The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.

The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.

This thesis investigates patterns and mechanisms of gene expression in P. falciparum. The rapidly cycling patterns of genes during the asexual stages confounds the analysis of gene expression in culture and in patients. In order to overcome this problem, we develop statistical models to estimate the temporal progression of malaria parasites by using the observed gene expression values and known reference sets. We extend this framework to account for lineage commitment, and show that, similar to asexual development, it is also possible to recover information about parasite sexual differentiation given observed gene expression values. Using datasets from our own lab as well as those available in the literature, we establish that the patterns of expression in patients are similar to those observed in culture but in additive mixtures whose proportions can vary between patients. We then investigate epigenetic and spatial factors that are important in regulating gene expression. Using second-generation DNA sequencing, we generate genomic maps of chromatin state and chromosome interactions. These maps provide the first global view of chromosome folding and locus-specific affinities in malaria. They also highlight the importance of epigenetic modifications in imposing structure on the spatial organization of the genome. After generating an initial set of genomic maps, we apply these tools to study chromatin reconfigurations during var gene switching. We show that when the active var gene changes, reconfigurations can be seen in the local three-dimensional chromatin structure of the activated locus. Overall, our results contribute new methods for analyzing malaria microarray data, highlight the importance of lineage mixtures in patient infections, generate an atlas of spatial interactions in the nucleus at a resolution of approximately 5 kilobases, and establish a link in malaria between the spatial configuration of active loci and the local chromatin environment.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

No description available.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

Microfluidics, the manipulation of fluids at nanoliter scale, has emerged to offer an ideal platform for biological analysis of a low number of cells. The technological advances in microfluidics have allowed both forming of valves, mixers and pumps and integrating of optic and electronic components into microfluidic devices to construct complete and functional systems. In this dissertation, I present novel microfluidic techniques and their applications in cellular probes delivery, cell separation and epigenetic study. In the first part of the dissertation, electroporation is implemented on microfluidic platform to generate uniform delivery of "exposed" nanoparticle or protein into cells. In contrast to endocytosis, electroporation is a physical method to breach cell membrane and does not involve vesicle encapsulation of delivered probes, which means these probes have exposed surface in the cytosol. Such trait enables the use of delivered nanoparticle and protein for intracellular targeting of native biomolecules. Laser-induced fluorescent microscopy was used for single particle illuminating to track single molecules in cells. Microfluidic device provide integrated platform for conducting electroporation, cell culture and imaging. In the second part, microfluidic immunomagnetic cell separation is introduced. I showed two new approaches to enhance immunomagnetic cell separation based on (1) uniquely microfabricated paramagnetic patterns inside separation channels; and (2) using combination of nonmagnetic beads and magnetic beads for selection of tumor initiating cells based on two markers of opposite preference in one step. Enhancement in cell isolation (high capture efficiency or high selection purity) is experimentally observed and the former is explained by computational model. In the final part of the dissertation, microfluidic device incorporating valves and mixers for sensitive study of chromosome conformation is presented. This device has small reaction chamber minimizing sample requirement, and allows multiple steps of biological analysis in a single chip avoiding sample loss during sample transfer. Several orders of magnitude improved detection sensitivity is achieved with our microfluidics based method. I envision all novel techniques discussed in this dissertation have great potential in application of disease prognosis, diagnosis and treatment. / Ph. D.

