The genes of eukaryotes exist as DNA-RNA-protein complexes known as chromatin. The structure of chromatin fluctuates to allow controlled access to genetic information while maintaining its important packaging function. Recent improvements in optics, image acquisition electronics, and live imaging techniques, as well as the introduction of fluorescent fusion proteins, have made it possible to use fluorescence light microscopy to study the dynamic nature of chromatin compaction in cells. Here we report the application of advanced fluorescence microscopy to characterize the effects of transcription on chromatin compaction in living yeast cells. Repressor protein-GFP fusion proteins which recognize specific operator sequences were used to fluorescently tag specific gene loci, and an OMX fluorescence light microscope was then used to track their positions in three dimensions. It was determined that image acquisition with the OMX microscope is rapid enough to track fluorescently tagged genomic loci in live yeast cells in 3D, and that it does so with a root mean squared (RMS) measurement error of 162 nanometers (nm). It was also determined that the OMX microscope can distinguish between strains with fluorescent spots separated by 40 or 70 kb genomic distances. Additionally, it was found that chromatin compaction of a 15 kb gene driven by the Gal1 promoter is correlated with the carbon source on which the cells are fed, and that three different carbon sources produce three different transcription-dependent chromatin structures. Reversible changes in end-to-end distance of ~500 nm within two seconds were detected in the induced strain. These findings indicate that improvements in light microscopy enable chromatin to be studied in living cells on a scale not previously possible.

In order to better characterize linker histone interactions with DNA, avian erythrocyte-specific linker H5 and the trypsin-resistant globular domain of H5 (GH5) were used in DNA binding studies. To begin, H5 displayed a considerably higher binding affinity for DNA than the isolated globular domain (GH5), supporting the importance of the terminal tail domains in binding. This conclusion is based upon binding curves conducted in low-salt solution, and on the considerably-higher salt concentration required to prevent protein-DNA contact. Linker histones also induce DNA-protein aggregation in a process that was found to result in protein insolubility in 2% SDS, and included protein-protein interactions that did not require the terminal tail domains. In addition, DNA supercoiling appeared to impede the aggregation process; this that may be attributable to binding of linker histones in isolated clusters, as gauged by a limit in the number of observed dithiobis (succinimidyl) propionate (DSP)-crosslinkable contacts. In a related study, the property of GH5 to bind, then organize onto DNA was investigated. GH5 crosslinked onto DNA with dithiobis (succinimidyl propionate), then cleaved with chymotrypsin, displayed highly uniform contacts that appeared to involve the C-terminal four amino acids, and suggests protein-protein interactions are important for binding. This finding may be relevant since GH5 (and H5) were observed to self-associate free in solution in an arguably specific manner. Finally, the exposure of Phe 93 to chymotrypsin was used to identify the surface of the globular domain that contacts DNA for the binding of intact H5. Results suggests that the side of the protein opposite to the recognition helix preferentially binds to DNA, supporting a novel winged-helix protein DNA-binding mechanism. Furthermore, parallel studies with octamers reconstituted onto a DNA fragment with twelve copies of the 208 b.p. rDNA 5s gene from Lytechinus variegatus, shows that H5 had a high binding affinity with all detectable protein binding to the reconstituted complex. H5 binding conferred protection to a site located near the dyad axis from endonuclease digestion, supporting the contention that H5 binds near or at the nucleosome dyad axis. H5 binding also was observed to condense fibers as observed from agarose gel electrophoresis, although velocity analytical sedimentation studies indicate that H5 in itself was not sufficient to fully compact chromatin fibers; rather H5 and 30 mM NaCl, in combination, were required. Results indicate that the chromatin-reconstituted "208-12 DNA" makes an excellent model for analyzing the effect of linker proteins on chromatin morphology. / Graduation date: 1998

The reversible process of histone acetylation and deacetylation is an important mechanism of epigenetic regulation in the control of gene expression and chromatin structure. In general, histone acetylation is related to gene activation, whereas histone deacetylation is associated with transcriptional gene silencing and maintenance of heterochromatin. A large number of histone deacetylases (HDACs), the enzymes that catalyze the reaction of histone deacetylation, have been identified in plants and other eukaryotes, and they were found to play crucial roles in plant growth and development. In Arabidopsis thaliana, histone deacetylase 1 (AtHD1) is a homolog of Saccharomyces cerevisiae Rpd3 that is a global transcriptional regulator. Downregulation of AtHD1 in transgenic Arabidopsis results in histone hyperacetylation and induces a variety of phenotypic and developmental defects, suggesting that AtHD1 is also a global regulator of many physiological and developmental processes. To characterize the expression pattern and distribution of AtHD1 in cells, the subcellular location of AtHD1 was determined by monitoring the expression of an AtHD1-GFP fusion protein in a transient expression assay and in transgenic Arabidopsis.The results show that AtHD1 is localized in the nucleus and appears to be excluded from the nucleolus. The histone deacetylase activity of AtHD1 was studied in an in vitro assay using radiolabeled histone peptides as a substrate. Recombinant AtHD1 produced by bacteria demonstrated a moderate but significant HDAC activity, whereas that produced by the baculovirus expression system did not have activity. This suggests that AtHD1 may require other cofactors or association with other proteins, rather than post-translational modifications, in order to have full HDAC activity. To study the possible interactions of AtHD1 with other proteins, a recombinant AtHD1 protein with two units of c-myc epitope fused to its C-terminus was expressed in transgenic Arabidopsis. We attempted to isolate proteins interacting with AtHD1 by co-immunoprecipitation (Co-IP). However, in the first few trials of Co-IP, a lot of contaminating proteins were present in the eluent along with the recombinant AtHD1-cmyc protein. Improvements in the experimental conditions are required for further investigation.

