sexual behavior and the olfactory recognition of females ready to mate (Swaney et al., 2008). The Peg3 gene product is a large zinc finger protein that plays a role in p53-mediated apoptosis and is therefore classified as a tumor suppressor gene (Deng and Wu, 2000; Relaix et al., 2000). In addition, it inhibits the growth-promoting Wnt signaling pathway in embryogenesis and is involved in the formation of glial cell tumors (Jiang et al., 2010) Regulation of imprinted genes Most known imprinted genes are not evenly distributed over the genome, but cluster in several in one 1 Mb each region (Reik and Walter, 2001; Edwards and Ferguson-Smith, 2007; Ideraabdullah et al., 2008; Bartolomei and Ferguson-Smith, 2011). The genes of an imprinted cluster are usually not shaped autonomously, but are subject to a common cis-regulatory DNA element. This GC-rich region, which controls monoallelic expression, is called the imprinting control region (ICR) or less often than 18

21 INTRODUCTION Imprinting control element (ICE). The parent-specific, differential methylation of these ICRs already takes place during gametogenesis (see chapter) and is also preserved in all somatic cells and developmental stages as well as in extraembryonic tissue (Hudson et al., 2010). A cluster usually contains both maternally and paternally expressed genes. However, these can be expressed differently in the various stages of development and differentiated tissues. Genes that are close to the ICR are mostly ubiquitously expressed monoallelically in all somatic and extraembryonic tissues, while genes lying further out in the cluster can never or, for example, only show pronounced expression in the placenta (Hudson et al., 2010). Furthermore, the shaping of the expression can also be canceled by the cell differentiation, as was shown, for example, on the basis of the biallelic expression of Igf2r in postmitotic neurons (Yamasaki et al., 2005). The differential methylation of the ICR itself, however, remains in all stages of development and differentiated cells. The controlled expression of the ICR-regulated genes can be controlled both by DNA methylation and by other epigenetic mechanisms such as repressive histone modifications, non-coding RNAs (ncrnas) or chromatin interactions. One possibility is the direct monoallelic silencing of the promoter of an imprinted gene by differential methylation, as is the case with the genes SNRPN (Horsthemke and Wagstaff, 2008) and Peg3 (Huang and Kim, 2009). A common mechanism of indirect control of imprinted genes, on the other hand, is the expression of ncrnas, the transcription of which often interferes in the opposite direction with that of an imprinted gene. An example of such an antisense transcript is Air, which regulates the monoallelic expression of Igf2r and the genes Slc22a2 and Slc22a3. The promoter of the Air transcript lies within an intron of Igf2r and corresponds to a maternally imprinted, differentially methylated ICR. The transcription of the Air ncRNA on the paternal chromosome overlaps with the Igf2r promoter and thus inhibits its transcription (Sleutels et al., 2002; Bartolomei and Ferguson-Smith, 2011). The prime example of an expression of expression regulated indirectly via secondary chromatin structures is the Igf2 / H19 locus on human chromosome 11p15.5 or on the distal end of mouse chromosome 7. The imprinting mechanism of this gene cluster is highly conserved and is also found in marsupials that represent a subclass of mammals (Mammalia) in addition to the higher mammals and the ancient mammals. The 19th

22 INTRODUCTION Igf2 / H19-ICR is methylated in the male germ line and is thus one of only four known primary DMRs that are paternalized (H19 / Igf2, Dlk1 / Dio3, Rasgrf1, Zdbf2; Bartolomei and Ferguson-Smith, 2011) . In humans it is specifically referred to as IC1 (Imprinting Center 1) and in mice as DMD (differentially methylated domain) (Jinno et al., 1996; Tremblay et al., 1997). The ICR lies intergenically between the two genes, Igf2 and H19, which are influenced by it, only a few kb upstream from the H19 promoter and approx. 90 kb downstream from Igf2 (Hark et al., 2000). Igf2 is expressed from the paternal chromosome, while H19 is expressed from the maternal chromosome. In the unmethylated state, the intergenic ICR acts as an isolator by binding the ubiquitous zinc finger protein CTCF (CCCTC-binding factor). On the maternal chromosome, the interaction of the Igf2 promoter is physically blocked via a chromatin loop with enhancer elements located downstream of H19. The methylation of the ICR on the paternal chromosome, on the other hand, prevents binding of the CTCF protein and the Igf2 promoter can be activated by the enhancer (Fig. 1.1; Bell and Felsenfeld, 2000; Hark et al., 2000; Engel et al., 2006). The identification of the bovine Igf2 / H19-ICR was part of this work. Igf2 CTCF ICR H19 Enhancer Igf2 ICR H19 Enhancer Fig. 1.1: Regulation and structure of the Igf2 / H19-ICR. Model of epigenetic regulation. The ICR is unmethylated on the maternal chromosome. This allows the CTCF protein to bind to the CTCF binding sites, thereby blocking the interaction of the enhancers downstream of H19 with the Igf2 promoter. The ICR acts as an isolator due to the CTCF binding. Due to the methylation of the ICR on the paternal chromosome, CTCF cannot bind, so that Igf2 can be activated. 20th

23 INTRODUCTION Imprinting diseases The monoallelic expression of imprinted genes poses a potential disease risk, since possible mutations of the expressed allele cannot be picked up by the second copy. Diseases that can be traced back to a loss of the functionality of the active allele of an imprinted gene are accordingly referred to as imprinting diseases.They often manifest themselves in growth anomalies, various malformations, mental retardation or cancer in childhood. The best-known imprinting diseases are Prader-Willi, Angelman, Silver-Russell and Beckwith-Wiedemann syndromes as well as transient neonatal diabetes mellitus (TNDM), pseudohypoparathyroidism type 1b (PHP-1b) or paternal or maternal UPD 14 Syndrome (Bartolomei and Ferguson-Smith, 2011; Tomizawa and Sasaki, 2012; Diedrich et al., 2013). The incidences of most imprinting diseases are in the range of 1 to 10 cases in births and are therefore very rare (Lidegaard et al., 2006). A mutagenic change in the unprinted allele manifests itself as an autosomal dominant form of inheritance. The inheritance of imprinting errors does not follow Mendel's rules, according to which the parental alleles segregate independently of one another, but rather depends on the respective parental transmission (Kelsey, 2007). Imprinting diseases are mostly due to chromosomal or genetic aberrations such as deletions, translocations, inversions, duplications and point mutations, which result in a change in the gene dose (Tomizawa and Sasaki, 2012). A moderate number of diseases, on the other hand, can be traced back to a uniparental disomy (UPD), in which one chromosome is present but both homologous specimens come from the same parent and thus have the same parent-specific character. UPDs usually arise in the zygote through a so-called trisomy rescue (the excess chromosome is removed from the zygote) or a monosomy rescue (duplication of the single chromosome; Buiting, 2010; Eggermann, 2010; Yamazawa et al., 2010). The imprinting disease rarely stems from an actual imprinting defect (primary epimutation) in the germline. The PWS / AS gene locus Among the most well-known human imprinting diseases are the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS), which despite being very different Phenotypes caused by dysregulation of the same imprinted locus on chromosome 15q11-q13 21

24 INTRODUCTION (Horsthemke and Wagstaff, 2008). The PWS is characterized by hypotonia in the newborn and a general failure to thrive. Later, the short children develop obesity due to hyperphagia and behavioral problems. In adulthood, obesity-associated diseases such as diabetes and cardiovascular diseases limit life expectancy. In contrast to AS, PWS patients only suffer from mild to moderate mental retardation and can manage their everyday life independently. In addition to severe cognitive disabilities and a lack of language development, AS is characterized by atactic, hyperactive movements and unmotivated fits of laughter, which is why it is sometimes referred to as happy puppet syndrome. The children usually suffer from epilepsy (characterized by a characteristic EEG) and sleep disorders. The imprinted PWS / AS locus contains numerous genes that are regulated by complex mechanisms, including cis- and trans-regulatory factors and ncrnas (Horsthemke and Buiting, 2006; Horsthemke and Wagstaff, 2008). The causes that can either lead to PWS and AS are correspondingly diverse and are therefore only explained in their basic features in the following. The maternally shaped ICR of the gene-rich cluster is in the promoter and exon1 area of ​​the small nuclear ribonucleoprotein-associated protein N gene (SNRPN) and is specifically referred to as IC (Imprinting Center) (Horsthemke and Buiting, 2006). The SNPRN protein is a polypeptide of the snrnp-smb / smn family and plays a role in the processing of pre-mrna and in tissue-specific alternative splicing (NCBI, 2014: Gene ID: 6638). The SNRPN transcript expressed by the paternal chromosome is part of a single transcript over 460 kb long, which contains over 70 small nucleolar RNAs (small nucleolar RNAs, snornas) and an antisense transcript, each of which is released individually by alternative splicing (Horsthemke and Wagstaff, 2008). The antisense transcript initiated from the SNRPN promoter presumably inhibits the expression of the UBE3A gene, which is dysregulated in AS patients (Chamberlain and Brannan, 2001; Fig. 1.2). Loss of paternal expression of the long transcript, for example through deletion or methylation of the ICR, leads to PWS, while in AS there is a loss of maternal expression of UBE3A (Horsthemke and Wagstaff, 2008; Bartolomei and Ferguson-Smith, 2011). The most common cause of both PWS and AS with approx. 70% of patients are de novo deletions of 15q11-q13. The remaining PWS cases (29%) almost all have a maternal UPD, while in AS a paternal UPD is only about 1-2% of the cases 22

25 INTRODUCTION. A truncating point mutation in the maternally inherited UBE3A gene was found in 10% of AS cases (Kishino et al., 1997; Matsuura et al., 1997; Ludwig et al., 2005; Horsthemke and Wagstaff, 2008). Imprinting defects of the ICR are responsible for the disease in only about 1% of PWS and about 3-5% of AS patients (Ludwig et al., 2005; Horsthemke and Wagstaff, 2008). The investigation of the imprinting of the SNRPN promoter ICR is the subject of this work. Embossed locus SNURF-SNRPN-sense- / UBE3A-antisense transcript xx C / D box snornas xxxx Fig. 1.2: Scheme of the embossed PWS / AS gene locus on human chromosome 15q11-q13 (modified from Horsthemke and Buiting, 2006) . Blue boxes represent genes that are only expressed by the paternal chromosome, while red boxes are only expressed by the maternal chromosome and black by both chromosomes (unprinted expression). The direction of transcription is shown in each case by an arrow. Methylated promoters on the maternal chromosome are indicated by filled black circles. Repressed expression is indicated by an X above the direction of expression. The long transcript expressed by the paternal chromosome contains, in addition to the sense transcript of SNURF-SNRPN (blue), the C / D box snornas (orange) and the antisense transcript (green), which represses UBE3A. The SRS / BWS gene locus is embossed Genes Igf2 and H19 are part of an imprinted locus on human chromosome 11p15.5, which is associated with the two imprinting diseases Silver-Russell (SRS) and Beckwith-Wiedemann syndrome (BWS). While the SRS is characterized by intrauterine and postnatal growth retardation, physical asymmetry and other heterogeneous clinical features (Eggermann, 2010), the thoracic spine shows pre- and postnatal tall stature, macroglossia, neonatal hypoglycemia and abdominal wall defects as well as an increased tumor risk (Weksberg et al., 2010; Tomizawa and Sasaki, 2012). 23