L'organisation de la chromatine a une importance fonctionnelle pour contrôler le programme d'expression des gènes. Par contre, les liens qui l'unissent au déroulement de la réplication de l'ADN sont beaucoup moins connus. Grâce à des approches basées sur la capture d'interactions chromosomiques et sur l'imagerie cellulaire, nous avons étudié les liens entre le repliement à grande échelle de la chromatine et le timing de réplication. Cette analyse, effectuée dans des cellules humaines lymphoblastoïdes, des cellules mononucléées du sang (PBMC) et des cellules issues d'une leucémie myéloïde à caractère érythrocytaire, a permis l'identification de domaines structuraux du noyau. Ces domaines sont relativement isolés les uns des autres et leurs frontières coïncident avec les zones d'initiation précoce. De plus, notre étude montre que ces zones d'initiation précoce interagissent préférentiellement, aussi bien entre voisins immédiats (séparation génomique de l'ordre de la mégabase) que le long du chromosome entier. Les loci répliqués tardivement interagissent eux-aussi avec leurs homologues, conduisant, dans l'espace nucléaire, à une ségrégation des loci en fonction de leur timing de réplication. Ces résultats sont soutenus par des mesures de distances sur des hybridations in-situ qui montrent que les loci répliqués en début de phase S sont plus proches qu'attendus. Nos travaux révèlent enfin que l'organisation de la chromatine est similaire dans des cellules en phase G0 (PBMC dormantes), démontrant qu'elle n'est pas spécifique des cellules en phase S. Pris ensemble, ces résultats apportent des preuves directes d'une organisation robuste de la chromatine, partagée par les cellules en cycle et dormantes, et corrélée au timing de réplication à différentes échelles. / Chromatin organization is of functional significance to control the gene expression program. However, its interplay with DNA replication program is less known. Though the capture of chromosomal interactions and cell imaging, we studied the links between the high-order folding of chromatin and the replication timing. This analysis, which has been performed in human lymphoblastoid cells, in peripheral blood mononuclear cells (PBMC), and in a myeloid leukemia cell line with erythroid properties, allowed the identification of structural domains in the nucleus. These domains are quite isolated from each other and their boundaries coincide with early-initiation zones. In addition, our study shows that these early-initiation zones preferentially interact with each other. These interactions have been observed between neighboring early-initiation zones (genomic separation around 1 to few megabases) but also along the whole chromosome. The late-replicated loci interact with their counterparts as well, leading to a nuclear segregation of the loci according to their replication timing. These results are sustained by distance measurements in in-situ hybridizations which show that loci replicated at the beginning of S-phase are closer than expected. Our work also reveals that chromatin organization is similar in cells blocked in G0 phase (quiescent PBMC), demonstrating that it does not result from the cells in S-phase. Taken together, these results provide direct clues for a robust chromatin organization, common to cycling and resting cells, and related to the replication timing at different scales.

La chromatine sert de support à de multiples processus biologiques, cependant son organisation spatiale diffère fortement selon l'échelle considérée. L'expression des gènes est ainsi coordonnée par des éléments régulateurs dispersés dans le génome mais capables d'interagir entre eux. Chez les métazoaires, des expériences de capture de conformation de chromosome (3C) combinées au séquençage haut-débit (Hi-C) ont permis la découverte de domaines d'association topologique (TAD), à l'échelle de la mégabase. Puisque la résolution du Hi-C reste limitée, nous avons utilisé la 3C-qPCR pour explorer, dans des cellules souches embryonnaires murines, la dynamique chromatinienne à l'intérieur de ces domaines ainsi qu'à leurs bordures. Nous identifions ainsi une modulation des fréquences de contacts, sur quelques centaines de kilobases. Cette modulation est plus ou moins importante en fonction du contenu en gènes des domaines, mais elle semble néanmoins universelle. Des modèles dérivés de la physique des polymères permettent de décrire cette modulation sous la forme d'une hélice statistique, que la chromatine adopterait en moyenne et en l'absence d'interactions spécifiques, à l'intérieur des TAD. Cette hélice reflète certaines contraintes que la chromatine subit à l'échelle supranucléosomale. Elle est très affectée par les bordures, qui bloquent la modulation, mais elle l'est beaucoup moins par le contenu en histone de liaison H1. Par ailleurs, grâce à des résultats de Hi-C à haute résolution, nous montrons que la modulation observée chez les souris n'est pas retrouvée chez la drosophile, où les caractéristiques des TAD semblent avant tout liées au paysage épigénétique local. Pour ces deux organismes, la dynamique chromatinienne à l'intérieur des domaines est donc sous le contrôle de phénomènes différents / The chromatin hosts various biological processes. However, its organization differs considerably depending on the scale. For example, gene expression is coordinated by regulatory elements that are dispersed in the genome but that are able to interact within the tridimensional space of the nucleus. In the Metazoa, chromosome conformation capture (3C) assays combined with high-throughput sequencing (Hi-C) uncovered the existence of topologically associating domains (TADs), at the mégabase scale. Due to the limited resolution of Hi-C, we used the 3C-qPCR method to explore, in murine embryonic stem cells, the chromatin dynamics inside TADs as well as at their borders. We found that contact frequencies undergo a periodic modulation over large genomic distances (few hundred kilobases). This modulation is weaker in gene-deserts than in gene-containing domains but it seems nevertheless to be universal. Using models derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt statistically, when no strong locus-specific interaction takes place, within the TADs. This statistical helix reflects some constraints that the chromatin undergoes at the supranucleosomal scale. It is affected by TADs borders, which disrupt the modulation, but linker histone H1 depletion only leads to subtle changes in the helix characteristics. Furthermore, using high-resolution Hi-C data, we found that chromatin dynamics is unconstrained in Drosophila where it seems mainly linked to the local epigenetics landscape. Therefore, distinct genome organization principles govern chromatin dynamics within mouse and Drosophila topologically associating domains.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20812号 / 医科博第83号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 斎藤 通紀, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM