Several factors direct the placement of specific nucleosomes, which in turn have the ability to regulate DNA accessibility. These factors include, but are not limited to, nucleotide sequence preference, nucleotide modifications, the type of histones present within the nucleosome, and the presence of additional transcription factor or chromatin remodelers. A combination of these and other factors are responsible for tightly controlled efficient transcription within the eukaryotic cell. In order to contribute to the understanding of these complicated processes, three separate hypothesis-driven investigations were carried out. First, we looked into nucleosome positioning and phasing within closely related cells lines. Second, we examined domain level nucleosome occupancy on various portions of the chromosome. Finally, we generated a novel method that significantly reduces data loss in in vitro nucleosome reconstitution experiments caused by nucleosome fragment-end bias. All three of our investigations yielded separate results. First, by examining positions and phasing patterns within similar cell types we find common patterns and minor differences within similar cell types. The presence of minor differences in nucleosome positions may cause unique expression patterns. Secondly, we found that decreased domain level nucleosome occupancy at the chromosome arms is not caused by the presence of a class of nucleosomes, termed fragile nucleosomes. Finally, we found that when our nucleosome recovery method is applied conservatively to our dataset, it is possible to recover 80% of the lost nucleosome reconstitution data.

<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>Chromatin structure plays an important role in gene regulation, especially in differentiating the diverse cell types in humans. In this dissertation, we analyze the nucleosome positioning and open chromatin profiles genome-wide and investigate the relationship with transcription initiation, the activity of regulatory elements, and expression levels. We mainly focus on the results of DNase-seq experiments, but also employ annotations from MNase-seq, FAIRE-seq, ChIP-seq, CAGE, and RNA microarrays. Our methods are based on computational approaches including managing large data sets, statistical analysis, and machine learning. We find that different transcription initiation patterns lead to distinct chromatin structures, suggesting diverse regulatory strategies. Moreover, we present a tool for comparing genome-wide annotation tracks and evaluate DNase-seq against a unique assay for detecting open chromatin. We also demonstrate how DNase-seq can be used to successfully predict rotationally stable nucleosomes that are conserved across cell types. We conclude that DNase-seq can be used to study genome-wide chromatin structure in an effort to better understand how it regulates gene expression.</p> / Dissertation

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 Kcnq1, Igf2 and H19 genes, located at the distal end of mouse chromosome 7. The H19 ICR, located in the 5' flank of the H19 gene represses paternal H19 and maternal Igf2 expression, respectively, but has no effect on Kcnq1 expression, which is controlled by another ICR located at the intron 10 of the Kcnq1 gene. This thesis demonstrates that the maternal H19 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 H19 ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the H19 ICR and CTCF. The Kcnq1 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 Kcnq1 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. 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.

Ly49 receptors are the canonical natural killer cell class-I major histocompatibility complex receptors expressed in mice. They have a well-defined role in natural killer cell self/non-self discrimination and in the developmental licensing of functional natural killer cells. In this thesis, I report two novel aspects of Ly49 receptor biology. First, I show that their expression may be regulated by specific nucleosome occupancy on AML-1 binding sites within the distal Ly49 promoter. This finding sheds light on a potential regulatory pathway that has thus far been unexplored in studies of the Ly49 receptor family, and highlights the Ly49 family as an ideal model system in which to study the impact of nucleosome occupancy in general. Second, I show that Ly49 receptors have a central and indispensable role in the emerging phenomenon known as adaptive natural killer cell memory. Natural killer cells have recently been observed displaying adaptive, long-lived, antigen specific memory responses comparable to T cell memory responses, but no explanatory mechanism has been discovered to describe how adaptive memory is possible in these ‘innate’ immune cells. Using Ly49-deficient mice, I show that the inhibitory, self-specific Ly49 receptors Ly49C and Ly49I are required for adaptive memory responses to chemical haptens or protein antigens. Moreover, I show that Ly49C/I binding capabilities are required during all stages of the memory response, as is antigen presentation in the context of class I major histocompatibility complex, again analogous to T cell memory responses. I present initial findings implicating these Ly49 receptors as key components of the antigen recognition process itself, and propose a mechanism based in evolutionarily ancient immunology to explain how this specificity could arise. Finally, I demonstrate that Ly49-dependent natural killer cell memory is capable of mediating powerful anti-cancer vaccination effects using an aggressive model of melanoma. Together, these findings in Ly49 family expression regulation and its functional role in adaptive NK cell responses open several new avenues of study in Ly49 receptor biology and natural killer cell immunology.