Le séquençage de nombreux génomes eucaryotes indique que l’augmentation de la «biocomplexité» au cours de l’évolution n’est pas directement corrélée à l’accroissement du nombre de gènes. En d’autres termes, nous sommes plus que la somme de nos gènes et l’ère post-génomique actuelle promet de cerner de façon plus précise les bases moléculaires de notre identité. A cet égard, il semble de plus en plus clair que l’épigénétique est riche d’une information qui se superpose à celle du code génétique. L’information épigénétique est principalement véhiculée par des modifications de l’ADN et des histones. La modification majeure de l’ADN est la méthylation de la cytosine qui est la marque d’une chromatine transcriptionnellement silencieuse. Quant aux histones, différentes modifications posttraductionnelles ont été décrites, comme l’acétylation, la phosphorylation, la méthylation et l’ubiquitinylation. L’ensemble de ces modifications constituerait un «code histone», dont le décryptage n’en est qu’à ses prémices, permettant d’associer à chaque combinaison de modifications un état particulier de la chromatine, et ainsi de l’expression génique. La méthylation des histones a longtemps été considérée comme irréversible mais l'identification récente de déméthylases des histones spécifiques de certains sites a révélé que cette modification est régulée de façon dynamique et réversible. La découverte de ces enzymes a ouvert de nouveaux axes de recherche dans le domaine de l'épigénétique (Klose and Zhang, 2007). Au cours de ma thèse de doctorat, nous nous sommes intéressés à la déméthylase PADI4 (peptidylarginine déiminase 4) qui convertit des résidus arginines des histones H3 et H4, associés à l'activation des gènes, en résidus citrullines, ce qui a pour conséquence d'entraîner une répression transcriptionnelle. Cette réaction porte le nom plus particulier de déimination/citrullination des histones. A l’heure actuelle, il est primordial de cerner comment la déméthylation des histones, et plus précisément la peptidylarginine deiminase 4 (PADI4), réprime la transcription. Dans un premier temps, nous avons mis en évidence un lien mécanistique entre la deméthylation et la désacétylation des histones. Nous avons montré que PADI4 interagit avec l’histone désacétylase HDAC1. Cette enzyme est responsable du décrochage des groupements acétyls des histones, conduisant à la fermeture de la chromatine. Des expériences d’immunoprécipitation de la chromatine indiquent que PADI4 et HDAC1 s’associent au promoteur du gène de réponse aux oetrogènes pS2 simultanément et de manière cyclique. L’utilisation d’une construction shRNA dirigée contre la protéine endogène HDAC1 indique que la liaison de PADI4 au promoteur du gène pS2 est dépendante de la présence de HDAC1. Dans la deuxième partie de notre travail, un lien mécanistique entre la déméthylation des histones par PADI4 et la méthylation de l’ADN a été mis en évidence. La méthylation de l’ADN est catalysée par des enzymes, appelées méthyltransférases de l’ADN (DNMTs), qui transfèrent des résidus méthyls sur les cytosines. Nous avons montré que la protéine DNMT3A interagit avec PADI4. Nous avons également démontré que l’enzyme PADI4 était capable de citrulliner/déiminer (convertir des résidus arginines en résidus citrullines) la méthyltransférase de l’ADN DNMT3A in vitro et que cette citrullination de la protéine DNMT3A par PADI4 stabiliserait DNMT3A in vivo. Enfin, nos récents travaux révèlent une relation mécanistique entre la protéine MeCP2, interprète des signaux de méthylation de l’ADN, et la protéine polycomb EZH2. Celle-ci possède une activité méthyltransférase d’histone sur les lysines 27 de l’histone H3. Nos données montrent que MeCP2 interagit avec EZH2 et que ces protéines fixent des régions promotrices communes. De plus, la déplétion en MeCP2 affecte la présence de EZH2 au niveau de ces régions communes. En conclusion, ce travail de thèse devrait permettre une meilleure compréhension des mécanismes moléculaires de l’épigénétique. Plus particulièrement, il devrait aider à mieux cerner comment la première histone déméthylase décrite, la peptidylarginine déiminase 4 ou PADI4, verrouille l’expression génique.

