Lysine-specific Demethylase 1B (KDM1B) is a chromatin regulator which functions as a histone eraser through the removal of the post-translational modifications mono and dimethylation of histone 3 on lysine 4 (H3K4me1/2). This process is enhanced by the formation of a complex with Nuclear Protein Glyoxylate Reductase (NPAC). NPAC resolves the sequestration of the nucleosome histone tail to allow robust demethylation of H3K4me2 by KDM1B, during transcriptional elongation by RNA polymerase 2 (RNAP II). KDM1B is involved in many crucial processes during development. Its physiological functions include the establishment of maternal genomic imprints, reset of the epigenome during somatic cell reprogramming, and regulation of brown adipogenic differentiation. In light of this, the role of KDM1B in human embryonic stem cells (hESCs) is examined through CRISPR/Cas9-editing to further dissect its biological functions during embryogenesis. CRISPR-induced knockouts of KDM1B exhibited similar cell proliferation rate and expression of OCT4 and NANOG pluripotency markers to wildtype cells. Furthermore, KDM1B-/- clones were able to maintain their pluripotency potential by differentiating to all germ layers in teratoma and embryoid body formation assays. In addition, RNA-seq of KDM1B-/- clones showed enrichment of mesoderm lineage-related gene ontology (GO) terms in the downregulated differentially expressed genes. Thus, KDM1B is believed to be dispensable during the pluripotent stage of the cell but proved fundamental during later stages of development.

No description available.

<p>Lysine specific demethylase 1 (LSD1) is a FAD-dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor &alpha; (ER&alpha;), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2-arylcyclopropylamines as irreversible mechanism-based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ER&alpha;-postive and ER&alpha;-negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis. </p><p>Furthermore, we observe that LSD1 and ER&alpha; work cooperatively to express certain estrogen-target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ER&alpha; recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ER&alpha;, we believe that CoREST may be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST <italic>in vitro</italic> in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity.</p> / Dissertation

Le mercure (Hg) est un polluant métallique préoccupant de par sa toxicité et son omniprésence dans les écosystèmes aquatiques. Sous sa forme méthylée, il est capable de se bioaccumuler dans les organismes et d’être bioamplifié le long de la chaîne trophique. La méthylation du Hg est un processus biotique principalement attribué aux microorganismes sulfato-réducteurs (MSR). La rhizosphère des plantes aquatiques a été récemment identifiée comme un compartiment privilégié de la méthylation du Hg dans certains écosystèmes tropicaux et boréaux. Les objectifs de cette étude étaient de déterminer l’influence des plantes aquatiques sur la biogéochimie et la bioaccumulation du Hg et le rôle que jouent les MSR dans ce processus au sein des écosystèmes aquatiques landais. L’utilisation de traceurs isotopiques stables du Hg a permis d’identifier le compartiment « plantes aquatiques » comme un lieu privilégié des transformations des espèces mercurielles (méthylation/ déméthylation du Hg) et comme la principale source de méthylmercure (MeHg) dans ces écosystèmes tempérés. La combinaison des approches moléculaires (T-RFLP, clonage, séquençage) et culturales (isolement, détection de MeHg par biosenseur) a démontré l’implication de MSR du genre Desulfovibrio dans le processus de méthylation du Hg au sein de la rhizoplane aquatique. D’après une expérience menée en microcosmes utilisant un traceur isotopique du Hg, le MeHg formé au niveau de la rhizosplane aquatique serait biodisponible pour la chaîne trophique. Cette dernière observation est à relier à des concentrations en Hg significatives, observées in situ, pour certains poissons de fin de chaîne alimentaire. / Mercury (Hg) is a metallic pollutant worrying because of its toxicity and ubiquity in aquatic ecosystems. Its organic form is easily bioaccumulated in organisms and biomagnified along food webs. Hg methylation is a biotic process mainly attributed to sulfate-reducing prokaryotes (SRP). The rhizoplane of aquatic plants has recently been identified as the principal compartment involved in Hg methylation in some tropical and boreal ecosystems. The objectives of this study were to determine the influence of aquatic plants on the biogeochemistry and bioaccumulation of Hg and the role of SRP in this process in the aquatic ecosystems of the Landes (South Western France). The use of Hg stable isotopic tracers allowed to identify the "aquatic plants" compartment as the main place for Hg species transformations (methylation / demethylation of Hg) and the main source of methylmercury (MeHg) in these temperate ecosystems. The combination of molecular (T-RFLP, cloning, sequencing) and cultural (isolation, MeHg detection by biosensor) approaches demonstrated the involvement of populations related to the genus Desulfovibrio in the process of Hg methylation in the aquatic rhizoplane. According to an experiment conducted in microcosms using a Hg isotopic tracer, MeHg formed in the aquatic rhizoplane seems to be bioavailable to the food chain. This last observation is linked to significant Hg concentrations, observed in situ, for some carnivorous fishes (end of the food chain).

