Non-coding antisense RNAs have been implicated in the epigenetic silencing of individual gene as well as chromosomal domains. While silencing of the overlapping gene by antisense RNAs has been well investigated, their functional role in silencing of chromosomal domains remains enigmatic. To elucidate mechanisms underlying the non-coding RNA mediated epigenetic silencing of chromosomal domains, we have chosen an antisense non-coding RNA, Kcnq1ot1, as a model system. Previously, a functional role of Kcnq1ot1 RNA and/or its transcriptional process has been implicated in silencing of multiple genes in the Kcnq1 imprinted cluster. However, these studies could not rule out the mechanisms involving other than Kcnq1ot1 RNA. Furthermore, it was also unclear how the Kcnq1ot1 promoter escapes silencing when its encoded RNA is capable of silencing flanking genes in cis. We have shown that NF-Y transcription factor plays a central role in the Kcnq1ot1 promoter activity, and that mutation of the NF-Y binding sites not only resulted in loss of silencing of flanking genes but also the ability of the Kcnq1ot1 promoter to protect against repressive chromatin marks, indicating that NF-Y maintains transcription-competent chromatin at the promoter through resisting the strong silencing effects of Kcnq1ot1 RNA. The Kcnq1ot1 RNA is an RNA Polymerase II encoded 91 kb long moderately stable nuclear transcript. We have demonstrated that it is the RNA not the act of transcription responsible for silencing and that the degree of silencing was proportional to the length of Kcnq1ot1 RNA. The kinetics of heterochromatin formation in relation to Kcnq1ot1 transcription revealed that overlapping gene was silenced initially by occlusion of basal transcription machinery and heterochromatin formation, whereas nonoverlapping gene was silenced subsequently by Kcnq1ot1-mediated heterochromatin spreading. This transcriptional silencing by Kcnq1ot1 RNA is mediated by an 890 bp region through promoting its interaction with the chromatin. Interestingly, we show that Kcnq1ot1 RNA establishes heterochromatin structures in a lineage-specific fashion by interacting with chromatin and chromatin remodelling complexes such as G9a and PRC2 complexes. More importantly, one of the parental chromosomes comprising Kcnq1 domain always found in the vicinity of perinucleolar region. Based on these data we proposed a mechanism whereby Kcnq1ot1 RNA establishes transcriptional silencing through recruitment of chromatin remodelling machinery and the maintenance of silencing achieved via targeting to the perinucleolar region.

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

In mammals, the most frequently observed hybrid dysgenesis effects are growth disturbances and male sterility. Profound defects in placental development have been described and our work on hybrids in genus Mus has demonstrated putative hybrid dysgenesis effects that lead to defects in lipid homeostasis and maternal behavior. Interestingly, mammalian interspecies hybrids exhibit strong parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit reciprocal phenotypes. Recent studies have provided strong link between epigenetic regulation and growth, behavior and placental development. Widespread disruption of genomic imprinting has been described in hybrids between closely related species of the genus Peromyscus. The studies presented in this thesis aim to investigate the effects of disrupted epigenetics states on altered growth, female infanticide and placental dysplasia observed in Mus hybrids. We showed that loss-of-imprinting (LOI) of a paternally expressed gene, Peg1, was correlated with increased body weight of F1 hybrids. Furthermore, we investigated whether LOI of Peg1 in F1 females would interfere with maternal behavior. A subset of F1 females indeed exhibited highly abnormal maternal behavior in that they rapidly attacked and killed the pups. By microarray hybridization, a large number of differentially expressed genes in the infanticidal females as compared to normally behaving females were identified. In addtion to Peg1 LOI, we studied allelic expression of numerous imprinted genes in adult Mus interspecies hybrids. In contrast to the study from Peromyscus, patterns of LOI were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Finally, we investigated the allelic interaction between an X-linked locus and a paternally expressed gene, Peg3, in placental defects in Mus hybrids. This study further strengthened the notion that divergent genetic and epigenetic mechanisms may be involved in hybrid dysgenesis in diverse groups of mammals.

Methylation of CpG islands located at the promoter regions is a mechanism which controls gene silencing and expression. Hyper or hypo methylation of these sites on promoter sequences have been associated with many diseases, like cancer. Even though promoter CpG islands and their methylation profiles are important regulators of gene expression, the exact mechanism of gene silencing through methylation is not known. Here, we have investigated the status of promoter CpG methylation under various evolutionary pressures by calculating the differences in promoter CpG content and methylation profiles at different pass points. In order to determine the list of genes under each category we have analyzed and compared the orthologs among 58 genomes available through ENSEMBL. The total number of CpG dinucleotides at the promoter regions of all groups of genes have been calculated and compared. Additionally, we have compared the experimentally determined methylation profiles of these CpG&#039 / s between human blood cells and fibroblast cells. While the promoter CpG content changed through common to newer genes, the number of the CpG units methylated found to be consistent. Here, we present the functional level analysis of common gene lists at different pass points and report the differences of the promoter CpG content and the methylation profiles among these groups with distinct evolutionary conservation status. We have also observed the conservation status of individual methylated CpG units on the low and high methylated genes. Our analysis revealed that the surrounding methylation content had a positive effect on the conservation of individual CpG&rsquo / s.

Genomic imprinting is an epigenetic phenomenon that causes a subset of mammalian genes to be expressed from only one allele in a parent-of-origin manner. The defects in the imprinting regulation result in disorders that affect development, growth and metabolism. We have used the Kcnq1 imprinted cluster as a model to understand the mechanism of imprinted gene regulation. The imprinting at the Kcnq1 locus is regulated by a long noncoding RNA, Kcnq1ot1, whose transcription on the paternal chromosome is associated with the silencing of at least eight neighboring genes. By destabilizing Kcnq1ot1 in an episomal system, we have conclusively shown that it is the RNA and not the process of transcription that is required for the gene silencing in cis. Kcnq1ot1 RNA interacts with the chromatin modifying enzymes such as G9a and Ezh2 and recruits them to imprinted genes to establish repressive chromatin compartment and gene silencing. Using the episomal system, we have identified an 890 bp silencing domain (SD) at the 5’ end of Kcnq1ot1 RNA, which is required for silencing of neighboring reporter genes. The deletion of the SD in the mouse resulted in the relaxation of imprinting of ubiquitously imprinted genes (Cdkn1c, Kcnq1, Slc22a18, and Phlda2) as well as reduced DNA methylation over the somatic DMRs associated with the ubiquitously imprinted genes. Moreover, Kcnq1ot1 RNA interacts with Dnmt1 and recruits to the somatic DMRs and this recruitment was significantly affected in the SD mutant mice. By using a transgenic mouse, we have conditionally deleted Kcnq1ot1 promoter at different developmental stages and demonstrated that Kcnq1ot1 maintains imprinting of the ubiquitously imprinted genes by regulating DNA methylation over the somatic DMRs. Kcnq1ot1 is dispensable for the maintenance of repressive histone marks and the imprinting of placental-specific imprinted genes (Tssc4 and Osbpl5). In conclusion, we have described the mechanisms by which Kcnq1ot1 RNA establishes and maintains expression of multiple imprinted genes in cis.

<p>The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic DNA replication initiates asynchronously from hundreds to tens of thousands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time determined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replication has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress. </p><p>We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone modifications and nucleosome positioning, are an attractive candidate for DNA replication control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The <italic>S. cerevisiae</italic> ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Next, we describe the <italic>D. melanogaster</italic> replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. </p><p>Taken together our data and analyses imply that the chromatin contains sufficient information to direct the DNA replication program.</p> / Dissertation

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

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.

In Saccharomyces cerevisiae, Set1p-mediated deposition of trimethylation on lysine 4 of histone H3 is a histone modification often associated with active transcription. Recently, it was discovered that members of the Jumonji family of proteins have the enzymatic ability to remove methylation on histone lysine residues. Here, I describe the function of the yeast Jumonji protein Jhd2p, the only yeast Jumonji with known demethylase activity towards histone H3 lysine 4 methylation. I find that during the development program of yeast sporulation, Jhd2p is responsible for demethylating lysine 4 on a global scale. Further, ChIP analysis examining lysine 4 methylation levels reveals that genes whose expression is dependent on JHD2 during sporulation are subject to what appears to be Jhd2p-mediated demethylation. Additionally, synthetic dosage lethality screens performed to identify genetic interactors of Jhd2p revealed that Jhd2p is a likely component of mitochondrial retrograde signaling, working alongside the transcription factors Rtg1p/Rtg3p.

In Saccharomyces cerevisiae, Set1p-mediated deposition of trimethylation on lysine 4 of histone H3 is a histone modification often associated with active transcription. Recently, it was discovered that members of the Jumonji family of proteins have the enzymatic ability to remove methylation on histone lysine residues. Here, I describe the function of the yeast Jumonji protein Jhd2p, the only yeast Jumonji with known demethylase activity towards histone H3 lysine 4 methylation. I find that during the development program of yeast sporulation, Jhd2p is responsible for demethylating lysine 4 on a global scale. Further, ChIP analysis examining lysine 4 methylation levels reveals that genes whose expression is dependent on JHD2 during sporulation are subject to what appears to be Jhd2p-mediated demethylation. Additionally, synthetic dosage lethality screens performed to identify genetic interactors of Jhd2p revealed that Jhd2p is a likely component of mitochondrial retrograde signaling, working alongside the transcription factors Rtg1p/Rtg3p.