HMGA1 Proteine sind kleine, basische, Nicht-Histon Proteine, die in Lösung keine Struktur aufweisen, durch drei AT-Haken, als DNA-Bindungsmotive, gekennzeichnet sind und präferentiell an die kleine Furche der DNA binden. Als differenziell exprimierte Architekturelemente des Chromatins erfüllen sie wichtige Funktionen bei der Regulation DNA abhängiger Prozesse in Zellen und während Entwicklungsprozessen. Aberrante Expressionen führen zu Entwicklungsdefekten und Krebs. In dieser Arbeit wurde der Einfluss von HMGA1 Proteinen auf die Organisation des Chromatins untersucht. Als Modell diente dabei zunächst die Differenzierung von C2C12 Muskelvorläuferzellen. Wie in einer früheren Arbeit gezeigt wurde, ist die Herunterregulation von HMGA1a essentiell für den Eintritt von C2C12 Zellen in die Myogenese. Eine konstante Überexpression von HMGA1a-eGFP hingegen verhindert die Muskeldifferenzierung durch Beeinflussung der Expression myogenesespezifischer Gene und Etablierung einer stabilen Chromatinstruktur. Wie in der vorliegenden Arbeit herausgefunden wurde, nimmt die differenzielle HMGA1a Expression nicht nur Einfluss auf die Expression muskelspezifischer Gene, sondern auch auf die globale Zusammensetzung des Chromatins durch eine reduzierte Expression von H1 Histonen und einer aberranten Expression von HMGB1, HMGN1 und HP1 Proteinen. HMGA1a wurde zusammen mit ORC Proteinen eine Funktion bei der Definition von Replikationsursprüngen in eukaryotischen Zellen zugesprochen. ORC Proteine wurden auch als Komponenten des Heterochromatins und als Interaktionspartner von HP1α identifiziert. Hier konnte mit Hilfe von Co-Immunpräzipitationen, Pull-down Assays und Verdrängungsexperimenten gezeigt werden, dass HMGA1 ein weiterer, direkter Interaktionspartner von ORC Proteinen im Heterochromatin ist und zusammen mit HP1α kooperiert. Pull-down-, Verdrängungs- und siRNA-Experimente zeigten zudem, dass HMGA1 zwar nicht direkt mit HP1α interagiert, die Kooperation der Proteine über ORC aber dennoch wichtig für die Aufrechterhaltung der Heterochromatinsstruktur ist. Damit erweisen sich HMGA1 Proteine als wichtige Stabilisierungsfaktoren des Heterochromatins. Bislang ging man davon aus, dass HMGA1 Moleküle linear, also eindimensional, an ein DNA Molekül binden. Das Vorhandensein von drei DNA-Bindungsmotiven und die eher struktur- als sequenzabhängige Bindung an die DNA lassen vermuten, dass HMGA1 Proteine auch gleichzeitig an benachbarte DNA-Stränge, also auch dreidimensional, binden könnten. Bekräftigt wurde diese Vermutung durch die Bildung von Chromatinaggregaten in Zellen die HMGA1a-eGFP überexprimierten. Dies wurde mittels konfokaler und hochauflösender Mikroskopie (dSTORM) analysiert. Um das Potential einer DNA-Quervernetzung durch HMGA1 Proteine nachzuweisen, wurde eine neue Methode entwickelt. Mit Hilfe eines neuartigen DNA Cross-linking Assays wurde nachgewiesen, dass HMGA1 Proteine in der Lage sind, zwei individuelle DNA Stränge zu vernetzen. Zudem wurde eine neue Domäne in HMGA1 entdeckt die maßgeblich zum Cross-linking beiträgt. Elektronenmikroskopische Analysen bestätigten, dass HMGA1 Proteine in der Lage sind Kreuzungen und Schleifen in DNA Molekülen zu erzeugen. Diese Ergebnisse unterstützen die Vermutung, dass HMGA1 Proteine im Zellkern ein DNA Gerüst bilden können, das Einfluss auf die zelltypische Chromatinorganisation nimmt und dadurch DNA abhängige Prozesse beeinflusst. In wie weit eine HMGA1 induzierte DNA Quervernetzung in vivo zum Beispiel in Chromozentren von C2C12 Zellen oder in Krebszellen, in denen HMGA1 Proteine stark überexprimiert sind, eine Rolle spielen, müssen künftige Untersuchungen zeigen. In dieser Arbeit konnte also gezeigt werden, dass HMGA1 