As células-tronco embrionárias (CTEs), extraídas da massa celular interna do blastocisto, tem como características principais a capacidade de auto-renovação e a pluripotência. Durante o desenvolvimento, as células perdem seu potencial de diferenciação e adquirem um perfil de expressão gênica mais restrito, modulado por mecanismos epigenéticos, assim como por microRNAs. Membros da família miR-29 têm como transcritos alvos enzimas responsáveis pela metilação da citosina em 5mC (DNMT3a e 3b) e também da desmetilação (TET1, 2 e 3) do DNA, pela hidroxilação de 5mC em 5hmC. Recentes trabalhos sugerem que a modulação do miR-29 sobre estes alvos teria um papel no início da diferenciação em CTEs de camundongos e no aumento de eficiência da geração de iPS em células humanas. No presente trabalho, buscou-se compreender o papel regulatório do miR-29a em seus alvos epigenéticos no contexto da pluripotência e no início da diferenciação com atRA. Para tanto, duas linhagens celulares pluripotentes humanas (H1 e NTera- 2) foram submetidas a indução de diferenciação com atRA e ao ganho de função do miR-29a durante quatro dias de cultivo para análises de expressão gênica. Ademais, em NT2, realizamos ensaios funcionais por microscopia de imunofluorescência quantitativa para avaliar os efeitos do ganho e perda de função do miR-29a, DNMT3b e TET1, sobre a expressão nuclear de OCT4 e os perfis globais de 5mC e 5hmC após 96 horas de transfecção. Neste ensaio, também avaliamos o papel específico da regulação pós-transcricional de DNMT3b e TET1 pelo miR-29a, utilizando moléculas bloqueadoras dos sítios alvo (TSB) do miR-29a nestes transcritos. Observamos que sob a indução do atRA, os níveis de expressão do miR- 29a e de seus genes alvos (com exceção de DNMT3b), assim como dos marcadores de endoderme e ectoderme, aumentaram, seguido da diminuição dos marcadores de pluripotência em ambas as linhagens. A transfecção de moléculas mímicas do miR-29a, reduziu os níveis de seus transcritos alvos após dois e quatro dias em NT2 e H1, além de reduzir os níveis nucleares de DNMT3b em NT2. Ainda, ocorreu um aumento na expressão de genes da endoderme, mesoderme e ectoderme em H1 e a queda da expressão gênica e nuclear de OCT4 em NT2. Com o uso de siRNA específicos, demonstramos que o knockdown dos níveis nucleares de DNMT3b foi acompanhado de uma queda nos níveis globais de 5mC e um aumento de OCT4 e de 5hmC. Já o knockdown de TET1, elevou os níveis de 5mC, mas também os níveis de 5hmC e OCT4 nuclear. As avaliações com o uso de TSB contra os sítios de ligação do miR-29a em seus transcritos alvo, TET1 e DNMT3b, demonstraram que em células NT2, o bloqueio da ligação do miR endógeno aos seus alvos resultam no aumento dos níveis globais de 5hmC, indicando que a regulação póstranscricional destes alvos pelo miR-29 teria um importante papel na regulação epigenética de células pluripotentes. / Embryonic stem cells (CTEs), extracted from the internal cell mass of the blastocyst, are main characterized by the capacity for self-renewal and pluripotency. During development, the cells lose their differentiation potential and acquire a restricter gene expression profile, modulated by epigenetic mechanisms, as microRNAs. Members of the miR-29 family have as target transcripts enzymes for cytosine methylation in 5mC (DNMT3a and 3b) and for DNA demethylation (TET1, 2 and 3), by hydroxylation of 5mC in 5hmC. Recent studies suggest that the modulation of miR-29 on these targets plays a role in early differentiation of mouse CTEs and in increasing human iPS cell generation efficiency. In the present study, we sought to understand the regulatory role of miR-29a in its epigenetic targets in the context of pluripotency and in early differentiation with atRA. For this, two human pluripotent cell lines (H1 and NTera-2) were submitted to differentiation induction with atRA and function gain of miR-29a during four days of culture for gene expression analysis. Furthermore, in NT2, we performed functional assays by quantitative immunofluorescence microscopy to evaluate the gain- and loss-of-function of miR-29a, DNMT3b and TET1 in the OCT4 nuclear expression and global profiles of 5mC and 5hC, 96 hours posttransfection. In this assay, we also evaluated the specific role of post-transcriptional regulation of DNMT3b and TET1 by miR-29a, using target site blocking molecules (TSB) of miR-29a. We observed that under the induction of atRA, the miR-29a expression levels and its target genes (except of DNMT3b), further the markers of endoderm and ectoderm, increased, followed by decreased pluripotency markers in both cell lines. Transfection of mimic molecules of miR-29a reduced the levels of their target transcripts after two and four days in NT2 and H1, and reduced nuclear levels of DNMT3b in NT2. In addition, the expression of endoderm, mesoderm and ectoderm genes increased in H1 and gene and nuclear expression of OCT4 decreased in NT2. With the use of specific siRNA, we demonstrated that the knockdown of nuclear levels of DNMT3b was accompanied by a drop in global 5mC levels and an increase of OCT4 and 5hmC. While, the knockdown of TET1 increased the levels of 5mC, 5hmC and nuclear OCT4. Evaluations using TSB against the miR- 29a binding sites in their target transcripts, TET1 and DNMT3b, show that in NT2 cells blocking the binding of endogenous miR to their targets results in an increase in global 5hmC levels, indicating that the post-transcriptional regulation of these targets by miR-29 would play an important role in the epigenetic regulation of pluripotent cells.

