L‘adaptation des bactéries à leur environnement résulte de régulations de l’expression génique pour optimiser leur physiologie aux conditions de culture. Le contrôle de la concentration des ARNm constitue l’une de ces régulations. Il dépend à la fois des variations de transcription et de dégradation des messagers. Si ces deux mécanismes sont bien étudiés à l’échelle moléculaire chez E. coli, leurs poids respectifs sur la régulation du niveau des transcrits à l’échelle du génome restent inconnus en raison de l’absence de données omiques relatives à la dégradation des ARNm lors de changements environnementaux. D’autre part, les paramètres déterminant la stabilité des messagers sont mal identifiés et n’ont jamais été hiérarchisés.Au cours de cette thèse, la stabilité de chacun des ARNm d’E. coli a été mesurée par la détermination du stabilome. Plus précisément, le temps de demi-vie de près de 70 % de tous les messagers a pu être déterminé de façon fiable pour quatre taux de croissance différents obtenus dans les mêmes conditions de culture à l’aide de chémostats. Pour la première fois, cette étude démontre qu’une croissance bactérienne plus rapide entraîne une augmentation globale de la dégradation des transcrits. L’intégration de ces données avec les données transcriptomiques montre que même si la transcription est le mécanisme principal de régulation du niveau des messagers, la dégradation exerce un effet inverse dans la plupart des cas. De plus, le rôle de la dégradation dans le contrôle de la concentration des ARNm s’accentue de façon significative avec l’augmentation du taux de croissance et affecte particulièrement les gènes impliqués dans le métabolisme carboné central. À partir des données de stabilité générées à différents taux de croissance, des approches de biologie intégrative ont permis d’identifier et de hiérarchiser les déterminants de la dégradation des ARNm. Ainsi, la concentration des messagers qui est le principal paramètre, mais aussi le biais de codon, la longueur de la séquence codante et la présence de certains motifs de séquence déterminent la stabilité d’un ARNm. Toutefois, si la hiérarchie des déterminants identifiés reste identique avec la variation du taux de croissance, la stabilité des ARNm de certaines catégories fonctionnelles en est dépendante. Cependant, d’autres déterminants du temps de demi-vie des messagers, en particulier à fort taux de croissance, restent encore à être identifiés. La protéine CsrA, appartenant au système Csr, est un exemple de régulateur post-transcriptionnel qui contrôle positivement ou négativement l’expression d’ARNm par divers mécanismes qui peuvent modifier leur stabilité. Toutefois, l’étendue de l’action de CsrA sur la stabilité des ARNm à l’échelle omique n’a jamais été étudiée. En comparant les stabilomes et transcriptomes d’une souche sauvage et d’une souche où l’activité de CsrA est diminuée, les effets indirects transcriptionnels de CsrA ont été mesurés et de nouveaux ARNm cibles de CsrA dont la stabilité est régulée par la protéine (en majorité stabilisés) ont été identifiés. De plus, la protéine CsrD, régulateur de la stabilité des ARN non codants CsrB/C, n’est pas impliquée dans la régulation de la stabilité des ARNm, mais agit sur la transcription de nombreux gènes indépendamment de son rôle au sein du système Csr. En conclusion, ces travaux ont permis de mieux appréhender les régulations de la stabilité des ARNm, en identifiant leurs déterminants et en caractérisant leur rôle et portée dans le contrôle de la concentration des messagers. Ils soulignent en particulier l’importance de ces régulations dans le processus d’adaptation bactérien / Bacterial adaptation to environment results from regulations of gene expression to optimize cell physiology to growth conditions. Control of mRNA concentration is one of those regulations. It depends on both variations of transcription and transcript degradation. Although these two mechanisms are well defined at the molecular level in E. coli, their respective impact on mRNA level regulation is still unknown at the genome scale because of a lack of omic data on mRNA stability during changing environment. Moreover, parameters determining messenger stability are not yet clearly identified and have never been ranked.During this PhD, the stability of each of the E. coli mRNAs was measured through stabilome determination. More precisely, the half-life of around 70 % of all messengers was reliably determined at four different growth rates obtained in the same growth conditions in chemostats. For the first time, this study demonstrated that increase of