In the last two decades the study of changes in the genome function that are not induced by changes in DNA has consolidated a strong research field called ”epigenetics”. Chromatin state changes play an essential role in the regulation of transcription of many genes, thus controlling cell differentiation. A large part of these changes is due to histone modifications that alter the accessibility of the DNA. Current state of the art visualization methods for the analysis of epigenetic data sets are not suited to represent the relationship between the combinatorial pattern of histone modifications and their regulatory effects.

The family of transcription factors TEAD regulates the expression of genes that affect cell proliferation, differentiation and apoptosis. Activity of TEAD1 is regulated via the Hippo signaling pathway. General mechanism of tumor cell suppression by the Hippo signaling pathway remains unclear. C-MYC and GLUT1, the two key regulators of glycolysis, were recently described as targets of the Hippo signaling pathway in human leukemia cells. In this diploma thesis, the interaction of TEAD1 with M-CAT binding motifs was experimentally confirmed in the first exon of C-MYC gene. In addition, a new interaction of TEAD1 with M-CAT binding motifs has been found in the enhancer of C-MYC promoter and enhancer of GLUT1 promoter by ChIP analysis. Regulation of glucose metabolism by the Hippo signaling pathway may represent a new mechanism of tumor cell suppression. Key words: Gene regulation, transcription factors, chromatin immunoprecipitation, bioinformatics

The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia

Genetic discoveries have highlighted the role of gene expression dysregulation in both rare and common diseases. In particular, a large number of chromatin modifiers, transcription factors, and RNA-binding proteins have been implicated in neurodevelopmental diseases, including epilepsy, autism spectrum disorder, schizophrenia, and intellectual disability. Elucidating the disease mechanisms for these genes is challenging, as the encoded proteins often regulate thousands of downstream targets. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing (scRNA-seq) to characterize a mouse model of HNRNPU-mediated epileptic encephalopathy. This gene encodes a ubiquitously expressed RNA-binding protein, yet we demonstrate that reduction in its expression leads to cell type-specific transcriptomic defects. Specifically, excitatory neurons in a region of the hippocampus called the subiculum carried the strongest burden of differential gene expression. In Chapter 3, we use scRNA-seq to identify convergent molecular and transcriptomic features in four different organoid models of a cortical malformation called periventricular nodular heterotopia. In Chapter 4, we build on these successes to propose a high-throughput drug screening program for neurodevelopmental genes that encode regulators of gene expression. This approach—termed transcriptomic reversal—attempts to identify compounds that reverse disease-causing gene expression changes back to a normal state. Finally, in Chapter 5, we focus on the role of synonymous codon usage in human disease. Codon usage can affect mRNA stability, yet its role in human physiology has been historically overlooked. We use population genetics approaches to demonstrate that natural selection shapes codon content in the human genome, and we find that dosage sensitive genes are intolerant to reductions in codon optimality. We propose that synonymous mutations could modify the penetrance of Mendelian diseases through altering the expression of disease-causing mutations. In summary, the work in this thesis broadly focuses on the role of gene expression dysregulation in disease. We provide novel frameworks for interrogating disease gene expression signatures, prioritizing mutations that may alter expression, and identifying targeted therapeutics.

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<div> <p>Mammalian embryos undergo a dramatic amount of epigenetic remodeling during the first week of development to establish the correct epigenetic status to support the developmental program. SWI/SNF chromatin remodeling complexes are multi-subunits complexes and utilize energy from ATP hydrolysis to modify chromatin structure non-covalently. The collection of subunits determines the identity of a given SWI/SNF chromatin-remodeling complex, directs its activity, and dictate where that complex will act. The aims of this study were to 1) determine the requirement of SNF5, a SWI/SNF core subunit found in BAF and PBAF complexes during preimplantation porcine embryo development, 2) determine the requirement of BRD7, a PBAF complex-specific subunit during preimplantation porcine embryo development, and 3) investigate the role of <i>CDH1</i>, a downstream gene regulated by ARID1A, another subunit found exclusively in BAF complexes, in cleavage stage porcine embryos. Our results indicate that the differential requirement for each subunit during early embryo development. Depletion of different subunits results in embryo arrest at distinct developmental stage. Together, our data suggest the SWI/SNF chromatin remodeling complexes are necessary for proper porcine embryo development and this requirement is associated with the composition of the complex.</p> </div> <br>

Multiple regulatory mechanisms work to ensure that eukaryotic transcription maintains mRNA pools and subsequent protein synthesis. When errors in transcription occur, deleterious effects on cellular fitness can develop. RNA degradation as well as histone modifications, specifically at promoter proximal nucleosomes, play a critical role in maintaining transcription, but, exact mechanisms are not fully understood. In this dissertation, I investigate the role of RNA degradation and chromatin dynamics in transcription regulation as well as further understand, through biochemical analysis, a critical histone deacetylase. Using various genome-wide methodologies in Saccharomyces cerevisiae, we find a functional interaction between the nuclear RNA exosome and histone variant H2A.Z that maintains mRNA levels. There is a reduction in RNA polymerase II nascent transcription following RNA exosome subunit Rrp6 depletion that is further globally accentuated with H2A.Z deposition loss. To understand the mechanism leading to this global reduction, we identify the mRNA of Sirtuin histone deacetylase Hst3 as a target of the RNA exosome, revealing a means to link degradation to the transcription machinery. These findings show that even slight changes in deacetylase or acetylase activity can have significant effects on transcription. Additionally, we reveal a global impact of H2A.Z on transcription. We further investigate the functional and structural significance of human surtuin histone deacetylase SIRT6 (yeast homolog Hst3). Using histone deacetylase assays, we confirm the significance of specific residues of SIRT6 in nucleosome binding and deacetylase activity. Additionally, we show SIRT6 has reduced deacetylase activity in vitro on acetylated lysine 56 as compared to acetylated lysine 9 on histone H3. Finally, we confirm structural findings that the histone tail of H2A impacts SIRT6 H3K9Ac deacetylation activity. Together, these findings indicate a critical importance of histone deacetylase activity in maintaining transcription, a novel role of H2A.Z in global transcription regulation that furthers our understanding of SIRT6 structure and function.

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28-1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32-24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.

Genome sizes for 36 species of cyclopoid copepods were determined by DNA-Feulgen cytophotometry of nuclei from adults collected from diverse habitats and locales in North America, South America, Europe, and Asia. Genome sizes are small, show a 20-fold range (C = 0.10-2.02 pg DNA), and vary in a discontinuous fashion. The genomes of cyclopoid copepods are remarkably small and constant within each species, unlike the large and variable genomes of marine calanoid species. These differences may reflect the evolutionary antiquity of marine copepods in relation to marine, brackish, and freshwater copepods, as well as differences in mechanisms used to modulate genome size. The small genome sizes of contemporary cyclopoids provide substantive evidence of evolutionary constraint, possibly favouring small genomes, rapid replication rates and accelerated development as adaptive strategies for survival in often fragmented, stressful, and changing habitats.