Zugl.: Göttingen, Univ., Diss., 2009

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 16, 2010). Includes bibliographical references. Also issued in print.

Thesis (M.S.)--West Virginia University, 1998. / Title from document title page. "December, 1998." Document formatted into pages; contains v, 40 p. Vita. Includes abstract. Includes bibliographical references (p. 32-39).

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 93-103). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

In most eukaryotes methylation of histone H3 on lysine 9 (H3K9me) is the key post-translational modification required for the assembly of constitutive heterochromatin at centromeres and other chromosomal regions. H3K9me is bound by the chromodomain proteins HP1/Swi6 and the Suv39/Clr4 H3K9 methyltransferase itself suggesting that, once established, H3K9me might act as an epigenetic mark that can transmit the chromatin state independently of the initiator signal. However, it has not been demonstrated that H3K9me does indeed act as an epigenetic mark. Fission yeast represents an excellent system to address this question since S. pombe lacks DNA methylation and H3K9me is catalysed by the unique, non-essential H3K9 methyltransferase Clr4. To determine whether H3K9me carries epigenetic properties it is important to uncouple H3K9me from genomic domains that have the intrinsic ability to recruit the heterochromatin machinery. One way to solve this problem is to isolate H3K9me from its original context and investigate whether at an ectopic site H3K9me can self-propagate through cell division. To accomplish this, we tethered regulatable TetR-Clr4 fusion protein at euchromatic loci in fission yeast. This resulted in the assembly of an extensive domain of H3K9me-dependent heterochromatin that is rapidly disassembled following TetR-Clr4 release. Strikingly, the inactivation of Epe1, a putative histone demethylase, is sufficient to maintain the silent H3K9me-dependent heterochromatin at the tethering sites through mitotic and meiotic cell divisions in absence of TetR-Clr4. These results indicate that H3K9me acts as an epigenetic mark to maintain heterochromatin domains; however, a regulatory mechanism dependent on Epe1 exists to actively remove H3K9me and thus prevent heterochromatin from being transmitted when assembled at inappropriate regions of the genome.