growth rate led to global increase of transcript degradation. Integration of these data with transcriptomic data showed that although transcription was the main mechanism which regulated mRNA level, messenger degradation exerted an opposite effect in most of the cases. The role of messenger degradation in the control of mRNA concentration was significantly accentuated with increasing growth rate and affected particularly genes involved in central carbon metabolism. Using mRNA stability data produced at different growth rates, integrative biology approaches allowed identification and ranking of the determinants of messenger stability. mRNA concentration which was the main parameter, but also codon bias, length of the coding sequence, sequence motifs contributed to transcript stability. However, although the hierarchy of determinants remained identical with variations of growth rate, the stability of mRNAs belonging to specific functional categories differed with the growth rate. Nevertheless, other determinants of messenger half-life, in particular at high growth rates still remain to be discovered. The CsrA protein, which belongs to the Csr system, is one example of a post-transcriptional regulator. CsrA positively or negatively controls expression of several mRNAs by mechanisms able to modify transcript stability. Nevertheless, the extent of CsrA effect on mRNA stability at the omic level has never been studied. By comparing stabilomes and transcriptomes of the wild type strain with a strain with reduced CsrA activity, the indirect transcriptional effects of CsrA were measured and new mRNAs whose stability was targeted by CsrA (mostly stabilized), were identified. Moreover, the CsrD protein, a regulator of CsrB/C small RNA stability, was not involved in mRNA stability regulation, but played a role in transcriptional regulation of many genes independently of its role in the Csr system. To conclude, this work provides a better understanding of the regulation of the mRNA stability. It identifies mRNA stability determinants and characterizes the role and extent of mRNA stability regulation in the control of messenger concentration. The study underlines the importance of this regulation in the process of bacterial adaptation

Alterações epigenéticas, como metilação do DNA e modificações pós traducionais em histonas, tem importante papel na carcinogênese mamária. A modulação de eventos epigenéticos constitui relevante alvo terapêutico, devido ao seu caráter reversível. Experimentalmente, o ácido docosahexaenoico (DHA), um membro da família dos ácidos graxos ômega-3, é capaz de diminuir proliferação, induzir morte celular e diminuir o potencial invasivo de células tumorais de mama. No entanto, os mecanismos antitumorais do DHA e sua capacidade de modular eventos epigenéticos ainda não estão totalmente elucidados. Nosso objetivo foi verificar, in vitro, a ação do DHA em eventos epigenéticos em diferentes linhagens de carcinoma mamário humano. Três linhagens celulares de câncer de mama (MDA-MB-231, SKBR-3 e MCF-7) foram tratadas durante 72 horas com 100 ?M de DHA ou etanol (controle). As modificações pós traducionais em histonas, acetilação no resíduo de lisina 9 da histona 3 (H3K9ac) e no resíduo 16 da histona 4 (H4K16ac), bem como trimetilação no resíduo de lisina 9 da histona 3 (H3K9me3) e no resíduo de lisina 27 da histona 3 (H3K27me3) foram avaliadas pela técnica de western blot. A análise da expressão do genes RASSF1A, DNMT1, DNMT3A e DNMT3B foi feita pela técnica da reação em cadeia da polimerase quantitativa via transcriptase reversa (RT-qPCR). A avaliação do padrão de metilação de região promotora do gene RASSF1A foi realizada pela técnica de reação em cadeia da polimerase metilação específica (MS-PCR). Foram também analisadas as fases do ciclo celular por citometria de fluxo. Comparado ao controle, o DHA induziu a acetilação no resíduo 16 da histona 4 (H4K16ac) nas linhagens MCF7 (p = 0,04) e MDA-MB-231 (p = 0,03). Observamos que a H3K9me3 foi parcialmente inibida nas linhagens MDA-MB-231 e SKBR-3, após o tratamento com DHA, mas sem alcançar valor estatisticamente significante. Encontramos também diminuição dos níveis de H3K27me3 após o tratamento com DHA nas três linhagens estudadas, porém não foi estatisticamente significativo. O DHA aumentou a expressão do gene RASSF1A na linhagem MCF-7 (1,98 vezes, p = 0,03), mas não nas linhagens MDA-MB-231 e SKBR-3. Não houve mudanças estatisticamente significativas na expressão dos genes DNMT1, DNMT3A e DNMT3B. As análises qualitativas da metilação demonstraram que a região promotora analisada do gene RASSF1A