Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Heidelberg, Univ., Diss., 2002.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 109-119).

The JmjC-histone lysine demethylases (JmjC-KDMs) are epigenetic regulators responsible for the demethylation of methylated lysine residues on the N-terminal histone tails. As Fe²⁺ and 2-oxoglutarate dependent oxygenases (2OG oxygenases), the JmjC-KDMs possess an absolute requirement for molecular oxygen and are related to the cellular oxygen sensing HIF hydroxylases, PHD2 and FIH. Several JmjC-KDMs are known HIF target genes, hence are upregulated in hypoxia. Moreover, a number of JmjC-KDMs have been shown to have differential oxygen dependences, while aberrant histone methylation has been observed in both hypoxic cells and disease states such as cancer and cardiovascular disease. The work described in this thesis aimed to investigate the impact of hypoxia on the JmjC-KDM, KDM4A. In vitro kinetic analyses revealed a K_m^app(O₂) for recombinant KDM4A of 173 ± 23 μM, which is higher than reported values for the 2OG oxygenases C-P4H, mPAHX and even FIH, and approaching those evaluated for the key oxygen sensor PHD2 (230-1746 μM). These results indicate that KDM4A activity is highly sensitive to oxygen availability, and has the biochemical potential to act as an oxygen sensor in the context of epigenetic regulation. Subsequent investigation of the cellular oxygen dependence of KDM4A, and found that the activity of ectopically expressed KDM4A in U2OS cells demonstrates a graded response to oxygen. Importantly, this trend correlates with the in vitro results, providing further evidence that hypoxia may impact upon epigenetic regulation by the JmjC-KDMs. The various factors that may contribute to the hypoxic inhibition of KDM4A were investigated both in vitro and in cells. The results of these studies suggested that altered concentrations of TCA cycle intermediates, comprising reduced levels of the 2OG oxygenase co-substrate 2OG and increased concentrations of the reported inhibitor 2HG, are likely to only minimally affect the activity of KDM4A in hypoxia. Interestingly, the 2OG oxygenase inhibitor IOX1 possessed increased inhibitory potency against KDM4A under conditions of low oxygen, implying that the use of mixed-mode inhibitors against KDM4A may be of therapeutic benefit in hypoxic disease states. This may be of particular pertinence to cardiac hypertrophy (CH), in which KDM4A activity is reported to have pathophysiological consequences. In a collaboration with Dr Tim McKinsey (University of Colorado, Denver), the KDM4 inhibitor CCT1 was tested in a phenotypic screen of cardiomyocyte hypertrophy, the results of which further support a role for KDM4A in this disease, and suggest that the use of small-molecule inhibitors of KDM4A may be a viable therapeutic strategy in CH. Finally, the effect of reactive oxygen species, levels of which may be increased in hypoxia, on KDM4A activity was explored. Recombinant KDM4A was found to be acutely sensitive to inhibition by hydrogen peroxide (H₂O₂) when compared to the HIF hydroxylases PHD2 and FIH. These results imply that KDM4A may act as a sensor of oxidative stress at the chromatin level, and further investigation in a more biologically relevant context is proposed. Overall, the work described herein demonstrates that the activity of KDM4A is sensitive to oxygen availability, a phenomenon that is likely to have significant implications for epigenetic regulation in hypoxia and the expression of KDM4A-regulated genes in ischaemic disease states.