In Saccharomyces cerevisiae, Set1p-mediated deposition of trimethylation on lysine 4 of histone H3 is a histone modification often associated with active transcription. Recently, it was discovered that members of the Jumonji family of proteins have the enzymatic ability to remove methylation on histone lysine residues. Here, I describe the function of the yeast Jumonji protein Jhd2p, the only yeast Jumonji with known demethylase activity towards histone H3 lysine 4 methylation. I find that during the development program of yeast sporulation, Jhd2p is responsible for demethylating lysine 4 on a global scale. Further, ChIP analysis examining lysine 4 methylation levels reveals that genes whose expression is dependent on JHD2 during sporulation are subject to what appears to be Jhd2p-mediated demethylation. Additionally, synthetic dosage lethality screens performed to identify genetic interactors of Jhd2p revealed that Jhd2p is a likely component of mitochondrial retrograde signaling, working alongside the transcription factors Rtg1p/Rtg3p.

In Saccharomyces cerevisiae, Set1p-mediated deposition of trimethylation on lysine 4 of histone H3 is a histone modification often associated with active transcription. Recently, it was discovered that members of the Jumonji family of proteins have the enzymatic ability to remove methylation on histone lysine residues. Here, I describe the function of the yeast Jumonji protein Jhd2p, the only yeast Jumonji with known demethylase activity towards histone H3 lysine 4 methylation. I find that during the development program of yeast sporulation, Jhd2p is responsible for demethylating lysine 4 on a global scale. Further, ChIP analysis examining lysine 4 methylation levels reveals that genes whose expression is dependent on JHD2 during sporulation are subject to what appears to be Jhd2p-mediated demethylation. Additionally, synthetic dosage lethality screens performed to identify genetic interactors of Jhd2p revealed that Jhd2p is a likely component of mitochondrial retrograde signaling, working alongside the transcription factors Rtg1p/Rtg3p.