Proteine die Chromatinstruktur auf drei Ebenen organisieren können: Durch Beeinflussung der Chromatinzusammensetzung durch Veränderung der Expression von Chromatinproteinen, durch Interaktion mit anderen Architekturelementen des Chromatins und durch Organisation eines potentiellen DNA Gerüsts. / HMGA1 proteins are small basic non-histone proteins characterized by three DNA binding domains, the AT-hooks, which bind to the minor groove of DNA. As differentially expressed architectural chromatin proteins, they perform important functions in the regulation of DNA dependent processes and in development. Aberrant expression leads to developmental defects and cancer. In this thesis the influence of HMGA1 proteins on chromatin organization is investigated. Initially C2C12 myogenic precursor cells were studied, which can be differentiated to myotubes. Previously it had been shown that down-regulation of HMGA1 proteins is crucial for the initiation of myogenic differentiation. Constant over-expression of HMGA1a-eGFP prevents myogenic differentiation by influencing the expression of myogenic genes and by the establishment of a stable chromatin structure. Here it was shown that the differential HMGA1 expression does not only influence the expression of myogenic specific genes but also affects total chromatin composition. This was shown by reduced and aberrant expression of chromatin proteins such as histone H1, HMGB1, HMGN1 and HP1 proteins. Recently it was demonstrated that HMGA1 together with ORC proteins function in origin definition in eukaryotic cells. ORC proteins were also identified as components of heterochromatin and direct interaction partners of HP1α. Here, it was shown by co-immunoprecipitation, pull-down assays, siRNA and displacement experiments that HMGA1 proteins can interact with ORC proteins directly and that they can cooperate with HP1α in heterochromatin. It could be shown that HP1α indeed does not directly interact with HMGA1 but together with ORC proteins is relevant for heterochromatin maintenance. Thus HMGA1 proteins turned out to be important stabilizers of heterochromatin. Until recently it was thought that HMGA1 proteins bind DNA collinearly. In principle the three independent DNA binding AT-hooks of HMGA1 also suggest a concomitant binding to neighboring DNA strands, which could lead to a three dimensional stabilization of DNA. This assumption was affirmed by the occurrence of chromatin aggregates in HMGA1a-eGFP overexpressing cells, which was analyzed by confocal and high resolution (dSTORM) microscopy. By using a newly developed DNA cross-linking assay, which allows the analysis of a DNA crosslinking capability of a protein, it was proven that HMGA1 proteins can bind two individual DNA fibers simultaneously. Furthermore a novel domain in HMGA1 proteins was discovered which is significantly involved in the DNA cross-linking. Electron microscopic analyses confirmed that HMGA1 proteins can specifically generate crossings and loops in DNA molecules. These results support the assumption that HMGA1 proteins can create a DNA scaffold that has influence on cell typical chromatin organization and possibly also affects DNA dependent processes. To what extent HMGA1 induced DNA cross-linking plays a role in vivo, for example in the organization of chromocenters of C2C12 cells or in cancer cells, where HMGA1 proteins are over-expressed, will need to be elucidated in further experiments In summary, this work shows, that HMGA1 proteins influence chromatin structure and composition by affecting the expression of chromatin proteins, by interacting with other architectural chromatin proteins or by producing a higher organization of chromatin on its own.