apresentou-se hipermetilada nas três linhagens celulares. Após o tratamento com DHA, houve tendência de desmetilação na região promotora do RASSF1A na linhagem MCF-7 e SKBR3, mas não na linhagem MDA-MB-231. Não houve diferença significativa na porcentagem de morte e distribuição das células MDA-MB-231, SKBR-3 e MCF-7 nas diferentes fases do ciclo celular após tratamento com DHA. Em conclusão, o DHA pode atuar em mecanismos epigenéticos e, ainda, reativar o gene supressor de tumor, como RASSF1A, anteriormente silenciado por hipermetilação, em células MCF-7. Espera-se que esses resultados contribuam para melhor compreensão do potencial papel anticâncer do DHA no câncer de mama / Epigenetic changes, such as DNA methylation and post-translational histone modifications, play an important role in mammary tumorigenesis. Epigenetic events are important as therapeutic targets, because of its reversible nature. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids family, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanism of DHA and its ability to modulate epigenetic events are not completely understood. Our objective was to verify, in vitro, the action of DHA in epigenetic events related to transcriptional reactivation of tumor suppressor gene, such as RASSF1A, in different human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated with DHA (100 ?M) or vehicle (ethanol) for 72 hours. Western blot was used to analyze histone modification, as histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation, H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3). Real time quantitative PCR (RT-qPCR) was performed for gene expression quantification of RASSF1A, DNMT1, DNMT3A and DNMT3B. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR (MS-PCR). Moreover, we evaluated the phases of the cell cycle by flow cytometry. Compared to control cells, DHA induced H4K16ac in MCF-7 (p=0.04) and MDA-MB-231 (p=0.03). We observed that H3K9me3 was partially inhibited in MDA-MB-231 and SKBR-3 cells, after treatment with DHA, but did not reach a statistically significant value. We also found decreased levels of H3K27me3 after treatment with DHA in the three cell lines studied, but not statistically significant. DHA increased RASSF1A expression on MCF-7 (1.98 fold; p=0.03), but not in MDA-MB-231 and in SKBR-3 cells. There were no statistically significant changes in expression of genes DNMT1, DNMT3A and DNMT3B. These three breast cancer cells lines show methylation in specific region of RASSF1A promoter. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7 and SKBR-3. No significant difference was observed in the percentage of cell death nor cell distribution of MDA-MB-231, SKBR-3 and MCF-7 at different stages of the cell cycle after treatment with DHA. In conclusion, we suggest that DHA may act beneficially in epigenetic mechanisms and reactivation of tumor suppressor gene, RASSF1A as previously silenced by hypermethylation. It is hoped that these results can contribute to better understanding of the anticancer role of DHA in breast cancer

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes. / Classical major histocompatibility complex class II (MHCII) molecules are cell-surface glycoproteins specialized in the presentation of peptides, mainly derived from extracellular pathogens, to the antigen receptors of CD4+ T cells in the adaptive immune system. They are encoded, with those of the MHC class I, by the most polymorphic genes known to date, with multiple loci and high allelic diversity at each one. Moreover, the polymorphism within MHCII genes is not restricted to coding sequences. It has also been observed in promoters where it was shown to affect the expression level of the genes. Variation in gene regulation is believed to be an important factor from which modification in morphology, physiology or behaviour can be observed in all organisms. Repeated DNA sequences with functional roles in this regulation have been identified within the non-coding parts of the genomes. On the other hand, pathogen-driven selection is also believed to be important in the evolution and maintenance of the polymorphism of the MHC genes in vertebrates. Studies have shown the implication of different MHC alleles in disease resistance or susceptibility. In this study, our aims were to characterize the polymorphism of the MHIIb gene in brook charr (Salvelinus fontinalis), to document its effects on the survival conferred by specific alleles and/or genotypes following an infection and on the variation of the expression level of the gene in different environmental conditions. In a first part, we identified a total of 6 MHIIb alleles, designated Safo-DAB*0101 to Safo-DAB*0601, showing a high similarity to coding sequences from teleost fish and human. Analysis of the b1 domain sequences indicates the effect of a positive selection pressure to select polymorphic mutations in that region of the molecule. Four of these alleles were tested in a challenge experiment against the pathogen Aeromonas salmonicida to evaluate their effect on fish survival. We found that one allele, DAB*0101, was significantly associated with resistance to furonculosis. In addition to homozygotes for this allele, its resistance effect was also detected in the heterozygote individuals of the DAB*0101/*0201 genotype. In contrast, other allelic combinations, namely heterozygous genotypes DAB*0201/*0301 and DAB*0301/*0401 were significantly associated with increased susceptibility. Given that its frequency was relatively low in the population, the negative frequency dependant selection hypothesis could explain the advantage associated with the allele DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr. In a second part, we reported the identification of a polymorphic minisatellite formed of a 32 nucleotides motif in the second intron of MHIIb gene, and for which distinctive repeat numbers of the motif were associated to each alleles (69, 27, 20, 40, 19 and 25 repeats for the DAB*0101 to DAB*0601 alleles respectively). Relative expression levels of four alleles were determined in heterozygous fish at temperature of 18 ºC and 6 ºC. Results indicate that alleles carrying the longest minisatellite showed a 1.67 to 2.56-fold reduction in the transcript expression relatively to the shortest one. In contrast, no significant differences were seen in the expression levels between alleles with comparable minisatellite length at both temperatures. Furthermore, the repressive activity associated to the longest minisatellite was more effective at temperature of 18 ºC in fish from three different genotypes. We finally observed a significant 2.08-fold up-regulation of the total MHII transcript amount at 6 ºC. The results support the implication of repeated DNA sequences in the regulation of the gene transcriptional activity and suggest that a temperature-sensitive minisatellite could potentially be submitted to selective forces and therefore play an important role in gene expression and evolution in ectothermic organisms.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Alterações epigenéticas, como metilação do DNA e modificações pós traducionais em histonas, tem importante papel na carcinogênese mamária. A modulação de eventos epigenéticos constitui relevante alvo terapêutico, devido ao seu caráter reversível. Experimentalmente, o ácido docosahexaenoico (DHA), um membro da família dos ácidos graxos ômega-3, é capaz de diminuir proliferação, induzir morte celular e diminuir o potencial invasivo de células tumorais de mama. No entanto, os mecanismos antitumorais do DHA e sua capacidade de modular eventos epigenéticos ainda não estão totalmente elucidados. Nosso objetivo foi verificar, in vitro, a ação do DHA em eventos epigenéticos em diferentes linhagens de carcinoma mamário humano. Três linhagens celulares de câncer de mama (MDA-MB-231, SKBR-3 e MCF-7) foram tratadas durante 72 horas com 100 ?M de DHA ou etanol (controle). As modificações pós traducionais em histonas, acetilação no resíduo de lisina 9 da histona 3 (H3K9ac) e no resíduo 16 da histona 4 (H4K16ac), bem como trimetilação no resíduo de lisina 9 da histona 3 (H3K9me3) e no resíduo de lisina 27 da histona 3 (H3K27me3) foram avaliadas pela técnica de western blot. A análise da expressão do genes RASSF1A, DNMT1, DNMT3A e DNMT3B foi feita pela técnica da reação em cadeia da polimerase quantitativa via transcriptase reversa (RT-qPCR). A avaliação do padrão de metilação de região promotora do gene RASSF1A foi realizada pela técnica de reação em cadeia da polimerase metilação específica (MS-PCR). Foram também analisadas as fases do ciclo celular por citometria de fluxo. Comparado ao controle, o DHA induziu a acetilação no resíduo 16 da histona 4 (H4K16ac) nas linhagens MCF7 (p = 0,04) e MDA-MB-231 (p = 0,03). Observamos que a H3K9me3 foi parcialmente inibida nas linhagens MDA-MB-231 e SKBR-3, após o tratamento com DHA, mas sem alcançar valor estatisticamente significante. Encontramos também diminuição dos níveis de H3K27me3 após o tratamento com DHA nas três linhagens estudadas, porém não foi estatisticamente significativo. O DHA aumentou a expressão do gene RASSF1A na linhagem MCF-7 (1,98 vezes, p = 0,03), mas não