Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.

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The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases. In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through an NF-κB-dependent signaling pathway. Functional studies reveal that NOR1 increases monocyte adhesion by inducing the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Transient transfection and chromatin immunoprecipitation assays identify VCAM-1 as a bona fide NOR1 target gene in endothelial cells. Finally, we demonstrate that NOR1-deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE-/- mice by decreasing the macrophage content of the lesion. In smooth muscle cells (SMC), NOR1 was previously established as a cAMP response element binding protein (CREB) target gene in response to platelet-derived growth factor (PDGF) stimulation. CREB phosphorylation and subsequent binding of phosphorylated CREB to the NOR1 promoter play a critical role in inducing NOR1 expression. In this dissertation, we further demonstrate that histone deacetylase (HDAC) inhibition potentiates and sustains PDGF-induced NOR1 mRNA and protein expression in SMC. This augmented NOR1 expression is associated with increased phosphorylation of CREB, recruitment of phosphorylated CREB to the NOR1 promoter, and trans-activation of the NOR1 promoter. Additionally, HDAC inhibition also increases NOR1 protein half-life in SMC. Collectively, these findings identify a novel pathway in endothelial cells underlying monocyte adhesion and expand our knowledge of the epigenetic mechanisms orchestrating NOR1 expression in SMC. Finally, we establish a previously unrecognized atherogenic role of NOR1 in positively regulating monocyte recruitment to the vascular wall.

The genetic information required for the existence of a living cell of any kind is encoded in the sequence information scripted in the double helix DNA. A modern trend in biology struggles to come to grip with the amazing fact that there are so many different cell types in our body and that they are directed from the same genomic blueprint. It is clear, that the key to this feature is provided by epigenetic information that dictates how, where and when genes should be expressed. Epigenetic states “dress up” the genome by packaging it in chromatin conformations that differentially regulate accessibility for key nuclear factors and in coordination with differential localizations within the nucleus will dictate the ultimate task, expression. In the imprinted Igf2/H19 domain, this feature is determined by the interaction between the chromatin insulator protein CTCF and the unmethylated H19 imprinting control region. Here I show that CTCF interacts with many sites genome-wide and that these sites are generally protected from DNA methylation, suggesting that CTCF function has been recruited to manifest novel imprinted states during mammalian development. This thesis also describes the discovery of an epigenetically regulated network of intra and interchromosomal complexes, identified by the invented 4C method. Importantly, the disruption of CTCF binding sites at the H19 imprinting control region not only disconnects this network, but also leads to significant changes in expression patterns in the interacting partners. Interestingly, CTCF plays an important role in the regulation of the replication timing not only of the Igf2 gene, but also of all other sequences binding this factor potentially by a cell cycle-specific relocation of CTCF-DNA complexes to subnuclear compartments. Finally, I show that epigenetic marks signifying active or inactive states can be gained and lost, respectively, upon exposure to stress. As many genes belonging to the apoptotic pathway are upregulated we propose that stress-induced epigenetic lesions represent a surveillance system marking the affected cells for death to the benefit of the individual. This important observation opens our minds to the view of new intrinsic mechanisms that the cell has in order to maintain proper gene expression, and in the case of misleads there are several check points that direct the cell to towards important survival decisions.

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

Orientador: Flavia Lombardi Lopes / Banca: Silvia Helena Venturoli Perri / Banca: José Fernando Garcia / Banca: Ricardo da Fonseca / Banca: José bento Sterman Ferraz / Resumo: O objetivo desse estudo foi identificar polimorfismos de nucleotídeo único (SNPs) potencialmente sujeitos a controle epigenético exercido por metilação do DNA via seus envolvimentos na criação, remoção ou deslocamento de sítios CpG (meSNPs) e a partir de tal identificação criar um banco de dados para meSNPs, bem como determinar a possível associação desses marcadores com ilhas CpG (CGIs) e com o perfil metilacional de tecidos submetidos ao ensaio de recuperação de ilhas CpG metiladas combinado com plataformas de sequenciamento de nova geração (MIRA-seq) em bovinos. Usando as variantes anotadas para os SNPs identificados no Run5 do projeto 1000 Bull Genomes e a sequência genômica bovina de referência UMD3.1.1, identificamos e anotamos 12.836.763 meSNPs de acordo com o padrão de variação criado por cada SNP em um sítio CpG. Também analisamos a distribuição genômica desses meSNPs, sendo a maioria deles localizados em regiões intergênicas (68,00%) e intrônicas (26,32%). Globalmente, os meSNPs representam 22,53% dos 56.969.697 SNPs descritos na base de dados e 12,35% deles estão localizados em CGIs. Comparando o número observado com o número esperado de meSNPs nas CGIs e nos tecidos submetidos ao MIRA-seq, verificamos um enriquecimento médio (P<0,01) para meSNPs de 2,47 vezes em CGIs relaxadas e 1,90 vezes em CGIs rigorosas. Nos tecidos, o enriquecimento foi de 1,52 vezes em longissimus dorsi e 2,09 vezes em intestino delgado. Dez meSNPs com metilação diferencial, sendo 1 em longi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to identify single nucleotide polymorphisms (SNPs) potentially subject to epigenetic control exerted by DNA methylation via their involvement in creating, removing or displacement CpG sites (meSNPs) and from this identification create a database for meSNPs, as well as to determine its possible association with CpG islands (CGIs) and the methylation profile of tissues submitted to the methylated-CpG island recovery assay combined with next generation sequencing platforms (MIRA-seq) in cattle. Using the variant annotations for SNPs identified in Run5 of the 1000 bull genomes project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the pattern of variation caused at the CpG site. We have also analyzed the genomic distribution of the meSNPs, with the majority being located in intergenic regions (68.00%) and then in introns (26.32%) and the remainder distributed among proximal promoters (3.93%), coding regions (1.27%), untranslated regions (UTRs) (0.29%), non-coding RNAs (0.11%) and splice regions (0.08%). Overall, meSNPs represent 22.53% of 56,969,697 SNPs described in the database of which 12.35% are located in CGIs. Comparing the observed number with the expected number of meSNPs in the CGIs and tissues submitted to the MIRAseq we found a mean enrichment (P<0.01) for meSNPs of 2.47 times in the relaxed CGIs and 1.90 times in the strict CGIs. In the tissues the enrichment was of 1.52... (Complete abstract click electronic access below) / Doutor

Biological molecules often undergo large structural changes to perform their function. Computational methods can provide a fine-grained description at the atomistic scale. Without sufficient approximations to accelerate the simulations, however, the time-scale on which functional motions often occur is out of reach for many traditional methods. Natural Move Monte Carlo belongs to a class of methods that were introduced to bridge this gap. I present three novel applications for Natural Move Monte Carlo, two on proteins and one on DNA epigenetics. In the second part of this thesis I introduce a new protocol for the testing of hypotheses regarding the functional motions of biological systems, named customised Natural Move Monte Carlo. Two different case studies are presented aimed at demonstrating the feasibility of customised Natural Move Monte Carlo.

La méthylation de l’ADN est la marque épigénétique la plus connue. Elle consiste en l’ajout d’un groupement méthyle au niveau de la cytosine, produisant la 5-méthyl-cystosine (5mC). Cette réaction chimique est catalysée par des ADN méthyltransférases : DNMT1, DNMT3A et DNMT3B. Peu de choses sont connues concernant les changements de 5mC au cours des lignages cellulaires dans l’embryon et comment cette marque contribue à l’établissement ou au maintien de l’identité cellulaire. Nous avons cherché à mieux comprendre ces mécanismes en étudiant la 5mC dans deux lignages cellulaires : les cellules primordiales germinales (PGCs) et les cellules souches hématopoïétiques (HSCs). Nous avons généré les premiers méthylomes de ces cellules au cours de leur développement chez la souris. Chez les PGCs, nous avons mis en évidence l’existence de deux phases de reprogrammation de la 5mC. Une première phase entre E9,5 et E13,5, où le génome des PGCs se déméthyle et une phase de reméthylation entre E14,5 et E17,5, chez les gamètes mâles uniquement. Néanmoins, certaines régions, dont notamment les éléments transposables sont résistants à la vague de déméthylation. L’utilisation de souris conditionnellement, nous a permis de mettre en évidence l’implication des protéines DNMT1 et UHRF2 dans le maintien de la 5mC au niveau de ces séquences. Concernant les HSCs, nous avons mis en évidence qu’elles acquièrent un profil de 5mC qui leur est propre lors de deux phases. La première a lieu dès l’apparition des HSCs dans l’organisme tandis que l’acquisition de la signature hématopoïétique définitive se déroule à l’âge adulte dans la moelle osseuse. L’utilisation de souris conditionnelles, nous a permis de mettre en exergue l’implication de DNMT3A et DNMT3B dans la mise en place de ces profils, avec un rôle prépondérant de DNMT3B lors de la phase d’acquisition précoce et de DNMT3A lors du verrouillage de leur profil de 5mC. / The methylation of DNA is a well-known epigenetic mark. It consists in adding a methyl group to a cytosine producing the 5-methylcytosine (5mC). This is catalysed by the DNA methyltransferase (DNMT) family: DNMT1, DNMT3A and DNMT3B. Little is known about the changes in DNA methylation that follow lineage decisions in the embryo and how these contribute, establish or maintain cellular identities. We are addressing these questions using as a model the specification of mouse primordial germ cells (PGCs) and mouse hematopoietic stem cells (HSCs) in the mouse embryo. We generate the first genome-wide maps of 5mC during their development. These maps highlight two waves of DNA methylation in PGCs. The first one takes place between E9,5 and E13,5, where the genome demethylates while the second one corresponds to a remethylation phase only in male PGCs between E14,5 and E17,5. Nevertheless, some regions, notably the transposable elements, are resistant to this demethylation wave. We demonstrate the implication of DNMT1 and UHRF2 in maintaining the 5mC on these regions using transgenic mice presenting specific deletion in PGCs. In HSCs, the 5mC maps highlight two wave of DNA methylation. The first one correlates with the first appearance of the HSCs in early embryos while the second one corresponds to their migration to the bone marrow and seems to act as a definitive lock for their hematopoietic identity. Using transgenic mice presenting specific deletions in HSCs, we prove the implication of DNMT3A and DNMT3B in hematopoietic stem cells, with a major role in locking HSC identity of DNMT3B during the first wave and a DNMT3A during the second one respectively.