Epigenetics refers to heritable changes in phenotype without alterations to the genotype. Epigenetic changes involve two main mechanisms: DNA methylation and histone modification. Methylation of DNA at cytosine bases is the best-studied epigenetic process to date. CpG methylation states are thought to be maintained throughout cell divisions. However, loss of DNA methylation or DNA demethylation has been observed in specific stages of mammalian development. Such prominent examples of developmental DNA demethylation processes occur in developing primordial germ cells and in preimplantation embryos. However, little is known about DNA methylation changes of other tissues in mammalian development. Therefore, the first aim of this PhD study was to investigate changing nuclear distributions and levels of DNA methylation during development in order to discover dynamic variations amongst developing mouse tissues. In addition, a transgenic MBD-GFP mouse was employed to visualise DNA methylation in tissues. Several hypothetical mechanisms for the enzymatic removal of 5mC have been proposed. One of the proposed candidates is Tet-mediated successive oxidation of 5mC to generate 5hmC, 5fC and 5caC. 5hmC has therefore been considered as a transient intermediate in an active cytosine demethylation pathway. Nevertheless, some studies suggest that 5hmC may also function as an epigenetic modification in its own right. Thus, the second aim of this study was to address the research question of how and where 5hmC originates during development. In order to be able to identify tissues undergoing dynamic nuclear changes in DNA methylation and hydroxymethylation states during early mouse development, new working protocols for immunodetection of 5mC and 5hmC on tissue cryosections were required. The protocol optimisation for 5mC immunodetection is discussed in greater detail in Chapter 3. It was found that DNA methylation immunostaining of cryosections required heat-mediated DNA denaturation, which was partly compatible with protein immunostaining. Next, Chapter 4 focuses on identifying tissues undergoing dynamic changes in 5mC and 5hmC patterns during development from E9.5 to E14.5 mouse embryonic stages, using optimised immunohistochemistry protocols. These protocols revealed interesting dynamic observations of 5mC and 5hmC in the developing cerebral neocortex, surface ectoderm, liver, red blood cells, diaphragm and heart. These findings suggested that dynamic changes of 5mC and 5hmC during neocortical and compact myocardial development were in good agreement with a model where the formation of 5hmC may correlate with the loss of old 5mC, but the observations were also consistent with an involvement of de novo methylation in the generation of 5hmC. In other developing tissues, including surface ectoderm, liver, red blood cells, diaphragm and cardiac trabeculae, dynamic changes in 5mC and 5hmC levels were in line with a model where the 5hmC may act as a new epigenetic mark that functions independently. The optimised protocol also confirmed DNA demethylation of the germ cells at E12.5. The presence of three Tet family enzymes (Tet1, Tet2, Tet3) and de novo methyltransferase DNMT3A in mouse E12.5 tissues is reported in the second part of Chapter 4. It was found that Tet1, Tet2, Tet3 and Dnmt3a were present at detectable levels in neocortex, liver, diaphragm and heart. Contrastingly, no apparent signals for Tet1, Tet2, Tet3 and Dnmt3a were observed in red blood cells. This result was expected due to the very low levels of 5hmC staining in E12.5 red blood cells. The third aim of the present study was to investigate the existence of crosstalk between various epigenetic mechanisms. Thus, Chapter 5 focuses on exploring the relationship between 5mC and repressive histone marks, H3K9me3 and H3K27me3. Histone methylation dynamics at H3K9 and H3K27 were observed during mouse fetal development in neocortex and heart. The overall distribution patterns of H3K9me3 and H3K27me3 demonstrated strong association with developmental changes in 5mC, suggesting that these three repressive epigenetic marks work in concert to establish a silenced state of heterochromatin. Chapter 6, on the other hand, focuses on visualising DNA methylation in tissues using mouse transgenic tools. It was found that brain, liver, heart and neural tube expressed high levels of GFP. But no apparent developmental dynamics of GFP was observed. In conclusion, this study will contribute scientific understanding of dynamic DNA methylation and nuclear heterochromatin organisation during mammalian development, and its role in the specification and maintenance of cell lineages forming tissues and organs. This knowledge will provide insight into current barriers to cell fate reprogramming, which will be of benefit to cell regenerative biomedical technologies.

