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NAD+-dependent histone deacetylase SIRT1 in the process of oogenesis, fertilization and early embryonic development
Valentová, Iveta ; Nevoral, Jan (advisor) ; Drutovič, David (referee)
SIRT1 is a histone deacetylase from the sirtuin family that affects epigenetic and non- epigenetic targets. We can assume that the known SIRT1 substrates are involved in the regulation of gametogenesis and early embryonic development. Our hypotheses say SIRT1 is present in oocytes and early embryos and it plays a physiological role in oocyte maturation, fertilization and early embryonic development. A mouse model of a conditional knock-out line producing Sirt1-deficient oocytes was developed to verify our hypotheses. Oocytes and embryos were analyzed for SIRT1, its selected substrates and other markers by immunocytochemistry. We found out that the presence of SIRT1 contributes to oocyte quality through modulation of the chromatin histone code and stabilization of the spindle. Furthermore, the purely maternal origin of SIRT1 presents in both zygote pronuclei. Last but not least we discovered a significant effect of SIRT1 on early embryonic development, probably mainly due to its role in the activation of the embryonic genome. The results confirm our hypothesis that SIRT1 is present in oocytes and embryos mainly around chromatin. The results show that SIRT1 is a maternal factor determining oocyte quality and it is necessary for the embryonic genome activation.
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The role of SIRT1 during in vitro maturation of oocytes
Landsmann, Lukáš ; Nevoral, Jan (advisor) ; Šolc, Petr (referee)
SIRT1 histone deacetylase acts towards many epigenetic and non-epigenetic targets. The involvement of SIRT1 in oocyte maturation is assumed and the importance of ooplasmic SIRT1 pool for further destiny of matured oocyte is strongly suggested. We hypothesized that SIRT1 play role of the signal molecule in mature oocyte through selected epigenetic and non- epigenetic regulation. We observed SIRT1 re-localization in mature oocyte and the association with spindle microtubules. In matured oocyte, SIRT1 shows a spindle-like pattern and spindle- specific SIRT1 action is supported decreasing α-tubulin acetylation. Based on the observation of histone code in immature and matured oocytes, we suggest that SIRT1 is mostly predestined for epigenetic mode of action in germinal vesicle (GV) of immature oocyte. Accordingly, SIRT1- driven trimethylation of histone H3 on lysine K9 in matured oocyte is considered to be an inheritance of GV epigenetic transformation. Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocyte capable to be switched from epigenetic to the non- epigenetic mode of action readily depending on meiosis progress. Keywords: oocyte, SIRT1, histone, developmental competence, tubuline, epigenetics
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Molecular composition and ultrastructure of holokinetic chromosomes
Šejgunovová, Nikola ; Král, Jiří (advisor) ; Dalíková, Martina (referee)
Holokinetic chromosomes are a specific type of chromosomes which differentiate from standard (monocentric) chromosomes especially by a diffuse form of domain which binds microtubules (holocentromere). It is related to changes on an ultrastructural and molecular level. These changes are shown in modifications in mitotic and meiotic division and in evolution of karyotypes. Holokinetic chromosomes don't have a primary constriction with a localized centromere and therefore neither an inner centromere domain which would connect sister chromatids. Kinetochore structure of holokinetic chromosomes seems to be simpler than kinetochore structure of monocentric chromosomes. Kinetochore covers most of the surface of mitotic chromosomes. There have been described several variants of meiosis of holokinetic chromosomes which differentiate by position of kinetochore on chromosomes. On a molecular level holokinetic chromosomes differentiate from monocentric chromosomes by a distribution of proteins of a centromere-kinetochore complex, which cover most of the surfaces of mitotic and meiotic chromosomes. This applies, for example, to centromeric histone H3 (CENH3), whose amount and distribution changes during interphase and nuclear division, which is unique in comparison to monocentric chromosomes. The distribution...