In eukaryotic cells, DNA wraps around histones to form nucleosomes, which are the basic units of chromatin. Chromatin is classified as active euchromatin or repressive heterochromatin, depending on the modifications on histones and DNA. Heterochromatin, which is defined by the presence of histone modifications such as H3K9 methylation, serves important functions in cells such as silencing transposable elements, preventing aberrant recombination, and regulating gene expression.The fission yeast, which shares basic chromatin modification pathways with higher eukaryotes, is a premier model system for study heterochromatin formation. One important heterochromatin regulator is the JmjC-domain protein Epe1. It contains a conserved JmjC domain, which is commonly found in active demethylases. Despite that no in vitro demethylase activity has been demonstrated, Epe1 has been regarded as an H3K9 demethylase based on genetic evidence. However, the mechanism of its regulation is unclear at the beginning of my studies. In this thesis, I investigated the regulation of Epe1 through an unbiased genetic screen to identify factors important for Epe1 functions. From the screen, I identified multiple subunits within a transcriptional coactivator SAGA complex. I determined that Epe1 physically recruits SAGA to heterochromatin to promote histone acetylation and transcription, which provides a mechanism for a long-standing paradox regarding heterochromatin at repetitive DNA elements: heterochromatin normally represses transcription but the formation of heterochromatin requires transcription of the repeats. While past results suggest a role of Epe1 in promoting transcription of repeats, our results demonstrate how Epe1 promotes transcription. From this screen, I also identified multiple genes in the cAMP signaling pathway that are important for Epe1 function. I demonstrated that the cAMP signaling pathway regulates Epe1 protein levels post-transcriptionally, and this effect was also seen in cells experiencing glucose starvation, which dampens the cAMP signaling. This study uncovers another layer of control of Epe1 and provides a critical link between nutrient conditions and heterochromatin regulation. Altogether, my studies identified both a mechanism by which Epe1 promotes transcription within heterochromatin and a layer of Epe1 regulation by the glucose-sensing cAMP signaling pathway. These results will help future studies on Epe1 functions and how it is involved in epigenetic adaptation to changing nutrient conditions.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The Anopheles gambiae complex is comprised of the most important vectors of malaria in Sub-Saharan Africa. Most current control methods involve the use of chemicals that help to limit potential contact with these mosquitoes. However, these control methods still have risks that include insect resistance, environmental toxicity, human health, as well as animal health. In order to develop new strategies that either produce novel targeted insecticides or transgenic mosquitoes that can replace current mosquito populations, it is important to acquire as much biological information about the vector as possible. The reduction in cost and speed of high-throughput sequencing has brought forth many new sequenced genomes that can provide a wealth of information about individual populations as well as their respective evolutionary histories. However, in order to fully understand a genome, these sequences must be assembled properly. One of the largest challenges toward fully assembling a genome is the abundance of repetitive sequences. These sequences, typically part of gene poor regions known as heterochromatin, are generally left as unassembled scaffolds that are neglected in many genomic studies. Heterochromatin is a biologically important chromatin state that has roles in gene regulation and genome stability. Exclusion of these chromatin domains from experimental assays can provide an incomplete picture in regards to organismal biology. A lack of information regarding heterochromatin, even in An. gambiae, necessitates further understanding and characterization of this chromatin type that can provide valuable information about the mosquito's biology. Heterochromatin is organized differently amongst different species. Some species with compact genomes, like Drosophila melanogaster, exhibit rigid organization of heterochromatin, with repetitive elements being confined to peri-centromeric and sub-telomeric regions of the chromosome. Larger genomes such as Aedes aegypti, have a much less structured heterochromatin pattern, with repetitive elements being dispersed across the genome. However, An. gambiae's genome is more intermediate in size as well as transposable element content. These factors may have an impact in controlling how heterochromatin is organized within the An. gambiae genome. Does An. gambiae compensate for the increased genome size by expanding past the peri-centromeric heterochromatin into new intercalary compartments? In An. gambiae, heterochromatin had yet to be identified separately from euchromatin. Morphologically, some regions of An. gambiae chromosomes exhibited characteristics similar to transcriptionally active puffs or peri-centromeric heterochromatin. We characterize these regions, as well as the rest of the genomic landscape, by using morphological and genetic features to identify various chromatin types. Peri-centromeric heterochromatin and new regions of intercalary heterochromatin were identified. Genomic coordinates representing the transition from euchromatin to heterochromatin were also identified. By finding these heterochromatin-euchromatin boundaries, various genetic features could be assigned to either heterochromatin or euchromatin. Critical genes associated with heterochromatin formation and basic genomic functions were identified. These data help to better understand features that are associated with the different environments created by chromatin compaction. This study also looks at the Piwi-interacting RNA (piRNA) pathway and its role in An. gambiae. The piRNA pathway is associated with transposable element (TE) suppression in many species, where clusters of vestigial TEs provide some of the RNA necessary for the pathway to function. These clusters are primarily associated with heterochromatin in Drosophila melanogaster. We identify piRNA clusters in An. gambiae and see a similar shift from primarily peri-centromeric compartmentalization toward the presence of intercalary regions located within the euchromatin. Transposable elements are maintained in secondary heterochromatin regions that exhibit similar morphology and features to peri-centromeric heterochromatin. The piRNA pathway also has implications in gene regulation, germline development, and anti-viral immunity. Three candidate genes associated with spermatogenesis and embryogenesis have been identified. These genes showed piRNA enrichment, and upon further analysis show up-regulation after a blood meal is taken. These genes could potentially prove useful in vector control as targets of transgenic experiments. Heterochromatin is an important, yet neglected aspect of the genome. These studies attempt to provide data to stimulate the study of heterochromatin through characterization of heterochromatin-related genomic features. / Ph. D.

Li, Chi Han Samson. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 158-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...