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

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

Epstein-Barr virus (EBV) is a gammaherpesvirus that persistently infects more than 90% of the human population and its latent infection is associated with several malignancies. EBNA1 is the only viral protein required for the persistence of EBV episomes through its contributions to the replication, mitotic segregation of EBV episomes, and transcriptional activation of viral latency genes. These functions require EBNA1 binding to the DNA elements within the EBV genome that are important for transcription and replication, and interactions of EBNA1 with cellular factors. The aim of this thesis is to characterize the functional significance of the interactions between EBNA1 and chromatin-associated proteins NAP1, TAF-I, and Brd4, particularly in EBNA1-mediated transcription and DNA replication. Here I investigated the interactions of EBNA1 with NAP1 or TAF-I that were previously discovered in our laboratory by proteomic methods. I found that EBNA1 binds directly to NAP1 and to the β isoform of TAF-I, resulting in increased solubility of EBNA1 in vitro. Brd4 was also found to bind to EBNA1 and this interaction is mediated by an EBNA1 sequence essential for transactivation. Brd4, NAP1 and TAF-I all localize with EBNA1 to the transcriptional element and alteration of their levels inhibits transcriptional activation, suggesting that these proteins contribute to EBNA1-dependent transactivation through protein complex formation at the transcriptional element. On the other hand, only TAF-I is recruited to the origin of replication by EBNA1 and acts as a repressor for EBNA1-mediated DNA replication. My data suggest that this negative regulation is linked to TAF-I’s ability to interfere with histone methylation and thus to alter chromatin structure at the origin of DNA replication. Finally, I showed that NAP1 and TAF-I are required for expression of lytic protein BZLF1 and that TAF-I associates with the BZLF1 promoter during lytic reactivation.

Epstein-Barr virus (EBV) is a gammaherpesvirus that persistently infects more than 90% of the human population and its latent infection is associated with several malignancies. EBNA1 is the only viral protein required for the persistence of EBV episomes through its contributions to the replication, mitotic segregation of EBV episomes, and transcriptional activation of viral latency genes. These functions require EBNA1 binding to the DNA elements within the EBV genome that are important for transcription and replication, and interactions of EBNA1 with cellular factors. The aim of this thesis is to characterize the functional significance of the interactions between EBNA1 and chromatin-associated proteins NAP1, TAF-I, and Brd4, particularly in EBNA1-mediated transcription and DNA replication. Here I investigated the interactions of EBNA1 with NAP1 or TAF-I that were previously discovered in our laboratory by proteomic methods. I found that EBNA1 binds directly to NAP1 and to the β isoform of TAF-I, resulting in increased solubility of EBNA1 in vitro. Brd4 was also found to bind to EBNA1 and this interaction is mediated by an EBNA1 sequence essential for transactivation. Brd4, NAP1 and TAF-I all localize with EBNA1 to the transcriptional element and alteration of their levels inhibits transcriptional activation, suggesting that these proteins contribute to EBNA1-dependent transactivation through protein complex formation at the transcriptional element. On the other hand, only TAF-I is recruited to the origin of replication by EBNA1 and acts as a repressor for EBNA1-mediated DNA replication. My data suggest that this negative regulation is linked to TAF-I’s ability to interfere with histone methylation and thus to alter chromatin structure at the origin of DNA replication. Finally, I showed that NAP1 and TAF-I are required for expression of lytic protein BZLF1 and that TAF-I associates with the BZLF1 promoter during lytic reactivation.

Folliculogenesis is a complex process which describes the maturation of the ovarian follicle, from the primordial stage all the way to the ovulation of the antral follicle, and its sequela, the formation of the corpus luteum (CL). Imitation switch (ISWI) proteins are a class of ATP-dependent chromatin remodelers which mobilize nucleosomes to regulate a number of cellular processes including transcription, replication, and DNA repair. The pattern of expression of the mammalian ISWI proteins SNF2H and SNF2L in the mouse ovary suggests a role in the coordination of the proliferation and differentiation of granulosa cells during folliculogenesis. Here, we report that SNF2H is associated with proliferating granulosa cells, while SNF2L expression is induced following the LH surge which triggers their terminal differentiation into luteal cells. Knockdown of Snf2l by siRNA is sufficient to downregulate the expression of StAR, an important steroidogenic enzyme, and marker of the CL. Furthermore, SNF2L is thought to directly regulate StAR expression by physically binding to its promoter as indicated by chromatin immunoprecipitation (ChIP). In order to identify additional targets regulated by SNF2L, an unbiased microarray screen was developed to look for genes induced by LH in a SNF2L-dependent manner. One of the candidates, Fgl2 is strongly induced at 8h post hCG only in granulosa cells with intact SNF2L activity. Furthermore overexpression of SNF2L is sufficient to induce FGL2, and SNF2L is present on its promoter in the SIGC rat granulosa cell line. Some of the SNF2L binding partners that may be important in this regulation are PR-A and FLI-I, which have been found to interact with SNF2L by IP. Finally we describe here the phenotype of a Snf2l KO mouse which includes multiple reproductive defects, including resistance to superovulation, low secondary follicle counts, and a high incidence of abnormal antral follicles. Taken together these data suggest an important role of ISWI proteins in folliculogenesis, particularly SNF2L, which may regulate multiple genes important for the terminal differentiation of granulosa cells into luteal cells following the LH surge.

DNA condensates have attracted the attention of biophysicists, biochemists and polymer physicists for more than thirty years. In the biological community, the quest to understand DNA toroid formation has been motivated by its relevance to gene packing in certain viruses and by the potential use of DNA toroids in artificial gene delivery (e.g. gene therapy). In the physical sciences, DNA toroids are appreciated as a superb model system for studying particle formation by the collapse of a semiflexible, polyelectrolyte polymer. The thesis includes an analysis of the kinetic and thermodynamic factors governing DNA condensate morphology in solution, and discusses implications for future applications of DNA condensation in vitro as a model system for testing theories of polyelectrolyte collapse. In addition, DNA condensation by folded bovine protamine, a naturally occurring multivalent oligopeptide responsible for packing genomic DNA in bovine sperm cells, has been studied as well. The analysis of morphology, size, DNA strand packing density, and the stability of structural integrity of DNA condensates obtained with folded bovine protamines suggests that we have reconstituted native sperm cell chromatin. The results of this study were used to model the local structure of bovine sperm cell chromatin.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Within the span of a single human lifetime, we have discovered that DNA is the basis of genetic inheritance, deciphered the genetic code, and determined the entire sequence of multiple human genomes. However, we still have only a basic understanding of many of the processes that regulate DNA structure, function, and dynamics. The work presented in this dissertation describes the roles of two sets of genes that regulate the expression of genetic information and its transmission from one generation to the next.The condensin II complex has been implicated in the maintenance of genomic integrity during cell division and in transcriptional regulation during interphase. These roles stem from its ability to regulate chromosome structure though the mechanisms of this regulation are unclear. Evidence suggests that it is important for chromosome condensation and segregation during mitosis and meiosis. We have shown that this complex regulates the condensation of chromosomes during interphase. Its ability to reduce chromosome axial length provides a mechanism for the establishment of chromosome territories. We have also shown that condensin II differentially regulates interactions between homologous and heterologous DNA sequences. These findings contribute to our understanding of the overall structure of the nucleus, the regulation of chromosome structure, and the regulation of gene expression.The function of the Drosophila gene, sticky, is poorly understood. It contributes to cytokinesis by phosphorylating myosin II, but it also has a role in the regulation of chromatin structure. Mutations in sticky are associated with a wide range of developmental abnormalities. We provide evidence that this gene regulates the expression of numerous other genes which contribute to the phenotypes observed when sticky is mutated. We also show that sticky function overlaps with that of dfmr1, an ortholog of the gene associated with the most common form of human mental retardation. These findings contribute to our understanding of transcriptional regulation in chromatin and its implications in development and disease.

No description available.