This thesis explores chromatin modification (CM) as a biological system and uses known CM factors in four model organisms; yeast, worm, fly, and human to explore how CM factors have consistently evolved across a diverse spectrum of 111 organisms by using the InParanoid homology algorithm. Using InParanoid, phylogenetic profiles are constructed for each model organism to highlight evolutionary trajectories and which CM factors are lost, expanded, and are specific to some lineages. Phylogenetic tree construction demonstrates that peripheral subunits of CM complexes evolve independently. Accurate mapping of domains to CM factors and their homologs reveals that the architecture of domains is very well conserved, with only one potential case of a domain swap. Homology, domain architecture, and protein-protein interaction is then combined to illustrate an interolog example and potential interaction candidates. The techniques highlighted in this thesis represent a generic and powerful approach to analyzing any biological system of interest.

This thesis explores chromatin modification (CM) as a biological system and uses known CM factors in four model organisms; yeast, worm, fly, and human to explore how CM factors have consistently evolved across a diverse spectrum of 111 organisms by using the InParanoid homology algorithm. Using InParanoid, phylogenetic profiles are constructed for each model organism to highlight evolutionary trajectories and which CM factors are lost, expanded, and are specific to some lineages. Phylogenetic tree construction demonstrates that peripheral subunits of CM complexes evolve independently. Accurate mapping of domains to CM factors and their homologs reveals that the architecture of domains is very well conserved, with only one potential case of a domain swap. Homology, domain architecture, and protein-protein interaction is then combined to illustrate an interolog example and potential interaction candidates. The techniques highlighted in this thesis represent a generic and powerful approach to analyzing any biological system of interest.

Gene expression is a process that involves changes in chromatin organization and structure. Chromatin is thought to be organized in a structure consisting of looped domains, which are fixed at their bases to the nuclear matrix or scaffold. SAFB has been identified as a nuclear matrix binding protein in humans. Human SAFBs contain an N-terminal DNA-binding SAP-Box, and an RNA recognition motif (RRM). However it is unknown how the features of SAFB are linked to gene expression and chromatin organization. I have identified a single homologue of SAFB in Drosophila. To understand the role of SAFB in gene expression and nuclear structure, I have begun to characterize Drosophila SAFB. I found two SAFB splice forms, a full length SAFB containing DNA and RNA binding domains, and a smaller splice form lacking the RNA binding domain. I have showed that SAFB is expressed throughout embryogenesis, in adult testis and ovaries, and larval and adult brains. In addition, I made SAFBGFP constructs to characterize the cellular localization of SAFB. In S2 cells, embryos and neuroblasts, GFP-SAFB was found throughout the nucleus and in nuclear speckles and is retained in the matrix after soluble proteins and DNA are removed. Using larval polytene chromosomes, I show that GFP-SAFB binds to specific DNA bands, some of them overlapping with RNA Polymerase II. After heat shock, GFP-SAFB is recruited to the highly expressed heat shock genes. Treatment of polytene chromosomes with RNAse caused the majority of bands to disappear, meaning that the binding of most of SAFB to chromosomes was mostly through RNA. To distinguish binding of SAFB to DNA from protein-protein interaction, I constructed a GFP-tagged version of SAFB lacking the SAP domain, which binds to fewer sites in the genome. RNAse treatment abolished nearly all binding. Together, my data show that Drosophila SAF-B is a component of the nuclear matrix, that localized to specific loci in the chromosomes, and is recruited to actively-transcribed genes.

The reversible process of histone acetylation and deacetylation is an important mechanism of epigenetic regulation in the control of gene expression and chromatin structure. In general, histone acetylation is related to gene activation, whereas histone deacetylation is associated with transcriptional gene silencing and maintenance of heterochromatin. A large number of histone deacetylases (HDACs), the enzymes that catalyze the reaction of histone deacetylation, have been identified in plants and other eukaryotes, and they were found to play crucial roles in plant growth and development. In Arabidopsis thaliana, histone deacetylase 1 (AtHD1) is a homolog of Saccharomyces cerevisiae Rpd3 that is a global transcriptional regulator. Downregulation of AtHD1 in transgenic Arabidopsis results in histone hyperacetylation and induces a variety of phenotypic and developmental defects, suggesting that AtHD1 is also a global regulator of many physiological and developmental processes. To characterize the expression pattern and distribution of AtHD1 in cells, the subcellular location of AtHD1 was determined by monitoring the expression of an AtHD1-GFP fusion protein in a transient expression assay and in transgenic Arabidopsis.The results show that AtHD1 is localized in the nucleus and appears to be excluded from the nucleolus. The histone deacetylase activity of AtHD1 was studied in an in vitro assay using radiolabeled histone peptides as a substrate. Recombinant AtHD1 produced by bacteria demonstrated a moderate but significant HDAC activity, whereas that produced by the baculovirus expression system did not have activity. This suggests that AtHD1 may require other cofactors or association with other proteins, rather than post-translational modifications, in order to have full HDAC activity. To study the possible interactions of AtHD1 with other proteins, a recombinant AtHD1 protein with two units of c-myc epitope fused to its C-terminus was expressed in transgenic Arabidopsis. We attempted to isolate proteins interacting with AtHD1 by co-immunoprecipitation (Co-IP). However, in the first few trials of Co-IP, a lot of contaminating proteins were present in the eluent along with the recombinant AtHD1-cmyc protein. Improvements in the experimental conditions are required for further investigation.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 155-169). Also available in print.

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