Nucleosomes, the primary unit of chromatin structure, are positioned either statistically or specifically. The statistical positioning denotes the arbitrary positioning of nucleosomes on DNA agreeing with the nucleosome’s broad coverage of the genome—however, there is evidence that nucleosomes are also positioned specifically at controlled positions. DNA sequences determine the specific nucleosome positions, and the presence or depletion of nucleosomes affects the availability of the DNA region to other proteins. The DNA sequences of H2A and H2A.Z nucleosomes in Drosophila were analysed in search of nucleosome positioning patterns. Dinucleotide patterns with 10 bp periodicity were identified from the DNA sequences of H2A nucleosomes. Compared with the yeast patterns, the Drosophila patterns had the same periodicity but different dinucleotides near the dyad, which was related to the different H3 structure between them. The nucleosome positioning patterns from the H2A.Z nucleosomes implied the specific histone-DNA interaction as a result of the deviations of the patterns where the different amino acids of H2A and H2A.Z interact with the DNA. The Ly49 gene cluster was selected as a model system to study the interplay between nucleosomes and transcription factors. Ly49 proteins, the surface receptors on NK cells, display variegated expression patterns, and the bidirectional promoter Pro-1 is known as a key determinant of the stochastic expression of each Ly49 gene. The systematic analysis of nucleosome positions based on the genome sequences in the Ly49 gene cluster revealed that the repressing Pro-1 reverse promoters are open, while the activating forward Pro-1 promoters were covered by nucleosomes. Furthermore, specific nucleosome positions covered transcription factor binding sites. The covered factor binding sites were further examined by their periodic appearances on the nucleosome-covered sequences, which revealed the accessibility to the sites. The sequence analysis predicted that the regulation by the transcription factor AML-1 would be sensitive to the nucleosome coverage; the prediction was confirmed by cell line experiments. The 10 bp periodic nucleosome positioning patterns interact with histones specifically. The long nucleosome positioning patterns coexist with the short sequence motifs for transcription factor binding sites adding another layer of the control to the transcriptional regulation.

Le positionnement des nucléosomes le long des génomes eucaryotes est crucial étant donné qu’il affecte l’accessibilité de l’ADN à des protéines impliquées dans la transcription, la réplication, ou encore la réparation de l’ADN. Si il est aujourd’hui admis que les remodeleurs de chromatine ainsi que les préférences des nucléosomes pour certains motifs d’ADN constituent les deux principaux déterminants du positionnement des nucléosomes in vivo, leur importance relative fait encore l’objet de controverses. Dans le cadre de cette problématique nous avons développé une stratégie d’assemblage de répétitions de la séquence 601 positionnante de nucléosome directement dans le génome de Saccharomyces cerevisiae. Cette technique assistée par la technologie CRISPR/Cas9 et des oligonucléotides chevauchants s’est révélée très efficace et a permis d’assembler des répétitions sur une étendue d’environ 15 kilobases. Nous avons ainsi pu isoler trois souches se caractérisant par trois longueurs d’ADN de liaison de respectivement 20, 50 et 90 paires de bases séparant deux 601 consécutifs tout le long des répétitions. Ces longueurs d’ADN de liaison ont été choisies du fait de leur compatibilité avec les modèles de la fibre de 30 nm étudiés in vitro et parce qu’elles sont fréquemment observées chez les eucaryotes. Nous avons ensuite regardé si ces répétitions de la séquence 601 suffisent à dicter la succession des nucléosomes de S. cerevisiae selon le pas de chromatine attendu. Pour cela, nous avons eu recours à une approche de MNase-seq afin d’analyser les positions des dyades des nucléosomes dans les répétitions. Les résultats de ces analyses révèlent de façon intéressante l’incapacité de la séquence 601 à positionner le nucléosome dans ce contexte cellulaire et cela malgré l’étendue de la région de 601 répétés constituée. Nous avons également analysé le positionnement des nucléosomes chez ces trois mêmes souches suite à l’inactivation de Chd1, l’un des deux principaux architectes du paysage nucléosomal chez la levure, afin de s’affranchir de son potentiel effet sur le positionnement des nucléosomes dans la région 601. Nos résultats montrent que l’absence de Chd1 ne permet pas de rétablir un positionnement des nucléosomes sur les monomères de 601, suggérant que le 601 n’est pas positionnant in vivo ou que la région répétée est sous l’influence d’autres facteurs de remodelage. D’un point de vue méthodologique, notre technique de construction de répétitions in vivo permet d’envisager des approches simplifiées de biologie synthétique pour la construction de librairies de répétitions dans le génome de S. cerevisiae. / Nucleosome positioning along eukaryotic genomes is crucial as it influences DNA accessibility for DNA binding proteins involved in DNA replication, transcription and repair. It is now accepted that both nucleosome preferences for some DNA sequences and remodeling factors play an important role in nucleosome positioning in vivo. However their relative importance remains a matter of debate. To investigate the role played by DNA sequence in nucleosome positioning in a cellular context we developped a strategy to assemble tandem DNA repeats of a nucleosome positioning sequence directly into Saccharomyces cerevisiae’s genome. This method is assisted by CRISPR/Cas9 and overlapping oligonucleotides and it turned out to be very efficient as it allowed to synthetize about 15 kilobases of tandem DNA repeats inside a yeast chromosome. Using this apporoach we obtained three yeast strains differing by the DNA linker length separating two consecutive monomeres of the 601 nucleosome positioning sequence. We chose three lengths of linker (20, 50 and 90 pb) for two reasons. First, they are compatible with the formation of a 30 nm chromatin fiber in vitro, and second, nucleosome repeat length of 167, 197 and 237 pb are found in eukaryotic genomes. We then verified if nucleosomes are effectively positioned according to the theoretic DNA linker lengths we designed in the “601” repeated region. To that goal we performed MNase-seq analysis to deduce nucleosomes dyads positions in the repeats. Interestingly our results show that the 601 sequence is not able to dictate strong nucleosomes positioning differing from the natural nucleosome repeat length of about 165 pb along the repeats in an in vivo context. We further investigated positions of dyads in the same three strains after inactivating the gene coding for the chromatin remodeler Chd1, which could potentially be responsible of the nucleosomes organization in the repeated area. Our results show no effect of Chd1, indicating that the “601” sequence has no positionning effect in vivo or that other trans-acting factors are implicated in nucleosome positioning in the engineered repeats. Finally, this work provides a new fast and simple approach for synthetic DNA repeats construction inside the yeast genome and could easily be applied for other synthetic chromatin engineering approaches.

Gene activity is regulated at two separate layers. Through structural and chemical properties of DNA – the primary layer of encoding – local signatures may enable, or disable, the binding of proteins or complexes of them with regulatory potential to the DNA. At a higher level – the structural layer of encoding – gene activity is regulated through the properties of higher order DNA structure, chromatin, and chromosome organization. Cells with abnormal chromosome compaction or organization, e.g. cancer cells, may thus have perturbed regulatory activities resulting in abnormal gene activity. Hence, there is a great need to decode the transcriptional regulation encoded in both layers to further our understanding of the factors that control activity and life of a cell and, ultimately, an organism. Modern genome-wide studies with those aims rely on data-intense experiments requiring sophisticated computational and statistical methods for data handling and analyses. This thesis describes recent advances of analyzing experimental data from quantitative biological studies to decipher the structural layer of encoding in human cells. Adopting an integrative approach when possible, combining multiple sources of data, allowed us to study the influences of chromatin (Papers I and II) and chromosomal aberrations (Paper IV) on transcription. Combining chromatin data with chromosomal aberration data allowed us to identify putative driver oncogenes and tumor-suppressor genes in cancer (Paper IV). Bayesian approaches enabling the incorporation of background information in the models and the adaptability of such models to data have been very useful. Their usages yielded accurate and narrow detection of chromosomal breakpoints in cancer (Papers III and IV) and reliable positioning of nucleosomes and their dynamics during transcriptional regulation at functionally relevant regulatory elements (Paper II). Using massively parallel sequencing data, we explored the chromatin landscapes of human cells (Papers I and II) and concluded that there is a preferential and evolutionary conserved positioning at internal exons nearly unaffected by the transcriptional level. We also observed a strong association between certain histone modifications and the inclusion or exclusion of an exon in the mature gene transcript, suggesting a functional role in splicing.

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

Eukaryotní DNA se váže kolem nukleozomů, čím ovplyvnuje vyšši strukturu DNA a přístup k vazebním mistům pro všeobecní transkripční faktory a oblasti genů. Je proto důležité vědet, kde se nukleozomy vážou na DNA, a jak silná tato vazba je, abychom mohli porozumět mechanizmům regulace genů. V rámci projektu byla implementována nová metoda pro predikci nukleozomů založená na rozšíření Skrytých Markovových modelů, kde jako trénovací a testovací sada posloužila publikována data z Brogaard et al. (Brogaard K, Wang J-P, Widom, J. Nature 486(7404), 496-501 (2012). doi:10.1038/nature11142). Správne predikováno bylo zhruba 50% nukleozomů, co je porovnatenlný výsledek s existujícimi metodami. Okrem toho byla provedena řada experimentů popisující vlastnosti sekvencí nukleozomů a ich organizace.