<p>Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification.</p><p>In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype.</p><p>In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21^WAF1/CIP1 (CDKN1A) and p27^KIP1 (CDKN1B).</p><p>TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.</p>

Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...

Regulation of gene expression is an essential process for all living organisms. Transcriptional regulation, associated with chromatin, is governed by: (1) DNA sequence, which creates regulatory sites (promoters, enhancers and silencers), where sequence motifs and features (e. g. CpG) can attract transcription factors (TFs) and influence chromatin structure or RNA polymerase II (Pol II) binding, initiation and elongation; (2) non-sequence, epigenetic factors - histone modifications, TF binding, chromatin remodelling (histone placement, eviction and reconstitution), and non-coding RNA regulation. These factors interact with each other, creating a complex network of interactions. In this thesis I describe computational studies of heterochromatin factors in regulation of gene and repeat expression, an analysis of active regulatory elements, and global analyses of big datasets in C. elegans. I first show that a team of heterochromatin factors - HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 - collaborates with piRNA and nuclear RNAi pathways to silence repetitive elements and protect the germline. I also found that the TACBGTA motif is particularly enriched on repeats and heterochromatin factors binding sites, and that repeat elements are derepressed in the soma during normal C. elegans ageing. I then describe the work on active regulatory regions. I show that CFP-1/CXXC1 binds CpG dense, nucleosome depleted promoters and, along SET-2, is required for H3K4me3 deposition at these loci. Using expression profiling I determined that the majority of CFP-1 binding targets are not significantly mis-regulated in cfp-1 mutants, but are weakly upregulated in bulk analyses. I also show that CFP-1 functionally interacts with the Sin3S/HDAC complex. In cfp-1 mutant I observed both loss and gain of SIN-3 binding, depending on chromatin context. Finally, I performed a data driven study on a large collection of ChIP-seq profiles using non-parametric sparse factor analyses (NSFA) and compared it to other, unsupervised machine learning algorithms. This study uncovered interactions and structure in genomic datasets. In addition, I present a collection of computational tools and methods I developed to facilitate processing, storage, retrieval, annotation, and analyses of large datasets in genomics.

Maternal age is a significant risk factor for adverse pregnancy outcomes, and is strongly associated with an increased risk of aneuploidy of the conceptus, as well as a significantly higher frequency of serious pregnancy complications known as the "Great Obstetrical Syndromes", including miscarriage, pre-eclampsia and fetal growth restriction. In the last 40 years average maternal age has increased considerably in many wealthy countries, and in the UK the number of babies born to women aged 35 and over is set to surpass those born to women under 25. The high incidence of aneuploidy in older mothers can be attributed to abnormalities in the oocyte and embryo, however the "Great Obstetrical Syndromes" do not appear to be related to the oocyte and may instead be linked to abnormal development of the placenta. In this thesis, I show that advanced maternal age in the mouse is associated with a drastically increased variability of developmental progression in utero, including developmental delays and growth restriction, severe embryonic abnormalities and higher resorption rates. I find that these embryonic defects are always accompanied by gross morphological and transcriptomic abnormalities in the placenta. Notably, I show that the increased risk of these complications can be rescued by transfer of embryos from aged females to a young surrogate mother, thus implicating the aged maternal uterus as the basis for embryonic and placental defects. Transcriptomic analysis of the decidua compartment in placentas from aged pregnancies revealed abnormal expression of genes involved in the decidualization process, which occurs during early pregnancy and facilitates implantation and development of the conceptus. I show that these defects are already obvious in the peri-implantation window, with endometrial stromal cells from aged females being unable to mount an adequate decidualization response due to a decline in their ability to respond to pregnancy hormones. This blunted decidualization reaction in turn may lead to abnormal development of the placenta. These age-associated decidualization defects are cell-intrinsic and can be recapitulated in vitro. The detected insufficient activation levels and abnormal intracellular distribution of phospho-STAT3, combined with highly variable progesterone receptor expression, may be possible causes of these defects. In addition, I examined the possible effects of ageing on the epigenome as a potential contributor to the decline in endometrial function. My results indicate that ageing of the uterus displays some of the common epigenetic hallmarks of tissue ageing. However, more importantly, decidual cells of aged females exhibit abnormal distributions of the histone modification H3K4me3, and are refractory to the profound DNA methylation remodeling that I find takes place during pregnancy. These age-related changes in the epigenome may underpin, or contribute to, the observed decline in uterine function during pregnancy. Understanding the mechanism underlying these epigenomic and functional changes in the ageing reproductive tract may pave the way for new therapeutic strategies to improve maternal and fetal outcomes of pregnancy in older mothers.

Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification. In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype. In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21WAF1/CIP1 (CDKN1A) and p27KIP1 (CDKN1B). TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.

The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

Nearly one half of the human genome is composed of transposable elements (TEs). Once dismissed as 'selfish' or 'junk' DNA, TEs have also been implicated in a numerous functions that serve the needs of their host genome. I have evaluated the role of TEs in mediating the epigenetic mechanisms that serve to regulate human gene expression. These findings can be broadly divided into two major mechanisms by which TEs affect human gene expression; by modulating nucleosome binding in the promoter regions and by recruiting epigenetic histone modifications that enable them to serve as promoters and enhancers. Thus. the studies encompassed in this thesis elucidate the contributions of TEs in epigenetically regulating human gene expression on a global as well as local scale.

No description available.

Epigenetics is an important layer of transcriptional control necessary for cell-type specific gene regulation. We developed computational methods to analyze the combinatorial effect and large-scale organizations of genome-wide distributions of epigenetic marks. Throughout this dissertation, we show that regions containing multiple genes with similar epigenetic patterns are found throughout the genome, suggesting the presence of several chromatin domains. In Chapter 1, we develop a hidden Markov model (HMM) for detecting the types and locations of epigenetic domains from multiple histone modifications. We use this method to analyze a published ChIP-seq dataset of five histone modification marks in mouse embryonic stem cells. We successfully detect domains of consistent epigenetic patterns from ChIP-seq data, providing new insights into the role of epigenetics in longrange gene regulation. In Chapter 2, we expand our model to investigate the genome-wide patterns of histone modifications in multiple human cell lines. We find that chromatin states can be used to accurately classify cell differentiation stage, and that three cancer cell lines can be classified as differentiated cells. We also found that genes whose chromatin states change dynamically in accordance with differentiation stage are not randomly distributed across the genome, but tend to be embedded in multi-gene chromatin domains. Moreover, many specialized gene clusters are associated with stably occupied domains. In the last chapter, we develop a more sophisticated, tiered HMM to include a domain structure in our chromatin annotation. We find that a model with three domains and five sub-states per domain best fits our data. Each state has a unique epigenetic pattern, while still staying true to its domain’s specific functional aspects and expression profiles. The majority of the genome (including most introns and intergenic regions) has low epigenetic signals and is assigned to the same domain. Our model outperforms current chromatin state models due to its increased domain coherency and interpretation.