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Redox signaling to chromatin
Plšková, Zuzana
Chromatin is a highly dynamic structure, which is constantly subjected to regulation in response to environmental conditions. This response is mediated by chromatin modifiers, such as histone acetyltransferases, which deposit histone post-translational modifications, resulting in changes in gene expression. One of such modifiers in plants is GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5). Despite its importance in plant growth and development, it remains poorly understood how GCN5 is regulated. Emergine evidence points towards redox regulation of chromatin remodeling that can be executed via redox modifications of chromatin modifiers. To investigate possible redox regulation of GCN5, we used redox insensitive lines, carrying GCN5 with mutated cysteine to serine. Even though gcn5 mutants displayed enhanced susceptibility to paraquat-induced oxidative stress, the mutated lines phenocopied the wild type. We further probed the interactome of GCN5 to identify putative functional partners, whose association with GCN5 could be altered under oxidative stress conditions, or affected by its redox status. Mutating of a single cysteine residue in GCN5 did not result in significant changes of its interactome, suggesting that additional single and higher order mutants need to be explored.
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The role of the AGO-hook domain of histone chaperone SPT6L in regulation of gene expression
Kašpar, Tomáš ; Čermák, Vojtěch (advisor) ; Převorovský, Martin (referee)
AGO-hook domains present in some eucaryotic proteins are crucial for a binding family of ARGONAUTE proteins (AGO). These AGO proteins are essential in many biological processes regulating gene expression by small RNA (sRNA), which is complementary to the gene that is supposed to be influenced. This thesis claims to find the function of the putative AGO-hook domain of the protein SPT6L. SPT6L is an elongation factor and histone chaperon of a complex of RNA polymerase II (Pol II) where it is acting in the epigenetic marking of histones. SPT6L of Arabidopsis thaliana is one of two paralogues of SPT6 proteins, that is characteristic of the presence of the AGO-hook domain. This is a plant specificity of this protein. What's more, the function of this domain remains unknown. Despite this, it could be assumed that this domain is necessary for binding of AGO proteins in the complex of Pol II, and by these interactions, it can enable guidance of regulation of chromatin modification, or it can co-transcriptionally influence nascent transcripts of Pol II by the sRNA. This thesis casts light on the function of the AGO-hook domain of SPT6L in A. thaliana in processes of gene regulation and protein interactions. It claims to confirm the interaction of the AGO-hook domain of SPT6L protein with AGO proteins and...
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Role of Smarca5 (Snf2h) during transcription of transfected DNA template.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Smetana, Karel (referee)
Cellular and tissue characteristics are results of dynamic regulation of gene expression. DNA wrapped into proteins, referred to as chromatin, requires involvement of mechanisms guiding accessibility of specific sequences. In higher organisms, chromatin remodeling proteins are indispensable in regulating chromatin structure including ISWI ATPase SMARCA5. SMARCA5 is involved in almost any transaction on DNA including transcription, however precise in vivo role of SMARCA5 in these processes remains unknown. To advance understanding of specific role of SMARCA5 in the development of chromatin structure during transcription we devised cellular model in which SMARAC5 level is manipulated while chromatin structure development and transcriptional response are monitored. Our data indicate that the transfected DNA template that is transcribed is enriched with histone H3 and its specific methylation of Histone H3 lysine (K) 4, a mark of active chromatin structure. Overexpression of SMARCA5 results within the reporter gene coding sequence in ~2,5-3 fold increase of both H3 occupancy an its modification H3K4Me3. Increased DNA template commitment into chromatinization is associated with repression of reporter gene expression. These results are supported by studies indicating dynamic development of nucleosomal...
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Production and analysis of cellular conditional inactivation models of the ISWI ATPase Smarca5
Tauchmanová, Petra ; Stopka, Tomáš (advisor) ; Burda, Pavel (referee)
The eukaryotic nuclear processes such as replication, DNA damage repair (DDR) and transcription are highly dependent on the regulation of chromatin structure. The dynamic changes in chromatin accessibility are controlled by a class of chromatin-remodeling factors which form multimeric complexes and use ATP as the source of their helicase activity. In this study we have established a mouse embryonic fibroblast in vitro model with conditional inactivation of chromatin remodeling ATPase Smarca5 and used this powerful tool to test the regulation of cell cycle, proliferation and DDR signaling in conditions with low Smarca5 activity. Our results show that decreased dosages lead to decreased proliferation apparent already within few days post induction of Smarca5 deletion that is accompanied with decrease of cells in S and M phases of cell cycle, increasing cell ploidy and accelerated cell senescence. Additionally, the Smarca5 depleted cells upregulated many protein markers associated with DNA damage and cellular stress. Our results thus indicate that Smarca5 has indispensable roles during cell proliferation including in the maintenance of genome integrity during S phase of cell cycle.
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Determinants of the splice site selection in protein-coding and long non-coding RNAs
Krchňáková, Zuzana ; Staněk, David (advisor) ; Svoboda, Petr (referee) ; Blažek, Dalibor (referee)
In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...
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The role of transcription factors PU.1 a GATA-1 during leukemia differentiation.
Burda, Pavel ; Stopka, Tomáš (advisor) ; Kořínek, Vladimír (referee) ; Machová Poláková, Kateřina (referee)
Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...
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Actin and the ARP 2/3 complex in the nucleus
Němcová, Barbora ; Bellinvia, Erica (advisor) ; Hála, Michal (referee)
The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. Actin constitutes a wide family of proteins that are major components of the cytoskeleton. Actin is one of the most abundant proteins in living organisms. Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes. Recent studies have shown that actin is actively transported from the cytoplasm to the nucleus, where it regulates transcriptional aktivity, regulates RNA polymerases, is involved in chromatin remodeling and repair damaged DNA. The presence of typical actin filaments in the nucleus has not been demonstrated directly.but nuclear actin occurs in many forms such as actin rods, short actin polymers, actin monomers, or actin complexes with profilin or cofilin. Most eukaryotic cells also contain at least eleven actin-related proteins (ARPs). Although many ARPs are cytoskeletal, recent biochemical and genetic work has demonstrated that some ARPs function largely or entirely in the nucleus. Nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the...
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Role of sequence context in DNA methylation
Polák, Jan ; Fischer, Lukáš (advisor) ; Širl, Marek (referee)
Cytosine methylation of DNA is a pivotal epigenetic mark, which contributes to the regulation of the gene expresion, silencing of transposable elements, and co-defines chromatine state. There are three cytosine contexts: CG, CHG and CHH (where H stands for C, A, or T). Arabidopsis thaliana (and plants in general) has an arsenal of molecular mechanisms capable of cytosine methylation in all of its contexts. That said, there are two tasks at hand: maintaining of pre-existing methylation and if need be, creating new methylated spots. The actual process of maintaining of the methylation depends on the cytosine context. Methylation of symmetrical contexts of CG and CHG can utilize the information about the methylation pattern from the second DNA strand. The aymmetrical context of CHH, and also CHG need to look for this information elsewhere: in the methylation of the lysine 9 of H3 histone. This creates a self-reinforcing loop and a crosstalk between two epigenetic mechanisms. Maintaince of methylation of CHH is also navigated by small RNA complementary to the locus in question. This mechanism of enzyme navigating by RNA is also used in establishing a new methylated site for all of the contexts. CG methylation is most prevalent in both heterochromatine and euchromatine. It also has a special functions...
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Epigenetic Aspects of normal and malignant hematopoiesis: role of chromatin remodeling ISWIATPase.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Dráber, Peter (referee) ; Otáhal, Pavel (referee)
Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...
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Determinants of the splice site selection in protein-coding and long non-coding RNAs
Krchňáková, Zuzana ; Staněk, David (advisor) ; Svoboda, Petr (referee) ; Blažek, Dalibor (referee)
In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...
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