nas linhagens MDA-MB-231 e SKBR-3. Não houve mudanças estatisticamente significativas na expressão dos genes DNMT1, DNMT3A e DNMT3B. As análises qualitativas da metilação demonstraram que a região promotora analisada do gene RASSF1A apresentou-se hipermetilada nas três linhagens celulares. Após o tratamento com DHA, houve tendência de desmetilação na região promotora do RASSF1A na linhagem MCF-7 e SKBR3, mas não na linhagem MDA-MB-231. Não houve diferença significativa na porcentagem de morte e distribuição das células MDA-MB-231, SKBR-3 e MCF-7 nas diferentes fases do ciclo celular após tratamento com DHA. Em conclusão, o DHA pode atuar em mecanismos epigenéticos e, ainda, reativar o gene supressor de tumor, como RASSF1A, anteriormente silenciado por hipermetilação, em células MCF-7. Espera-se que esses resultados contribuam para melhor compreensão do potencial papel anticâncer do DHA no câncer de mama / Epigenetic changes, such as DNA methylation and post-translational histone modifications, play an important role in mammary tumorigenesis. Epigenetic events are important as therapeutic targets, because of its reversible nature. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids family, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanism of DHA and its ability to modulate epigenetic events are not completely understood. Our objective was to verify, in vitro, the action of DHA in epigenetic events related to transcriptional reactivation of tumor suppressor gene, such as RASSF1A, in different human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated with DHA (100 ?M) or vehicle (ethanol) for 72 hours. Western blot was used to analyze histone modification, as histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation, H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3). Real time quantitative PCR (RT-qPCR) was performed for gene expression quantification of RASSF1A, DNMT1, DNMT3A and DNMT3B. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR (MS-PCR). Moreover, we evaluated the phases of the cell cycle by flow cytometry. Compared to control cells, DHA induced H4K16ac in MCF-7 (p=0.04) and MDA-MB-231 (p=0.03). We observed that H3K9me3 was partially inhibited in MDA-MB-231 and SKBR-3 cells, after treatment with DHA, but did not reach a statistically significant value. We also found decreased levels of H3K27me3 after treatment with DHA in the three cell lines studied, but not statistically significant. DHA increased RASSF1A expression on MCF-7 (1.98 fold; p=0.03), but not in MDA-MB-231 and in SKBR-3 cells. There were no statistically significant changes in expression of genes DNMT1, DNMT3A and DNMT3B. These three breast cancer cells lines show methylation in specific region of RASSF1A promoter. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7 and SKBR-3. No significant difference was observed in the percentage of cell death nor cell distribution of MDA-MB-231, SKBR-3 and MCF-7 at different stages of the cell cycle after treatment with DHA. In conclusion, we suggest that DHA may act beneficially in epigenetic mechanisms and reactivation of tumor suppressor gene, RASSF1A as previously silenced by hypermethylation. It is hoped that these results can contribute to better understanding of the anticancer role of DHA in breast cancer

In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

Breast cancer, as the most prevalent cancer in women, is responsible for more than 15% of new cancer cases and about 6.9% of all cancer-related death in the US. A major cause of therapeutic failure in breast cancer is the development of resistance to chemotherapy, especially for triple-negative breast cancer (TNBC). Therefore, how to overcome chemoresistance is the major challenge to improve the life expectancy of breast cancer patients. Our studies demonstrate that TNBC cells surviving the chronic treatment of chemotherapeutic drugs show significantly higher expression of the dual serine/threonine and tyrosine protein kinase (DSTYK) than non-treated parental cells. In our in vitro cellular models, DSTYK knockout via the CRISPR/Cas9-mediated technique results in apoptotic cell death of chemoresistant cells upon drug treatment. Moreover, DSTYK knockout promotes chemotherapeutic drug-induced tumor cell death in an orthotopic mouse model. These findings suggest that DSTYK exerts an important and previously unknown role in promoting chemoresistance. Our studies provide fundamental insight into the role of DSTYK in chemoresistance in TNBC cells and lay the foundation for the development of new strategies targeting DSTYK for improving TNBC therapy.