26 INTRODUCTION The embossed region is divided into two imprinting domains. The distal domain 1 consists of the imprinted genes Igf2 and H19 and is regulated by the paternal methylated imprinting center 1 (IC1). Domain 2 comprises the imprinted genes KCNQ1, KCNQ1OT1 and CDKN1C. The differentially methylated IC2 contains the promoter of KCNQ1OT1 (also known as LIT1), which codes for a paternally expressed ncrna and thus inhibits the expression of the maternally expressed genes KCNQ1 and CDKN1C (FIG. 1.3). The IC2 is also often referred to as the KvDMR. Fig. 1.3: Scheme of the imprinted BWS / SRS gene locus on human chromosome 11p15.5 (taken from Weksberg et al., 2010). As with PWS and AS, the causes of these two diseases are diverse. BWS is mostly caused by hypomethylation (loss of methylation) of the KvDMR / IC2 on the maternal chromosome (50%), but also by paternal UPD (20%), mutations in the paternal allele of CDKN1C (5%), maternal transmission of de novo translocations / inversions (1%), which mostly interrupt the KCNQ1 gene, as well as paternal duplications of 11p15.5 (1%; Weksberg et al., 2010). Hypermethylation of IC1 (Igf2 / H19-ICR) is causal in approx. 5-10% of cases (Sparago et al., 2004; Eggermann, 2009; Tomizawa and Sasaki, 2012). In contrast, SRS patients show hypomethylation of IC1 on the paternal chromosome in at least 44% of the cases. The SRS and BWS thus show opposing clinical pictures, which, interestingly, can be traced back to opposing epimutations (hypermethylation of IC1 in the thoracic spine and hypomethylation in the SRS) (Eggermann, 2009). 24

27 INTRODUCTION Epigenetic Genome Reprogramming Genomic imprinting is a cyclical process that requires phases of imprint establishment, maintenance and deletion. In the course of this cycle there are two waves of global demethylation and remethylation, which are referred to as epigenetic genome reprogramming (Fig. 1.4). Due to the extensive amount of data, the sequences and processes involved in genome reprogramming are mainly explained below using the mouse model. The first genome-wide epigenetic reprogramming takes place during the early embryonic development and serves to eradicate the parental patterns in the as yet undifferentiated germline. This resetting of the epigentic modifications ensures the totipotency of the later gametes and prevents the inheritance of possible epimutations into the next generation (Hajkova et al., 2008; Smallwood and Kelsey, 2012). Some of the posterior epiblast cells develop into primordial germ cells (PGCs). During the migration of PGCs into the germinal ridge, a massive removal of DNA methylation takes place (Hackett et al., 2012). Typically, demethylation is preceded by changes in the histone modifications (Gifford et al., 2013). In mice, imprint erasure begins on day 11.5 and is completed on day 13.5 dpc (days post concetion) (Hajkova et al., 2002; Yamazaki et al., 2003). The reprogramming seems to take place preferentially on imprinted and unembossed single copy genes, while the demethylation of repetitive elements is delayed and incomplete. This presumably serves to protect against activation of transposable elements, which could lead to germline mutations through dysregulation of neighboring genes or transposition (Hajkova et al., 2002; Allegrucci et al., 2005). Since the demethylation of DNA takes only a few hours, the rapid and comprehensive eradication of the methyl groups must be mediated by active processes and cannot be traced back to passive demethylation through DNA replication. A likely mechanism of active demethylation is the exchange of methylcytosines and histones by the DNA repair machinery (Hajkova et al., 2002; Hajkova et al., 2008). Demethylation takes place despite the presence of the methyltransferase Dnmt1 (Hajkova et al., 2002), which has a strong affinity for hemimethylated DNA and is responsible for maintaining and thus inheriting the methylation pattern on daughter cells (Yoder et al., 1997a). Following the global demethylation, the methylation patterns are re-established in a gender-specific manner (de novo methylation). This is done in the male 25