Barley (Hordeum vulgare) is an economically important crop species with a large diploid genome. Around a half of the barley genome and a fifth of the genes are constrained within a low-recombining pericentromeric (LR-PC) region. I explored the LR-PC gene component with a genomic investigation of gene expression, diversity and evolution. Chromatin environments were also explored in the LR and high recombining (HR) regions by surveying the genic and genomic distributions of nine histone modifications. Firstly, regions of HR and LR were identified and compared for gene evolution, expression and diversity. LR regions of the barley genome were found to be restrictive for gene evolution and diversity, but not gene expression. I employed a bioinformatics approach to identify ancient gene pairs in barley to determine the long-term effects of residency in those regions upon gene evolution. Gene pair loss in LR regions was found to be elevated relative to the HR regions. Applying the same method to rice and Brachypodium distachyon revealed the same situation, suggesting a universal process in the grasses for loss of gene pairs in LR regions. The chromosomal distributions of transposable elements (TEs) were also explored and examined for correlations with recombination rate. Secondly, I developed a chromatin immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) protocol for the investigation of histone modifications in barley seedlings. A protocol was optimised for the fixation, extraction and sonication of barley chromatin. The protocol was applied using antibodies against 13 different histone modifications. Following DNA library construction and Illumina sequencing, a bioinformatics pipeline was devised to analyse the sequence data. NGS reads were mapped to a custom assembly of the barley cultivar Morex reference genome sequence before peak calling. Genomic and genic locations were determined for the covalently modified histones. Four modifications were discarded from further study on the basis of low peak numbers or unexpected chromosomal locations. The remaining nine modifications were classified into four groups based on chromosomal distributions. Groupings were closely mirrored by peak sharing relationships between the modifications except histone H3 lysine-27 tri-methylation (H3K27me3). In addition, chromatin states representing local chromatin environments were defined in the barley genome using the peak sharing data. Mapping the states onto the genome revealed a striking chromatin structure of the gene-rich chromosome arms. A telomere-proximal region bearing high levels of H3K27me3-containing states was found adjacent to an interior gene-rich region characterised by active chromatin states lacking H3K27me3. The LTR retroelement-rich interior was found to be associated with repressive chromatin states. The histone modification status of TE classes were also probed revealing unexpected differences relating to the genomic and genic distributions of these elements. Finally, a genome browser was created to host the information publicly.

Epigenetic modifications, transcription factor (TF) availability and chromatin conformation influence how a genome is interpreted by the transcriptional machinery responsible for gene expression. Enhancers buried in non-coding regions are associated with significant differences in histone marks between different cell types. In contrast, gene promoters show more uniform modifications across cell types. In this report, enhancer identification is first carried out using an enhancer associated feature in mouse erythroid cells. Taking advantage of public domain ChIP-Seq data sets in mouse embryonic stem cells, an integrative model is then used to assess features in enhancer prediction, and subsequently locate enhancers. Significant associations with multiple TF bound loci, higher expression in the closest genes, and active enhancer marks support functionality and tissue-specificity of these enhancers. Motif enrichment analysis further determines known and novel TFs regulating the target cell type. Furthermore, the features identified can facilitate more accurate enhancer prediction in other cell types.

Epigenetic modifications, transcription factor (TF) availability and chromatin conformation influence how a genome is interpreted by the transcriptional machinery responsible for gene expression. Enhancers buried in non-coding regions are associated with significant differences in histone marks between different cell types. In contrast, gene promoters show more uniform modifications across cell types. In this report, enhancer identification is first carried out using an enhancer associated feature in mouse erythroid cells. Taking advantage of public domain ChIP-Seq data sets in mouse embryonic stem cells, an integrative model is then used to assess features in enhancer prediction, and subsequently locate enhancers. Significant associations with multiple TF bound loci, higher expression in the closest genes, and active enhancer marks support functionality and tissue-specificity of these enhancers. Motif enrichment analysis further determines known and novel TFs regulating the target cell type. Furthermore, the features identified can facilitate more accurate enhancer prediction in other cell types.

There is a genome in a cell, as everyone knows, but there is also an epigenome. The epigenome regulates the transcription of the underlying genome. In the last decade, it was discovered that the epigenome state and its regulation are important for differentiation and development. Correlation studies with aging samples had led to the hypothesis that misregulation of the epigenome causes aging and cancer. Furthermore, diseases were identified which are caused by errors in the epigenome state and its regulation. Identification of erroneous epigenome states and misregulation requires the prior knowledge of the common state. Several studies aim at measuring epigenome states in different organisms and cell types and thus, provide a huge amount of data. In this dissertation, a pipeline is developed to analyze and characterize histone modifications with respect to different cell types. Application of this pipeline is shown for a published data set of mouse consisting of data for H3K4me3, H3K27me3, and H3K9me3 measured in embryonic stem cells, embryonic fibroblasts and neuronal progenitors. Furthermore, methods for the detection of the epigenetic patterns are presented in this dissertation. Therefore, a segmentation method is developed to segment the genome guided by the data sets. Based on this segmentation, the epigenome states as well as epigenetic variation can be studied. Different visualization methods are developed to highlight the epigenetic patterns in the segmentation data. Application of the segmentation AND visualization methods to the mouse data set had resulted in not only colorful squares but also in biological conclusions! It demonstrate the power of the developed methods. Although the studied data set in this dissertation contains only ordinary tissue cells, the methods are not restricted to study the reference epigenome state. Comparison of normal and disease cells as well as comparison with aged cells are possible with all of the methods. Finally, the methods are compared based on the obtained results. It shows that all methods highlight different aspects of the data. Thus, applying all methods to the same data sets, deep insights into the epigenome in murine embryonic stem cells, embryonic fibroblasts and neuronal progenitor cells are gained. For example, it had been found that several mechanisms exist setting H3K4me3 marks. Furthermore, not all mechanisms are found in all cell types. Strong evidence had been found that catalysis of H3K4me3 and H3K27me3 is coupled.

The phaseolin (phas) promoter drives the copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues when a nucleosome is positioned over its three phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis leaves was accomplished by ectopic expression of the transcription factor PvALF (Phaseolus vulgaris ABI3-like factor), and application of abscisic acid (ABA). PvALF belongs to a family of seed-specific transcriptional activators that includes the maize viviparious1 (VP1) and the Arabidopsis abscisic acid-insensitive3 (ABI3) proteins. The major goal of the study is to gain insight to the regulation of seed-specific gene expression in three different aspects. First, since ABI3 (homolog of PvALF) is involved in ABA-mediated expression of several seed-specific protein genes in Arabidopsis, understanding its transcriptional regulation will provide insight to the mechanism by which PvALF expression is controlled. To achieve this, ABI3 promoter deletion analysis using either $-glucuronidase (gus) or green fluorescent protein (gfp) reporter gene fusions have identified various regulatory regions within the ABI3 promoter including two upstream activating sequences and a minimal seed specific expression region. In addition, a 405 bp 5' UTR was shown to play a negative role in ABI3 expression, possibly through post-transcriptional mechanisms. Second, placement of PvALF expression under control of an estradiol-inducible promoter permitted chronological ChIP analysis of changes in histone modifications, notably increased acetylation of H3-K9, as phas chromatin is remodeled (potentiated). A different array of changes (trimethylation of H3-K4) is associated with ABA-mediated activation. In contrast, H3-K14 acetylation decreased upon phas potentiation and increased on activation. Whereas decreases in histone H3 and H4 levels were detected during PvALF-mediated remodeling, slight increases occurred following ABA-mediated activation, suggesting the restoration of histone-phas interactions or the redeposition of histones in the phas chromatin. The observed histone modifications thus provide insight to the factors involved in euchromatinization and activation of a plant gene. Finally, ectopically expressed ABI5 and PvALF renders the activation of phas ABA-independent, suggesting ABI5 acts downstream of ABA during phas activation.

Spt6 is a conserved eukaryotic transcription factor, known to interact with both nucleosomes and RNA polymerase II (RNAPII) to control transcription. We have initiated study of Spt6 in S. pombe in order to identify both novel and conserved roles in regulation of transcription and chromatin. We first constructed and analyzed spt6 mutants by several approaches. As Spt6 is known to be required for histone H3K36 methylation in both Saccharomyces cerevisiae and human cells, we examined the global levels of several histone modifications; we found that in S. pombe, Spt6 is required for both H3K4 and H3K36 trimethylation. We examined the chromatin state at two highly expressed genes, \(act1^+\) and \(pma1^+\), and found that there is a defect in recruitment of the methyltransferases responsible for those marks, Set1 and Set2, respectively. We also observed loss of nucleosomes, as well as a decrease in histone H2B monoubiquitylation. These results suggest that Spt6 plays an important role in chromatin regulation during transcription. We also conducted transcriptional analysis of an spt6 mutant by both microarray and high-throughput sequencing (RNA-seq) and discovered that Spt6 plays a critical role in maintaining the integrity of transcription genome-wide. We found that Spt6 is required to repress antisense transcription, with nearly 70% of genes having antisense transcripts increased by at least two-fold in an spt6 mutant. We also found that transcription of most long terminal repeats (LTRs) is derepressed. Finally, we found that a major class of transcripts elevated in the spt6 mutant is derived from heterochromatin, which is normally silenced. To study the heterochromatic silencing defect in greater detail, we analyzed the chromatin state of the pericentric repeats and found a decrease in H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several known silencing factors and a loss of siRNA production. We also see a very modest increase in RNAPII recruitment. Based on this combination of phenotypes, Spt6 is likely to contribute to both transcriptional and post-transcriptional silencing mechanisms. Taken together, we have found that Spt6 plays several important roles to control transcription in both euchromatin and heterochromatin in S. pombe.

Repo-Man is a chromatin-associated PP1 targeting subunit that coordinates chromosome re-organisation and nuclear envelope reassembly during mitotic exit. At the onset of mitosis, Repo-Man association with the chromosomes is very dynamic; at anaphase, Repo-Man targets to the chromatin in a stable manner and recruits PP1 to de-phosphorylate histone H3 at Thr3, Ser10 and Ser28. Previous studies have suggested that CDK1 and AuroraB are the kinases responsible for the inactivation of the complex and for its dispersal at the onset of mitosis respectively. We have previously shown that the binding of Repo-Man to PP1 is decreased in mitosis and we have identified a region adjacent to the RVTF motif that contains multiple mitotic phosphosites (RepoSLIM). This region is conserved only in another PP1 targeting subunit: Ki-67. In order to understand the importance of this region for the complex formation and stability, we have conducted mutational analyses on several residues, and addressed their contribution towards Repo-Man chromosome targeting and PP1 binding in vivo. We have identified new sites in Repo-Man that, when phosphorylated, contribute to the weakening of the binding between Repo-Man and PP1. Interestingly, our results also indicate that several kinases are involved in the mitotic regulation of the complex. We have also identified Lamin A/C as a Repo-Man substrate and introduced a new model for Lamin A/C regulation at interphase. Furthermore, we identified Repo-Man as a marker of malignancy in tripe egative breast cancer, which controls cell movement and levels of important oncogenic markers Aurora A and C-Myc, and propose Repo-Man/PP1 complex as a therapeutic target for the treatment of triple negative breast cancer through the newly identified RepoSLIM.

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.

DNA methylation refers to an addition of a methyl group to the 5 position of the cytosine pyrimidine ring. As the best characterized epigenetic mark, DNA methylation plays an important role in a plethora of biological functions, including gene repression, genomic imprinting, silencing of retro-transposons and X chromosome inactivation. Genomic imprinting refers to the mono-allelic expression of certain genes according to their parent-of-origin. In mammals, the expression of imprinted genes is controlled by the cis-acting regulatory elements, termed imprinted control regions (ICRs). ICRs are marked by parent-of-origin-specific DNA methylation and loss of DNA methylation at ICRs also causes aberrant expression of imprinted genes. Therefore it is believed that the genomic imprinting is a DNA methylation-associated epigenetic phenomenon. As accurate expression of imprinted genes is essential for normal embryonic growth, energy homeostasis, development of the brain and behaviour and abnormal expression of imprinted genes leads to numerous clinical phenotype and human disorders, it is important to investigate how the imprinted DNA methylation is stably maintained in mammals. DNA methyltransferases (DNMTs) are the main enzymes that play a in the establishment and maintenance of imprinted DNA methylation. In primordial germ cells (PGCs), DNMT3A and DNMT3L are involved in the establishment of imprinted DNA methylation. Whereas once established, the imprinted DNA methylation is maintained by DNMT1, DNMT3A and DNMT3B, but mainly by DNMT1. In addition, some other enzymes and DNA binding proteins also play a role in this process. One of the best examples is ZFP57, which forms a complex with KAP1 and SETDB1. ZFP57 maintains imprinted DNA methylation by recognizing a methylated hexa-nucleotide and recruits DNMTs to the ICRs in mammalian embryonic stem (ES) cells. Interestingly, DNA methylation analysis combined with promoter microarrays carried out in our lab suggested that imprinted DNA methylation is absent from some of the maternal ICRs in ES cells genetically null for G9a, a histone H3 lysine 9 methylase. This indicates that G9a might also play a role in the maintenance of imprinted DNA methylation. In my work, I found that the repressive H3K9me2 and imprinted DNA methylation are absent from several analysed ICRs in embryonic stem (ES) cells genetically null for either G9a or its partner histone methyltransferase GLP. A knockdown of G9a in ES cells reproduced these observations suggesting that G9a/GLP complex is required for the maintenance of imprinted DNA methylation. I also found that neither wild type nor catalytically inactive G9a can restore the loss of imprinted DNA methylation in G9a-/- ES cells. Chromatin immunoprecipitation (ChIP) combined with bisulfite DNA sequencing showed that imprinted DNA methylation was present on the H3K9me2-marked allele indicating a direct role for G9a in maintenance of genomic imprinting. Using a pharmacological inhibitor of G9a and mutagenesis analyses, I found that G9a maintains the imprinted DNA methylation independently of its catalytic activity and recruits DNMTs to the ICRs via its ankyrin repeat domain. Dimerization of G9a with GLP is also essential for the maintenance of genomic imprinting in ES cells. In summary, in addition to establish H3K9me2, histone methyltransferases G9a and GLP also play an essential role in the maintenance of genomic methylation imprints in ES cells.