Trimethylation of histone H3 lysine 9 (H3K9me3) and DNA methylation mark heterochromatin, contributing to gene silencing and normal cellular functions. My research investigated the control of H3K9me3 and DNA methylation in the filamentous fungus Neurospora crassa. The H3K9 methyltransferase complex, DCDC, consists of DIM-5, DIM-7, DIM-9, DDB1, and CUL4. Each component of DCDC is required for H3K9me3. The DIM-9/DDB1/CUL4 subunits are reminiscent of known cullin E3 ubiquitin ligases. I showed that core features of CUL4-based E3 ubiquitin ligases are not required for H3K9me3 and DNA methylation in Neurospora. H3K9me3 is bound by heterochromatin protein 1 (HP1) to recruit the DIM-2 DNA methyltransferase and the HCHC histone deacetylase complex. HCHC consists of HP1, CDP-2, HDA-1, and CHAP. Both HP1 and CDP-2 harbor conserved chromodomains that bind H3K9me3, and CHAP contains two putative AT-hook domains that bind A:T-rich DNA. To test the contributions of these domains to HCHC function, I deleted the chromodomains of HP1 and CDP-2. Deletion of the HP1 chromodomain resulted in a reduction of DNA methylation, which was not exacerbated by deletion of the CDP-2 chromodomain. A strain with deletions of chap and the HP1 chromodomain showed a DNA methylation phenotype comparable to the loss of the HDA-1 catalytic subunit. These findings support a model in which recognition of H3K9me3 and A:T-rich DNA by HP1 and CHAP, respectively, are required for proper HCHC function. To examine the relationships between H3K9me3, DNA methylation, and histone acetylation, I utilized in vivo protein tethering of core heterochromatin components. The requirement of DIM-7 for native heterochromatin, previously implicated in localizing the H3K9 methyltransferase DIM-5, was not bypassed by DIM-5 tethering, indicating that DIM-7 has additional roles within the DCDC. Artificial localization of the HCHC histone deacetylase, by tethering HP1 or HDA-1, resulted in induction of H3K9me3, DNA methylation, and gene silencing, but silencing did not require H3K9me3 or DNA methylation. HCHC-mediated establishment of H3K9me3 was not required for de novo heterochromatin formation at native heterochromatic loci suggesting a role in heterochromatin spreading. Together, this work implicates HDA-1 activity as a key driver of heterochromatin spreading and silencing. This dissertation includes previously published co-authored material.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-14T18:17:57Z No. of bitstreams: 1 Lais da Silva Pereira. Avaliação das histonas...2013.pdf: 2452594 bytes, checksum: 808d7633f6d2684df54ea8b640e282c0 (MD5) / Made available in DSpace on 2013-10-14T18:17:57Z (GMT). No. of bitstreams: 1 Lais da Silva Pereira. Avaliação das histonas...2013.pdf: 2452594 bytes, checksum: 808d7633f6d2684df54ea8b640e282c0 (MD5) Previous issue date: 2013 / Universidade Federal da Bahia. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A leishmaniose visceral é uma doença infecciosa grave, causada por protozoários intracelulares obrigatórios do gênero Leishmania. Vários antígenos de Leishmania têm sido avaliados como candidatos vacinais, destacando-se as proteínas de histonas (HIS), antígenos altamente conservados. A exposição de HIS pela Leishmania induz uma resposta imune potente no hospedeiro vertebrado. Desse modo, neste estudo, avaliamos, em hamsters, a capacidade imunoprotetora dos antígenos de histonas contra a infecção por Leishmania infantum. Os animais foram vacinados com estratégia homóloga, utilizando-se plasmídeos de DNA que codificam para HIS (pcDNA3LiH2A-H3, pcDNA3LiH2B-H4) ou heteróloga (DNA/proteínas HIS) mais 1nM de CpG. Quinze dias após a última imunização, os animais foram desafiados pela via intradérmica com 105 Leishmania infantum metacíclicas mais 0,5 par de glândula salivar de Lutzomya longipalpis. Após a última imunização e durante a infecção, realizaram-se dosagens de citocinas por PCR em tempo real (linfonodo e baço), sorologia por ELISA (soro), carga parasitária por diluição limitante e análise histopatológica de tecidos (linfonodo, baço e fígado). Detectou-se produção de anticorpos IgG anti HIS nos grupos imunizados com a estratégia homóloga e heteróloga, quando comparados aos hamsters não imunizados. As imunizações homóloga e heteróloga diferiram na razão IFN-γ/IL-10 no linfonodo em relação ao grupo controle. Não houve diferença significativa na expressão dessas citocinas no baço após a imunização, entretanto, cinco meses após o desafio o grupo homólogo apresentou um aumento na produção de IL-10 nesse órgão. Na análise histopatológica do baço, verificou-se formação de mais folículos com centro germinativo, evidentes nos animais imunizados independentemente do grupo analisado. Observou-se, também, leucocitose intrasinusoidal e periportal no fígado, e folículos reativos no linfonodo. Nenhuma das estratégias de imunizações com antígenos de histonas acarretou em diminuição da carga parasitária no linfonodo, baço e fígado. As estratégias de imunização homóloga e heteróloga, com antígenos de histonas nucleossomais, não foram capazes de proteger contra infecção por L. infantum no modelo do hamster. / Visceral leishmaniasis is a serious infectious disease caused by obligatory intracellular protozoan of the Leishmania genus. Many antigens from Leishmania have been evaluated as vaccine candidates mainly the histones (HIS) that are highly conserved. Exposure to HIS from Leishmania elicits a strong host immune response in the vertebrate host. Threfore, in this study, we evaluated the immunoprotective ability of HIS antigens against infection by Leishmania infantum in hamsters. The animals were immunized with homologous strategy using plasmid of the DNA coding for HIS (pcDNA3LiH2A-H3, pcDNA3LiH2B-H4) or heterologous strategy using DNA and recombinant protein plus CpG. Fifteen days after last immunization, the animals were challenged by the intradermal route with 105Leishmania infantum metacyclics plus 0,5 pair of Lutzomya longipalpis salivary gland. After the last immunization and in the course of the infection, we determined cytokine production by Real time PCR (lymph node and spleen), serology by ELISA (sera), parasite burden was estimated by limiting dilution assay and histopathological analysis (lymph node, spleen and liver). Production of antibody anti HIS IgG was detected in the immunized groups treated with the homologous and heterologous strategies when compared to non-immunized hamsters. Homologous and heterologous immunizations altered the ratio of IFN-γ/IL-10 in the lymph node compared to the control group. There is not a significant difference in the expression of this cytokine in the spleen after the immunization, however, five months after the challenge the homologous group presented a higher expression of IL-10 in this organ. The histopathological analysis of the spleen showed the formation of high number of follicles with a germinative center in the immunized animals independently of the immunization employed. Intrasinusoidal and periportal leukocytosis were observed in the liver and reactive follicles in the lymph node. Both immunization strategies with histone antigen did not result in reduction of parasite burden in the lymph node, spleen and liver. Immunization with homologous and heterologous strategies using histone antigen were not able to confer protection in the hamster model against L. infantum infection.

The dynamic nature of eukaryotic chromatin enables the packaging of large amounts of genetic material in a small space. At the same time, it provides controlled access to genomic DNA for a variety of nuclear processes for example, transcription and DNA repair. The transition between open and closed chromatin states is largely governed by ATP-dependent chromatin remodeling complexes, which operate on nucleosomes in concert, to modulate chromatin structure and composition. Exchange of the canonical and variant forms of histones in nucleosomes, and altering the spacing between consecutive nucleosomes, are two major ways which regulate chromatin-based processes and chromatin higher-order organization. The evolutionarily conserved INO80 and ISW1a complexes mediate these two aspects of nucleosome remodeling, respectively. Despite sharing conserved domain architecture of the core remodeling machinery, chromatin remodelers differ significantly in their modes of interaction with nucleosomes, and how they alter histone-DNA contacts. In this study, we have used a site-specific photocrosslinking approach coupled with peptide mapping to determine the interactions of subunits and domains of the S. cerevisiae INO80 and ISW1a complexes with nucleosomes. We find that specific interactions of remodelers with different regions of the nucleosome largely dictate their specialized functions and mechanisms. The ATP-dependent helicase-like (ATPase) domains of remodelers belonging to the ISWI and SWI/SNF families translocate along DNA close to the center of nucleosomes in order to mobilize, space or disassemble nucleosomes. In contrast, we observed that INO80 has a strikingly distinct mechanism, which is different even from its paralog SWR1. INO80 mobilizes nucleosomes as well as catalyzes the exchange of histone variant H2A.Z for the canonical histone H2A, while SWR1 mediates the reverse exchange of H2A for H2A.Z, without being able to mobilize nucleosomes. We have found that INO80, in order to promote H2A-H2B dimer exchange, translocates along DNA at the H2A-H2B interface close to the edge of nucleosomes and persistently displace DNA from H2A-H2B. Blocking either DNA translocation or the accumulation of DNA torsions close to the edge of the nucleosome interferes with this dimer exchange by INO80. SWR1 and other SWI/SNF and ISWI remodeling complexes translocate along DNA at the H3-H4 interface and do not persistently displace DNA from the histone octamer as does INO80. This study shows for the first time an ATP-dependent chromatin remodeler that invades nucleosomes at the DNA entry site instead of the center − a more logical approach for the displacement of H2A-H2B. We also investigated nucleosomal DNA interactions of other INO80 subunits and domains to understand the architecture of INO80 bound to nucleosomes. We found that the HSA (helicase-SANT-associated) domain of Ino80 along with actin-related protein (Arp) subunits Arp8 and Arp4 bind to the extranucleosomal DNA and is potentially involved in a coupling mechanism with the ATPase domain to regulate its activity. We also mapped the DNA binding regions of Arp8 and Arp4, which might be involved in recruiting INO80 to genomic sites. The ISWI remodeler ISW1a regulates the distance (spacing) between nucleosomes in an array by simultaneously interacting with two nucleosomes and directionally remodels one of them. We mapped DNA interactions of ISW1a subunits in mono- and di-nucleosomes. Our results show that the catalytic Isw1 subunit specifically interacts with the region of DNA translocation and DNA entry site of the asymmetrically positioned nucleosome in a di-nucleosome, which is preferentially mobilized. In contrast, the Ioc3 subunit interacts extensively with the linker DNA as well as the extranucleosomal DNA of the un-remodeled nucleosome. This bias in nucleosomal DNA interactions of ISW1a enables directional remodeling, which reveals the molecular basis of nucleosome spacing. We have identified a novel domain within the non-catalytic Ioc3 subunit of ISW1a that regulates nucleosome spacing. We found that when this domain is deleted, the catalytic Isw1 subunit loses its specificity and interacts with both the nucleosomes of a di-nucleosome substrate. This is consistent with the domain-deleted ISW1a mobilizing both nucleosomes efficiently, leading to the loss of its nucleosome spacing activity. In summary, this dissertation explores how different remodeling complexes have customized and regulated modes of nucleosome interaction in order to accomplish specialized remodeling outcomes. INO80 places its ATPase domain for translocation at the H2A-H2B dimer interface and persistently displaces DNA from its surface to promote H2A.Z exchange. Nucleosome spacing by ISW1a requires the catalytic Isw1 subunit to engage with and reposition one out of two consecutive nucleosomes in an array, while the Ioc3 subunit likely monitors the distance between them.