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Molecular composition of constitutive heterochromatin
Pajpach, Filip ; Král, Jiří (advisor) ; Holá, Dana (referee)
Constitutive heterochromatin of eukaryotes includes various types of repetitive DNA and transposons characteristic for given region. DNA of centromeric and telomeric re- gions is usually highly methylated and transcribed to RNA transcripts, which participate in formation, functions and spreading of heterochromatin along with histones, their mod- ifications and non-histone proteins. The most typical histone modification in heterochro- matin is methylation, which forms the binding site for protein HP1. This protein (and his paralogues in other eukaryotes except for S. cerevisiae) participates in formation of com- plexes including other proteins like histone methylases SUV39H and their paralogues. Es- sential are also telosome proteins regulating telomeric heterochromatin, Polycomb group proteins and many others, for instance MBD1, Epe1, SUMO and DNA methylases DNMT. Many proteins form complexes, which partake in mechanisms necessary for heterochro- matin maintenance, for example RDRC and RITS complexes in RNA interference, SHREC complex in heterochromatin spreading, and PRC complexes forming heterochromatin in specific situations. Key words: centromere, DNA, histone, HP1, constitutive heterochromatin, methylation, modification, protein, RNA, specific, telomere
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Use of antibodies for the characterization of chromatin modifications in Saccharomyces cerevisiae
Kovaľová, Libuša ; Hodek, Petr (advisor) ; Malík, Radek (referee)
Transcription of precursor mRNA (pre-mRNA) and its splicing were originally conceived as two separate processes. Using Saccharomyces cerevisiae as a model, it was shown that the assembly of the complex catalyzing pre-mRNA splicing (spliceosome) can occur cotranscriptionally, i. e., during the time before the termination of transcription by RNA polymerase II. Research on cotranscriptional splicing revealed that proteins involved in transcription and specific chromatin modifications may affect pre-mRNA splicing and its regulation. It is also possible that spliceosome assembly and chromatin modifications can affect each other. Prp45, the yeast ortholog of the human transcription coregulator SKIP/SNW1, has been previously associated only with splicing. The results obtained in our laboratory suggest that Prp45 could be used as a regulator coupling the processes of transcription and splicing. We have shown that PRP45 has genetic interactions with factors important for transcription elongation, as well as chromatin modifications, and that it affects early stage of spliceosome assembly. The aim of this bachelor project was document the relationship between the physiological role of Prp45 and H3K4 trimethylation using chromatin immunoprecipitation. It was found that prp45(1-169) mutation does not markedly...
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Histone code and its regulation during early embryonic development in pigs
Jelínková, Pavla ; Žalmanová, Tereza (advisor) ; Miriama, Miriama (referee)
Both pronuclei of the zygote undergo epigenetic changes after fertilization, which determines the quality of the zygote and successful early mammalian embryonic development. Shortly after fertilization epigenetic asymmetry among the pronuclei of the zygote is evident, while the paternal pronucleus undergoes active DNA demethylation, the DNA of the maternal pronucleus remains methylated. The male pronucleus in addition undergoes histone acetylation, whereas the histones of the female pronucleus remain methylated. Asymmetry of pronuclei and their epigenetic status predicts successful reprogramming of the genome, and thus the success of embryonic development. For the successful development of the embryo is therefore required correct formation of both of these pronuclei of the zygote and this formation of pronuclei is regulated by post-translational histone modifications called histone code. It was hypothesized that the histone code is regulated by the activity of NADP+ - dependent histone deacetylases, sirtuins.
In the experiment were used fully grown in vitro maturated pig oocytes that were fertilized with pig spermatozoa in vitro. After isolation of zygotes cultured with addition of the activator sirtuin resveratrol was performed immunofluorescence analysis of acetylated and methylated histone H3 at lysine K9 of pronuclei of the zygotes. From the results of control group asymmetry between the pronuclei of the zygote is evident; wherein the male pronucleus exhibits higher acetylation intensity contrast female pronucleus exhibits higher methylation intensity. After adding resveratrol to all experimental groups female pronucleus showed a significant increase of the methylated histone H3 at lysine K9, and contrary to the male pronucleus significant decrease of acetylated histone H3 at lysine K9.
Sirtuins are involved in the regulation of histone code in porcine zygote and it can be assumed that they also play a role during subsequent embryonic development, which is the subject of further study.
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