UHRF1 est une protéine nucléaire responsable du maintien et de la régulation de l'épigénome des cellules. Elle favorise la prolifération cellulaire et est surexprimée dans la plupart des cancers. TIP60, l'un des partenaires le plus important d’UHRF1, est impliqué dans le remodelage de la chromatine et la régulation transcriptionnelle grâce à son activité acétyltransférase. Ensemble, les deux protéines régulent la stabilité et l'activité d'autres protéines telles que la DNMT1 et la p53. Le but de cette étude était d'explorer le mécanisme d'interaction entre UHRF1 et TIP60 en visualisant cette interaction dans les cellules. La microscopie par imagerie à temps de vie de fluorescence et d'autres techniques de biologie moléculaire ont été utilisées. Les résultats ont montré que UHRF1 interagit directement avec le domaine MYST de TIP60 et cette interaction se produit dans la phase S du cycle cellulaire. Les deux protéines ont également montré une réponse similaire aux dommages à l'ADN, ce qui prédit une cohérence dans leur fonction dans le mécanisme de réparation de l'ADN. La surexpression de TIP60 a également induit la baisse du niveau d’UHRF1 et de DNMT1 ainsi qu’une induction d'apoptose dans les cellules ce qui suggère un rôle de TIP60 dans la régulation des fonctions oncogéniques d’UHRF1. / UHRF1 is a nuclear protein maintaining and regulating the epigenome of cells. Its promotes proliferation and is found upregulated in most of cancers. TIP60 is one of the important interacting partner of UHRF1 and is involved in chromatin remodeling and transcriptional regulation through its acetyltransferase activity. Together they regulate the stability and activity of other proteins such as DNMT1 and p53. The aim of this thesis was to explore the mechanism of interaction between UHRF1 and TIP60 by visualizing this interaction in cells. Fluorescent lifetime imaging microscopy and other molecular biology techniques were employed for this purpose. Results of this study showed that UHRF1 interacts directly to the MYST domain of TIP60 and this interaction prevails in the S-phase of cell cycle. Both proteins also showed a similar response to DNA damage predicting a coherence in their function in DNA repair mechanism. Overexpression of TIP60 also downregulated UHRF1 and DNMT1 and induced apoptosis in cells suggesting a role of TIP60 in regulation of oncogenic functions of UHRF1.

Les océans couvrent plus de 70% de la surface de la Terre (planète bleue) et la productivité primaire nette (PPN) marine est équivalente à celle terrestre. Alors qu‟il ne représente que 1% de la biomasse totale d‟organismes photosynthétiques de la planète, le phytoplancton est responsable d‟environ 45% de la PPN globale. Le terme phytoplancton décrit un assemblage polyphylétique comprenant des eucaryotes et procaryotes photosynthétiques dérivant avec les courants. Dans les océans contemporains, les diatomées constituent un groupe d‟eucaryotes unicellulaires autotrophes très abondant, responsable de 40% de la PPN marine. Les diatomées appartiennent à la lignée des straménopiles qui sont issus d‟un évènement d‟endosymbiose entre une algue rouge et un hôte hétérotrophe. Elles sont classifiées en deux groupes majeurs : les centriques qui son apparues il y a environ 200 millions d‟années (Ma), et les pennées qui ont évolué il y a environ 90 Ma. Deux génomes de diatomées ont récemment été séquencés : celui de la diatomée centrique Thalassiosira pseudonana (32 Mb), et celui de la diatomée pennée Phaeodactylum tricornutum (27 Mb). Mon sujet de doctorat s‟est focalisé sur l‟étude de différents aspects de la régulation de l‟expression génique ainsi que sur la dynamique et l‟évolution de ces génomes. L‟expression des gènes est régulée à différents niveaux: trancriptionel, post-transcriptionel, et épigénétique. Dans le cadre de mon doctorat, une étude de la régulation transcriptionelle chez les diatomées a été effectuée et comprend l‟identification et l‟analyse in silico des facteurs de transcription (FT). Cela a permis par exemple d‟établir qu‟une classe spécifique de FT, les Heat Shock Factors, sont particulièrement abondants chez les diatomées par rapport aux autres eucaryotes. L‟analyse de la représentation des FT identifiés dans différentes librairies d‟EST élaborées à partir de cultures ayant subi divers stress a permis de détecter certaines spécificités d‟expression. L‟évolution des génomes eucaryotes est largement impactée par les effets directs et secondaires des éléments transposables (ET) qui sont des éléments génétiques mobiles se trouvant dans le génome de la plupart des organismes. Dans le but d‟étudier la dynamique des génomes de diatomées, la recherche de différents types d‟ET a permis d‟établir qu‟une certaine classe, les rétrotransposons de type Copia, est la plus abondante dans ces génomes et constitue un part significativement plus importante du génome de P. tricornutum (5,8%) par rapport à T. pseudonana (1%). D‟autre part, des analyses phylogénitiques ont montré que les rétrotransposons de type copia forment deux classes distinctes et éloignées de la lignée Copia. L‟analyse de leurs niveaux d‟expression a montré que la transcription de deux éléments s‟active en réponse à des stress spécifiques comme la limitation en nitrate dans le milieu de culture. Cette activation est accompagnée par un hypométhylation de l‟ADN et l‟analyse de profils d‟insertions chez différents écotypes de P. tricornutum ainsi que l‟étude d‟autres phénomènes suggèrent que les rétrotransposons de type Copia ont joué un rôle important dans l‟évolution des diatomées. Mon grand intérêt pour les ET m‟a ensuite amené à chercher à les caractériser dans d‟autres génomes récemment séquencés tels celui de l‟algue brune Ectocarpus siliculosus. La recherche in silico de différents gènes codant des protéines capables d‟introduire ou de stabiliser des états épigénétiques telle que la modification des histones et la méthylation de l‟ADN a montré leur présence chez P. tricornutum ainsi que leurs particularités. La présence de certaines modifications d‟histones spécifiques d‟une conformation compacte ou ouverte de la chromatine dans le proteome de P. tricornutum a été montrée. De plus, la mise au point de la technique d‟immunoprécipitation de la chromatine chez P. tricornutum a permis d‟établir que les nucléosomes enrobés d‟éléments transposables étaient marqués par des modifications spécifiques. D‟autres expériences ont permis d‟établir que l‟ADN de différents types d‟éléments transposables est marqué par la méthylation de cytosines chez P. tricornutum. Une expérience permettant l‟analyse du profil de méthylation à l‟échelle de génome en utilisant une puce à ADN a été lancée et permettra de découvrir si certains gènes portent aussi des traces de méthylation. Enfin, les ARN interférents constituent un troisième mode de régulation de l‟expression se situant à l‟interface de la régulation transcriptionelle, post-transcriptionelle et épigénétique. Les mécanismes d‟interférences chez les diatomées ont été étudié par la recherche in silico d‟enzymes clés impliquées dans ce processus ainsi qu‟en établissant expérimentalement un lien direct avec la méthylation de l‟ADN.

The imprinting status of the SLC22A18AS gene, located in the human chromosome segment 11p15.5, was studied using PCR-SSCP analysis and fetal tissues from a battery of 17 abortuses. This gene showed monoallelic expression (genomic imprinting) in different tissues from two abortuses which were heterozygous for an SNP (c.473G>A) in its coding region. This gene was found to be paternally imprinted (maternally expressed) in five tissues namely lung, liver, brain, kidney and placenta from an abortus. The parental origin of the expressed allele could not be determined in the second abortus as both the mother and the abortus were heterozygous for the SNP. Since paternal blood samples from none of the 17 abortuses could be collected for DNA isolation, the mother's genotype was used to find the origin of the expressed allele. In order to understand the mechanism underlying imprinting of this gene, it was important to understand the nature of the epigenetic marks (imprints) on the two alleles of this gene. Since these epigenetic marks are generally observed in promoters or CpG islands associated with the imprinted genes, the promoters of the SLC22A18AS gene was characterized using transient transfection of putative SLC22A18AS promoter fragments cloned in the pGL3-Basic vector in human cells followed by luciferase reporter assay. Since the promoter of a gene lies upstream to the transcription start site (TSS), TSS of this gene was mapped. In silico approach revealed an EST (CB129046) which had an additional 39 bases upstream to the known mRNA sequence. TSS was then identified by the 5’ primer extension analysis. TSS was found to be 166 bases upstream to the 5’ end of this EST. In order to select cell lines for transient transfection of putative promoter constructs for promoter charaterization, RT-PCR analysis was used to see the expression of this gene in the following available cell lines in the lab: HuH7, HepG2, A549, HeLa, LNCaP and PC3. This gene was found to be maximally expressed in HepG2 cells. Expression of this gene was also observed in A549, HeLa, LNCaP and PC3 cells. HuH7, on the other hand, did not show any detectable expression of this gene. Based on the above data, HepG2 and A549 cells were selected for promoter characterization. Seven putative promoter constructs were transiently transfected in these cells and the promoter activity of different constructs was measured by luciferase assay. The assay identified two promoters for the gene: P1 promoter in a region from -855 to -254 bp and P2 promoter in a region from -1441 to -855. In order to see the presence of putative transcription factor binding sites in the upstream region of the gene, the MatInspector Professional program was used. The gene was found to be devoid of TATA and CCAAT boxes. Most of the putative transcription factor binding sites were present in a region from -855 to -254 bp which spans the P1 promoter, including a binding site for the Sp1 transcription factor. In order to see if Sp1 binds to the promoter of this gene, ChIP assay was performed. Sp1 was shown to bind the region harboring the P1 promoter. In order to see if Sp1 has a role in the regulation of this gene, Sp1 constructs were co-transfected with the SLC22A18AS P1 promoter construct in HepG2 and Sp1-null Drosophila SL2 cells. The results showed that the Sp1 has a positive regulation on the SLC22A18AS promoter activity. As stated earlier, epigenetic marks such as differential methylation of CpG dinucleotides in two alleles are associated with promoters of the genes. Since the promoters for SLC22A18AS were characterized, the presence of allele-specific differentially methylated regions (DMRs) associated with the promoters was investigated. In order to differentiate the two alleles in the promoter regions by SNPs, DNA sequence analysis of the promoter regions was performed in a battery of 17 abortuses to search for SNPs. Abortus no. 3 showed heterozygosity for a C to A change at nucleotide position -445 in the P1 promoter region, while abortus no. 2 showed heterozygosities for G to A and A to G changes at nucleotide positions -919 and -1321 respectively in the P2 promoter region. The alleles in the abortus no. 3 were designated as allele C and allele A. The alleles in abortus no. 2 were designated as allele GG and allele AA. Once the two alleles were differentiated by these SNPs, identification of DMRs was performed using sodium bisulfite genomic DNA sequencing. Genomic DNA from the abortus no. 3 was taken for the identification of DMR in the P1 promoter region, while genomic DNA from abortus no. 2 was taken for the identification of DMR in the P2 promoter region. Sodium bisulfite genomic DNA sequencing of the P1 promoter region showed heavy methylation of both the alleles. No DMR was observed in this region. Sodium bisulfite genomic DNA sequencing of the P2 promoter region using DNA from abortus no. 2 did not show any differential methylation of the two alleles. However, like the P1 promoter region, the P2 promoter region was also heavily methylated. In order to see the methylation status of both the promoter regions in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. A dense methylation was observed in both the promoter regions of the gene. Heavy methylation of CpG dinucleotides in these regions corroborates the imprinting result for this gene. Since the methylation epigenetic mark is also known to be associated with CpG islands, CpG Plot/CpG Report analysis was used to identify CpG islands in this gene. The analysis showed the presence of two CpG islands, CpG I and CpG II, in the second intron of the gene. As the CpG I island is known to lack methylated CpGs (Ali et al., unpublished result from our lab), a DMR was sought for the CpG II island region. Heterozygosity was ascertained in this region by sequencing DNA from 17 abortuses. However, none of the abortuses showed heterozygosity. It was reasoned that if there is a differential methylation of the two alleles in this region, half of the clones (alleles) should be unmethylated, and the other half should show methylation. Therefore, DNA from abortus no. 3 was randomly chosen for sodium bisulfite genomic sequence anaylsis to identify DMR. The CpG II island showed heavy methylation. However, a DMR was not identified. In order to see the methylation status of the CpG II island in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. Almost all the CpG sites showed methylation. The observation of a dense methylation of both the promoters and CpG II island suggested that methylation has a role in the expression of this gene. In order to confirm this observation, A549 and HuH7 cells were treated with a methyltransferase inhibitor, 5-aza-2’-deoxycytidine. 5-Aza-2’-deoxycytidine treatment in HuH7 cells restored the expression of this gene. Further, the expression of this gene was increased in A549 cells following the drug treatment. These results suggested that DNA methylation has a definite role in the modulation of expression of the SLC22A18AS gene. Histone acetylation is another key epigenetic player which is known to have a role in the expression of genes. In order to study the role of the histone acetylation, HuH7 and A549 cells were treated with TSA, a histone deacetylase inhibitor. Treatment of HuH7 and A549 cells with TSA didn’t have any effect on the expression of this gene. On the other hand, the expression of TPA, a gene shown to be regulated by TSA earlier, increased following the TSA treatment in both cell lines. These results suggested that histone acetylation doesn’t have any effect on the expression of this gene. Based on this observation, it was reasoned that histone acetylation is not associated with the imprinting of this gene. Therefore, we did not look for the allele-specific acetylation of histones in this gene. The SLC22A18AS gene has a weak ORF of 253 amino acids as the translation intiation site does not contain a consensus Kozak sequence for efficient translation. In order to determine if it codes for a protein, Western blot analysis was performed using lysates from A549 cells and human fetal liver tissue, and a polyclonal antibody raised in a rabbit against a bacterially expressed SLC22A18AS protein fragment from amino acids 138 to 245. The Western blot result was negative. It was reasoned that this gene might be expressed at a low level and therefore its expression could not be detected by Western blot analysis. Immunoprecipitation was then performed to enrich the SLC22A18AS protein in the lysates followed by Western blot analysis. SLC22A18AS was shown to be expressed as a 30 kDa band in the immunoprecipiates from A549 cell and human fetal liver tissue lysates. The subcellular localization of this gene was studied by immunofluorescence. The fluorescence immunolocalization was performed on A549 cells with anti-SLC22A18AS antibody. The SLC22A18AS protein was found to be localized in the cytoplasm of A549 cells.

Eukaryotic genomes are packed into the confines of the nucleus through a nucleoproteic structure called chromatin. Chromatin is a dynamic structure that can respond to developmental or environmental cues to regulate and orchestrate the functions of the genome. The fundamental unit of chromatin, the nucleosome, consists of a protein octamer, which contains two molecules of each of the core histone proteins (H2A, H2B, H3, H4), around which 147 bp of DNA is wrapped. The post-translational modifications (PTMs) of histones and methylation of the cytosine residues in DNA (DNA methylation) constitute primary epigenomic markers that dynamically alter the interaction of DNA with nucleosomes and participate in the regulation and control access to the underlying DNA. The main objective of my thesis was to understand the spatial and temporal dynamics of chromatin states in Arabidopsis by investigating on a genome-wide scale, patterns of DNA methylation and a set of well-characterized histone post-translational modifications. DNA methylation, a hallmark of epigenetic inactivation and heterochromatin in both plants and mammals, is largely confined to transposable elements and other repeat sequences. I show in this thesis that in Arabidopsis, methylated TE sequences having no or few matching siRNAs, and therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery, acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, I propose that this spreading of DNA methylation through promoter regions can explain, at least in part, the negative impact of siRNA-targeted TE sequences on neighbouring gene expression. In a second part, I have contributed to integrative analysis of DNA methylation and eleven histone PTMs. I have shown through combinatorial and cluster analysis that the Arabidopsis epigenome shows simple principles of organisation and can be distinguished into four primary types of chromatin that preferentially index active genes, repressed genes, TEs, and intergenic regions. Finally, in a third part, I integrated epigenomics with transcriptome data at three different time points in a developmental window to investigate the temporal dynamics of chromatin states in response to an external stimulus. This used the light-induced transcriptional response as a paradigm to assess the impact of histone H2B monoubiquitination (H2Bub), and showed that this PTM is associated with active transcription and implicated in the selective fine-tuning of gene expression. Taken together, the work presented here contributes significantly to our understanding of the spatial organisation of chromatin states in plants, its dynamic nature and how it can contribute to allow plants to respond to a signal from the environment.

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.

Primates are one of the best characterized phylogenies with vast amounts of comparative data available, including genomic sequences, gene expression, and epigenetic modifications. Thus, they provide an ideal system to study sequence evolution, regulatory evolution, epigenetic evolution as well as their interplays. Comparative studies of primate genomes can also shed light on molecular basis of human-specific traits. This dissertation is mainly composed of three chapters studying human and non-human primate evolution. The first study investigated evolutionary rate difference between sex chromosome and autosomes across diverse primate species. The second study developed an unbiased approach without the need of prior information to identify genomic segments under accelerated evolution. The third study investigated interplay between genomic and epigenomic evolution of humans and chimpanzees. Research advance 1: evolutionary rates of the X chromosome are predicted to be different from those of autosomes. A theory based on neutral mutation predicts that the X chromosome evolves slower than autosomes (slow-X evolution) because the numbers of cell division differ between spermatogenesis and oogenesis. A theory based on natural selection predicts an opposite direction (fast-X evolution) because newly arising beneficial mutations on the autosomes are usually recessive or partially recessive and not exposed to natural selection. A strong slow-X evolution is also predicted to counteract the effect of fast-X evolution. In our research, we simultaneously studied slow-X evolution, fast-X evolution as well as their interaction in a phylogeny of diverse primates. We showed that slow-X evolution exists in all the examined species, although their degrees differ, possibly due to their different life history traits such as generation times. We showed that fast-X evolution is lineage-specific and provided evidences that fast-X evolution is more evident in species with relatively weak slow-X evolution. We discussed potential contribution of various degrees of slow-X evolution on the conflicting population genetic inferences about human demography. Research advance 2: human-specific traits have long been considered to reside in the genome. There has been a surge of interest to identify genomic regions with accelerated evolution rate in the human genome. However, these studies either rely on a priori knowledge or sliding windows of arbitrary sizes. My research provided an unbiased approach based on previously developed “maximal segment” algorithm to identify genomic segments with accelerated lineage-specific substitution rate. Under this framework, we identified a large number of human genomic segments with clustered human-specific substitutions (named “maximal segments” after the algorithm). Our identified human maximal segments cover a significant amount of previously identified human accelerated regions and overlap with genes enriched in developmental processes. We demonstrated that the underlying evolutionary forces driving the maximal segments included regionally increased mutation rate, biased gene conversion and positive selection. Research advance 3: DNA methylation is one of the most common epigenetic modifications and plays a significant role in gene regulation. How DNA methylation status varies on the evolutionary timescale is not well understood. In this study, we investigated the role of genetic changes in shaping DNA methylation divergence between humans and chimpanzees in their sperm and brain, separately. We find that for orthologous promoter regions, CpG dinucleotide content difference is negatively correlated with DNA methylation level difference in the sperm but not in the brain, which may be explained by the fact that CpG depleting mutations better reflect germline DNA methylation levels. For the aligned sites of orthologous promoter regions, sequence divergence is positively correlated with methylation divergence for both tissues. We showed that the evolution of DNA methylation can be affected by various genetic factors including transposable element insertions, CpG depleting mutations and CpG generating mutations.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Made available in DSpace on 2015-05-14T12:56:02Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1722534 bytes, checksum: d8557488d35ade21ba45951fd8412a2f (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / DNA methylation, characterized by the addition of a methyl group in cytosines within CpG dinucelotides can modified gene transcription, leading to decrease or even silence a gene. The ability of the environmental factors to induce epigenetic changes has been investigated and many studies have shown a relationship between them. Studies show that pesticides, metal ions, drugs, diet, alcohol dependence and smoking are associated with epigenetic changes. Smoking is often associated with the risk of cancer in various tissues and cardiovascular diseases, being considered the leading cause of preventable death. The MLH1 gene is related to the repair of badly paired bases of DNA (DNA mismatch repair (MMR)). The hTERT gene comprises the catalytic subunit of telomerase enzyme, which is considered a biological clock, a marker indicating that the cellular senescence can be installed inevitably form. The TP53 is a tumor suppressor gene and its hypermethylation is related to the development of various cancers. The aim of this work was to investigate the smoking habit influence on DNA methylation status in the promoter of cancer-related genes, MLH1, hTERT and TP53 in oral epithelial cells of healthy subjects. Samples of oral epithelium of smokers, nonsmokers and former smokers were collected by rinsing and DNA was extracted. After, DNA Methylation analysis was performed by Methylation Sensitive Restriction Enzymes, using two restriction enzymes, the HpaII and HhaI, which cleave different sites. Following the enzymatic digestion, DNA was amplified by PCR, subjected to electrophoresis on a 6% polyacrylamide gel and stained with silver nitrate. Statistical analysis was performed by Chi-square test at a significance level of 5%. The investigated CpG dinucleotides located at HhaI and HpaII sites in the MLH1 gene promoter were observed to be fully methylated in DNA majority samples from the smoker group and statistical differences were found between nonsmokers and smokers and between smokers and former smokers (p<0.05). The same was observed in the hTERT gene promoter at HhaI site (p<0.05) and for HpaII site the unmethylated condition was more frequent in smoker in comparison to nonsmokers (p<0.05). For TP53 no differences were found among groups (p>0.05) which the fully methylated condition was found to be an usual event in healthy oral epithelial cells. We conclude that smoking may induce changes in DNA methylation status in cancer-related genes, such as MLH1 and hTERT of healthy oral epithelial cells and the cessation of smoking reversed the process. / A metilação de DNA é uma modificação química na molécula de DNA, e consiste na presença de um radical metil em dinucleotídeos CpG, presente principalmente em regiões promotoras do gene. Uma das principais funções da metilação de DNA é regular a transcrição gênica, sendo que a presença do radical metil pode suprimir por completo a expressão gênica. Estudos mostram que o meio ambiente pode modular a metilação de DNA. Como exemplo de fatores ambientais temos: a radiação ultravioleta, agrotóxicos, dieta, fármacos, uso crônico do álcool e o hábito de fumar. O fumo é frequentemente associado ao risco de câncer em diversos tecidos e doenças cardiovasculares, sendo considerado a maior causa de morte evitável. O gene MLH1 está relacionado ao reparo de bases mal pareadas do DNA (DNA mismatch repair (MMR)). O gene hTERT compõe a subunidade catalítica da enzima telomerase, a qual é considerada um relógio biológico, um marcador que indica que a senescência celular poderá se instalar de forma inevitável. O TP53 é um gene supressor tumoral e sua hipermetilação está relacionada ao desenvolvimento de diversos tipos de câncer. Assim, o objetivo deste estudo foi investigar o efeito do tabagismo no perfil de metilação de DNA em genes relacionados ao câncer, MLH1, hTERT e TP53 em células da mucosa bucal de indivíduos saudáveis. Para tanto, amostras de epitélio da mucosa bucal de indivíduos fumantes, não fumantes e ex-fumantes foram coletadas por bochecho e o DNA dessas células foi extraído. Após esse processo, a análise de metilação de DNA foi feita utilizando o método de Digestão Enzimática Sensível à Metilação, utilizando-se de duas enzimas de restrição, a HhaI e a HpaII, as quais clivam sítios diferentes. Em seguida à digestão enzimática, DNA foi amplificado por PCR, submetido à eletroforese em gel de poliacrilamida a 6% e corado pelo nitrato de prata. A análise estatística foi realizada pelo Teste de Qui-Quadrado ao nível de significância de 5%. Os dinucleotídeos CpG localizados nos sítios HhaI e HpaII no promotor do gene MLH1 mostraram-se totalmente metilados na maioria dos indivíduos do grupo fumante e diferenças significativas foram observadas entre fumantes e não fumantes e entre fumantes e ex-fumantes (p<0,05). O mesmo foi observado para o sítio HhaI no promotor do gene hTERT (p<0,05) e para o sítio HpaII a condição não metilada foi mais frequente em fumantes em comparação com não fumantes (p<0,05). Para o gene TP53 não foram encontradas diferenças entre os grupos (p>0,05), sendo a condição totalmente metilada um evento usual das células saudáveis da mucosa bucal. Assim, concluímos que o fumo está associado a alterações no perfil de metilação de DNA em genes relacionados ao câncer, como MLH1 e hTERT em células epiteliais saudáveis da mucosa bucal e a cessação do hábito de fumar reverteu o processo.

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

La protéine UHRF1 est impliquée dans le maintien et la transmission des modifications épigénétiques. Lors du processus de réplication, elle recrute la méthyltransférase de l’ADN Dnmt1 au niveau des sites CpG hémi-méthylés via son domaine SRA (SET and RING Associated), favorisant la duplication des profils de méthylation. La structure tridimensionnelle du complexe SRA/ADN révèle que la protéine induit un basculement de la méthylcytosine qui permet un ancrage spécifique de la protéine sur les sites hémim éthylés, facilitant le recrutement de la Dnmt1 au niveau de ces positions stratégiques. Dans ce contexte, notre projet vise à comprendre les mécanismes d’interaction du domaine SRA de UHRF1 avec l’ADN hémi-méthylé. Des oligonucléotides doubles brins ont été marqués à la 2-aminopurine, un analogue nucléosidique fluorescent sensible à l’environnement, à différentes positions au voisinage d’un unique site de reconnaissance CpG hémi-méthylé. Les mesures de spectroscopie de fluorescence à l’état stationnaire et résolues en temps de ces duplexes liés au domaine SRA nous ont permis de caractériser de manière site spécifique les changements conformationnels induits par la liaison du domaine SRA. En accord avec la structure tridimensionnelle du complexe SRA/ADN, nos données suggèrent que le domaine SRA est capable de basculer la méthylcytosine tout en préservant la structure des autres bases dans le duplexe. Le domaine SRA semble se lier selon le même mécanisme aux duplexes hémi-méthylés, bi-méthylés et non-méthylés. La protéine UHRF1 jouerait ainsi un rôle de “lecteur“ capable de scanner la séquence d’ADN à la recherche de sites hémi-méthylés. / The UHRF1 protein plays a key role in the maintenance and transmission of epigenetic modifications. Duringthe replication process, it recruits the DNA methyltransferase Dnmt1 to hemi-methylated CpG sites via itsSRA (SET and RING Associated) domain, promoting the duplication of the methylation profiles. Thetridimensional structure of the SRA/DNA complex revealed that the protein induces a base-flipping of themethylcytosine that enables a specific anchoring of the protein to hemi-methylated sites facilitating therecruitment of Dnmt1 to this strategic position. In this context, our project was aimed to further understand themechanism of interaction of the SRA domain with hemi-methylated DNA. To this end, oligonucleotideduplexes were labeled by 2-aminopurine, a fluorescent nucleoside analogue sensitive to environment, atvarious positions close to the single hemi-methylated CpG recognition site. Steady-state and time-resolvedfluorescence spectroscopy measurements of these duplexes bound to the SRA domain enabled us to sitespecificallycharacterize the conformational changes induced by the binding of this domain. In agreement withthe tridimensional structure of the SRA/DNA complex, our data suggest that the SRA domain is able to flip themethylcytosine while preserving the structure of the surrounding bases in the duplex. The SRA domain wasshown to bind with the same mechanism to hemi-methylated, fully-methylated and non-methylated duplexes.Our data suggest the UHRF1 protein plays a role of “reader” that scans the DNA sequence for hemimethylatedsites.