Repo-Man is a chromatin-associated PP1 targeting subunit that coordinates chromosome re-organisation and nuclear envelope reassembly during mitotic exit. At the onset of mitosis, Repo-Man association with the chromosomes is very dynamic; at anaphase, Repo-Man targets to the chromatin in a stable manner and recruits PP1 to de-phosphorylate histone H3 at Thr3, Ser10 and Ser28. Previous studies have suggested that CDK1 and AuroraB are the kinases responsible for the inactivation of the complex and for its dispersal at the onset of mitosis respectively. We have previously shown that the binding of Repo-Man to PP1 is decreased in mitosis and we have identified a region adjacent to the RVTF motif that contains multiple mitotic phosphosites (RepoSLIM). This region is conserved only in another PP1 targeting subunit: Ki-67. In order to understand the importance of this region for the complex formation and stability, we have conducted mutational analyses on several residues, and addressed their contribution towards Repo-Man chromosome targeting and PP1 binding in vivo. We have identified new sites in Repo-Man that, when phosphorylated, contribute to the weakening of the binding between Repo-Man and PP1. Interestingly, our results also indicate that several kinases are involved in the mitotic regulation of the complex. We have also identified Lamin A/C as a Repo-Man substrate and introduced a new model for Lamin A/C regulation at interphase. Furthermore, we identified Repo-Man as a marker of malignancy in tripe egative breast cancer, which controls cell movement and levels of important oncogenic markers Aurora A and C-Myc, and propose Repo-Man/PP1 complex as a therapeutic target for the treatment of triple negative breast cancer through the newly identified RepoSLIM.

Le génome des cellules eucaryotes est empaqueté dans la chromatine, dont l’établissement et la maintenance nécessitent des processus d’assemblage et de remodelage. Ce travail de thèse a été consacré à la caractérisation de deux facteurs de la machinerie d’assemblage de la chromatine. Le premier facteur étudié dans ce travail était HIRIP3, un homologue mammifère de la levure H2A.Z chaperon Chz1. Nous voulions vérifier si HIRIP3 est une chaperon d'histone par elle-même. Pour commencer, nous avons décrit l'interaction de HIRIP3 avec les histones in vivo. Ensuite, nous avons étudié la spécificité structurale de cette interaction in vitro. Nous avons caractérisé HIRIP3 comme une nouvelle chaperon d'histone H2A qui utilise le motif CHZ pour sa fonction. La deuxième partie de ce travail a été axée sur le complexe de remodelage de la chromatine SRCAP. Nous avons cherché à décoder son réseau d'interaction et à décrire ses sous-complexes. Nous avons reconstitué le complexe de base YL1, SRCAP, TIP49A, TIP49B et H2A.Z / H2B en utilisant le système d'expression chez baculovirus. Notre protocole nous a permis de purifier un complexe de base adapté aux futures études structurelles par microscopie cryo-électronique. / The genome of eukaryotic cells is packaged into chromatin, which establishment and maintenance require mechanisms of assembly and remodelling. This thesis work was dedicated to the characterization of two factors of chromatin assembly machinery. The first factor studied in this work was HIRIP3, a mammalian homologue of yeast H2A.Z chaperone Chz1. We aimed to test whether HIRIP3 is a histone chaperone by itself. At first, we established HIRIP3 interaction with histones in vivo. After then, we studied the structural specificity of this interaction in vitro. We have characterized HIRIP3 as a novel H2A histone chaperone that utilizes the CHZ motif for its function. The second part of this work was focused on SRCAP chromatin remodelling complex. We aimed to decipher its interaction network and to describe its sub-complexes. We have reconstituted YL1, SRCAP, TIP49A, TIP49B and H2A.Z/H2B core complex using baculovirus expression system. Our protocol allowed us to purify core complex suitable for future structural studies by cryo-electron microscopy.