Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

DNA damage repair is an essential and complex cellular process. Although the basic mechanisms of nucleotide excision repair (NER) have been studied for decades, some mechanistic details remain elusive. The lesion detection step remains one of the most elusive in the process of NER in the contest of chromatin. The work described herein addresses the initial events in the lesion detection step of chromatin repair, also referred to as global genome repair (GG-NER). Both the role of post-translational modifications of lesion identification proteins, and the initial sequence of events in recruitment of repair and remodeling factors are investigated. First, the controversial role of ubiquitination of DDB2 (a human lesion detection protein) is investigated. Due to documented DDB2 function in alternative physiological processes, its direct role in GG-NER is hard to study in human cells. To overcome this obstacle, we established the budding yeast, Saccharomyces cerevisiae as an alternative, simplified model organism to study DDB2-mediated GG-NER. Using this system, we show that inconsistent with the widely accepted model, rapid degradation of DDB2 post-UV irradiation is not an absolute requirement for progression of GG-NER. However, interestingly, our data suggest a role for ubiquitination in the release of DDB2 from chromatin. In both UV and mock treated samples, ubiquitin deficient cells had significantly higher amounts of DDB2 remaining bound to the chromatin compared to the isogenic parent cells. The discussion focuses on the possible physiological relevance of these observations. Additionally, the recruitment of the SWI/SNF chromatin remodeling complex to the silent HML (Hidden MAT Left) locus was also investigated. SWI/SNF is known to require recruitment for its role in transcription; therefore we investigate this requirement in GG-NER. Based on previously published data that indicate an UV-stimulated association of SWI/SNF and Rad4 (a lesion detection protein), we hypothesized that Rad4 is involved in recruitment of SWI/SNF to damaged DNA. Interestingly, our data suggest that Rad4 is not an absolute requirement for recruitment of Snf6 to the HML locus following UV irradiation. However, Rad16 appears to be. These data present an interesting insight into the lesion detection step in GG-NER and this will be discussed.

Chromatin insulators play a role in gene transcription regulation by defining chromatinboundaries. Genome-wide studies in Drosophila have shown that a large proportion of insulator sites are found in intergenic DNA sequences, supporting a role for these elements as boundaries. However, approximately 40% of insulator sites are also found in intragenic sequences, where they can potentially perform as yet unidentified functions. Here we show that multiple Su(Hw) insulator sites map within the 110 kb sequence of the muscleblind gene (mbl), which also forms a highly condensed chromatin structure in polytene chromosomes. Chromosome Conformation Capture assays indicate that Su(Hw) insulators mediate the organization of higher-order chromatin structures at the mbl locus, resulting in a barrier for the progression of RNA polymeraseII (PolII ), and producing a repressive effect on basal and active transcription. The interference of intragenic insulators in PolII progression suggests a role for insulators in the elongation process. Supporting this interpretation, we found that mutations in su(Hw) and mod(mdg4) also result in changes in the relative abundance of the mblD isoform, by promoting early transcription termination. These results provide experimental evidence for a new role ofintragenic Su(Hw) insulators in higher-order chromatin organization, repression of transcription, and RNA processing.

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. In this study we demonstrate that mutation of Chd2 renders cells susceptible to inefficient DNA repair and genomic instability. Homozygous and heterozygous Chd2 mutant mouse embryonic fibroblast accumulates higher levels of gamma-H2AX after DNA damage. Chd2 mutant cells show inefficiency in DNA repair of DNA lesions induced by X-rays and UV irradiation as assessed by single cell gel electrophoresis assays. These cells also exhibit increased chromosomal aberrations after treatment with low doses of X-ray irradiation (2 Gy) and show increased radiosensitivity in a clonogenic survival assay. At the molecular level, endogenous CHD2 protein level is induced after exposure to X-ray radiation. In addition, we have also demonstrated in this study that CHD2 is phosphorylated after DNA damage and is a potential substrate for phosphoinositide 3-kinase-related kinases (PIKK) - ATM/ATR. Additionally, mass spectrometric analysis showed possible association of CHD2 with the paraspeckle family of proteins known to be involved in an array of cellular processes specifically in RNA processing and DNA repair. An in vivo splicing assay demonstrated that CHD2 played a role in modulation of pre-mRNA splicing event. Collectively, our findings suggest that CHD2 is a multi-functional protein working with the paraspeckle protein complex to facilitate both the pre-mRNA splicing process and the initial DNA repair process. CHD2 may also be involved in the later stages of DNA damage response pathway by influencing p53’s transcriptional activity.

An overall coordination between the expressions of genes is required for the proper development of an individual. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of H19, known as the imprinting control region (ICR). This region shows differential methylation, with hypermethylation specifically on the paternal allele. On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF with the region. This association was investigated in both an in vitro episomal system and in an in vivo mouse model system by mutating the CTCF target sites at the H19 ICR. The importance of CTCF for the insulator property of the region was confirmed in both instances. In the mouse model, the disruption of the binding was also observed to affect the methylation profile of the ICR, which ultimately resulted in the de-repression of the maternal Igf2 allele. The relevance of multiple CTCF target sites in higher vertebrates for the proper insulator function was investigated using another knock-in mouse model with mutation at a single CTCF target site in the H19 ICR. The investigation confirmed the cooperation between the target sites for the establishment of a functional insulator on the maternal allele. Target sites in the ICR were also analysed for their differential binding affinity for the CTCF protein. The utilisation of the CTCF target sites was examined in different human tumours and cell lines. Methylation analysis conveyed a lack of correlation between the loss of insulator function and methylation status of the ICR with the loss of imprinting (LOI) of IGF2. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the H19 ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of IGF2 imprinting observed in some instances. In conclusion, this thesis confirms the importance of CTCF in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of H19 and Igf2. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of IGF2 imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.

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.

The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.

The genetic information encoded by the DNA sequence, can be expressed in different ways. Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of imprinted genes in a parent of origin-dependent manner. Imprinted genes are frequently found in clusters and can share common regulatory elements. Most of the imprinted genes are regulated by Imprinting Control Regions (ICRs). H19/Igf2 region is a well known imprinted cluster, which is regulated by insulator function of ICR located upstream of the H19 gene. It has been proposed that the epigenetic control of the insulator function of H19 ICR involves organization of higher order chromatin interactions. In this study we have investigated the role of post-translational modification in regulating insulator protein CTCF (CCCTC-binding factor). The results indicated novel links between poly(ADP-ribosyl)ation and CTCF, which are essential for regulating insulators function. We also studied the higher order chromatin conformation of Igf2/H19 region. The results indicated there are different chromatin structures on the parental alleles. We identified CTCF-dependent loop on the maternal allele which is different from the paternal chromatin and is essential for proper imprinting of Igf2 and H19 genes. The interaction of H19 ICR with Differentially Methylated Regions (DMRs) of Igf2 in a parent-specific manner maintains differential epigenetic marks on maternal and paternal alleles. The results indicate that CTCF occupies specific sites on highly condensed mitotic chromosomes. CTCF-dependent long-range key interaction on the maternal allele is maintained during mitosis, suggesting the possible epigenetic memory of dividing cells. In this study, we developed a new method called Circular Chromosome Conformation Capture (4C) to screen genome-wide interactions with H19 ICR. The results indicated there are wide intra- and inter-chromosomal interactions which are mostly dependent on CTCF-binding site at H19 ICR. These observations suggest new aspects of epigenetic regulation of the H19/Igf2 imprinted region and higher order chromatin structure.

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