As células-tronco embrionárias (CTEs), extraídas da massa celular interna do blastocisto, tem como características principais a capacidade de auto-renovação e a pluripotência. Durante o desenvolvimento, as células perdem seu potencial de diferenciação e adquirem um perfil de expressão gênica mais restrito, modulado por mecanismos epigenéticos, assim como por microRNAs. Membros da família miR-29 têm como transcritos alvos enzimas responsáveis pela metilação da citosina em 5mC (DNMT3a e 3b) e também da desmetilação (TET1, 2 e 3) do DNA, pela hidroxilação de 5mC em 5hmC. Recentes trabalhos sugerem que a modulação do miR-29 sobre estes alvos teria um papel no início da diferenciação em CTEs de camundongos e no aumento de eficiência da geração de iPS em células humanas. No presente trabalho, buscou-se compreender o papel regulatório do miR-29a em seus alvos epigenéticos no contexto da pluripotência e no início da diferenciação com atRA. Para tanto, duas linhagens celulares pluripotentes humanas (H1 e NTera- 2) foram submetidas a indução de diferenciação com atRA e ao ganho de função do miR-29a durante quatro dias de cultivo para análises de expressão gênica. Ademais, em NT2, realizamos ensaios funcionais por microscopia de imunofluorescência quantitativa para avaliar os efeitos do ganho e perda de função do miR-29a, DNMT3b e TET1, sobre a expressão nuclear de OCT4 e os perfis globais de 5mC e 5hmC após 96 horas de transfecção. Neste ensaio, também avaliamos o papel específico da regulação pós-transcricional de DNMT3b e TET1 pelo miR-29a, utilizando moléculas bloqueadoras dos sítios alvo (TSB) do miR-29a nestes transcritos. Observamos que sob a indução do atRA, os níveis de expressão do miR- 29a e de seus genes alvos (com exceção de DNMT3b), assim como dos marcadores de endoderme e ectoderme, aumentaram, seguido da diminuição dos marcadores de pluripotência em ambas as linhagens. A transfecção de moléculas mímicas do miR-29a, reduziu os níveis de seus transcritos alvos após dois e quatro dias em NT2 e H1, além de reduzir os níveis nucleares de DNMT3b em NT2. Ainda, ocorreu um aumento na expressão de genes da endoderme, mesoderme e ectoderme em H1 e a queda da expressão gênica e nuclear de OCT4 em NT2. Com o uso de siRNA específicos, demonstramos que o knockdown dos níveis nucleares de DNMT3b foi acompanhado de uma queda nos níveis globais de 5mC e um aumento de OCT4 e de 5hmC. Já o knockdown de TET1, elevou os níveis de 5mC, mas também os níveis de 5hmC e OCT4 nuclear. As avaliações com o uso de TSB contra os sítios de ligação do miR-29a em seus transcritos alvo, TET1 e DNMT3b, demonstraram que em células NT2, o bloqueio da ligação do miR endógeno aos seus alvos resultam no aumento dos níveis globais de 5hmC, indicando que a regulação póstranscricional destes alvos pelo miR-29 teria um importante papel na regulação epigenética de células pluripotentes. / Embryonic stem cells (CTEs), extracted from the internal cell mass of the blastocyst, are main characterized by the capacity for self-renewal and pluripotency. During development, the cells lose their differentiation potential and acquire a restricter gene expression profile, modulated by epigenetic mechanisms, as microRNAs. Members of the miR-29 family have as target transcripts enzymes for cytosine methylation in 5mC (DNMT3a and 3b) and for DNA demethylation (TET1, 2 and 3), by hydroxylation of 5mC in 5hmC. Recent studies suggest that the modulation of miR-29 on these targets plays a role in early differentiation of mouse CTEs and in increasing human iPS cell generation efficiency. In the present study, we sought to understand the regulatory role of miR-29a in its epigenetic targets in the context of pluripotency and in early differentiation with atRA. For this, two human pluripotent cell lines (H1 and NTera-2) were submitted to differentiation induction with atRA and function gain of miR-29a during four days of culture for gene expression analysis. Furthermore, in NT2, we performed functional assays by quantitative immunofluorescence microscopy to evaluate the gain- and loss-of-function of miR-29a, DNMT3b and TET1 in the OCT4 nuclear expression and global profiles of 5mC and 5hC, 96 hours posttransfection. In this assay, we also evaluated the specific role of post-transcriptional regulation of DNMT3b and TET1 by miR-29a, using target site blocking molecules (TSB) of miR-29a. We observed that under the induction of atRA, the miR-29a expression levels and its target genes (except of DNMT3b), further the markers of endoderm and ectoderm, increased, followed by decreased pluripotency markers in both cell lines. Transfection of mimic molecules of miR-29a reduced the levels of their target transcripts after two and four days in NT2 and H1, and reduced nuclear levels of DNMT3b in NT2. In addition, the expression of endoderm, mesoderm and ectoderm genes increased in H1 and gene and nuclear expression of OCT4 decreased in NT2. With the use of specific siRNA, we demonstrated that the knockdown of nuclear levels of DNMT3b was accompanied by a drop in global 5mC levels and an increase of OCT4 and 5hmC. While, the knockdown of TET1 increased the levels of 5mC, 5hmC and nuclear OCT4. Evaluations using TSB against the miR- 29a binding sites in their target transcripts, TET1 and DNMT3b, show that in NT2 cells blocking the binding of endogenous miR to their targets results in an increase in global 5hmC levels, indicating that the post-transcriptional regulation of these targets by miR-29 would play an important role in the epigenetic regulation of pluripotent cells.

DNA methylation and demethylation are involved in regulation of gene expression. CpG clusters have been identified as hotspots of oxidative damages and mutagenesis. DNA base excision repair can remove oxidative DNA damage on CpG clusters and mediate an active DNA demethylation pathway. In this study, we examined the molecular mechanisms underlying interactions among DNA methylation, demethylation and BER. Our results demonstrated that a single 5-methylcytosine did not exhibit a significant effect on BER. Surprisingly we found that the abasic site completely inhibited the activity of thymine DNA glycosylase (TDG) leading to the sustainment of the mismatch efficiently extended by pol β. Interestingly, APE1 3’-5’ exonuclease could removed the mismatch. Our results demonstrate a molecular mechanisms underlying DNA base lesion and BER in maintenance of a normal DNA methylation pattern and a critical role of APE1 to combat pol β extension of the mismatch thereby reducing the introduction of mutagenesis.

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第13369号 / 論理博第1573号 / 新制||理||1666(附属図書館) / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM