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abstract: Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than deoxyribose nucleic acid (DNA) sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. In 2011, Haynes et al. showed that a synthetic regulator called the Polycomb chromatin Transcription Factor (PcTF), a fusion protein that binds methylated histones, reactivated an artificially-silenced luciferase reporter gene. These synthetic transcription activators are derived from the polycomb repressive complex (PRC) and associate with the epigenetic silencing mark H3K27me3 to reactivate the expression of silenced genes. It is demonstrated here that the duration of epigenetic silencing does not perturb reactivation via PcTF fusion proteins. After 96 hours PcTF shows the strongest reactivation activity. A variant called Pc2TF, which has roughly double the affinity for H3K27me3 in vitro, reactivated the silenced luciferase gene by at least 2-fold in living cells. / Dissertation/Thesis / Masters Thesis Biological Design 2019

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA) has been linked to biofilm formation in B. henselae. BadA is a protein of 3036 amino acids and a member of the TAAs found in Bartonella and other Gram-negative bacteria. The function of BadA has been studied in vitro and is critical for agglutination, host cell adhesion and activation of a pro-angiogenic host response. However, very little is known about badA gene regulation or the molecular basis of biofilm formation. This work aims to determine whether BadA is necessary for the establishment of biofilms and how the bacteria regulate badA expression. Using genetic mutations, real-time cell adhesion assay, RT-qPCR, and microscopy, it was shown that BadA is required for biofilm formation. Using an in-frame complete deletion strain of badA, a reduced ability to form a biofilm was observed which was restored in the deletion strain complemented with a partial badA. Analysis of the B. henselae transcriptome shows nine highly transcribed, homologous RNAs, termed Bartonella regulatory transcript (Brt1-9). The Brts are short-sized (<200 >nucleotides), highly expressed, and located in an intergenic region indicative of small RNAs (sRNA). The Brts are predicted to form a stable stem and loop structure with a potential terminator/riboswitch region on the 3′ end. Located ~20 nucleotides downstream of each Brt is a poorly transcribed helix-turn-helix DNA binding protein gene termed transcriptional regulatory protein (trps 1-9). High brt transcription stops just before the start of the trp implicating the 3’ loop of the Brt as a terminating loop. Replacement of the trp with a gfp reporter gene shows that in the absence of the 3′ end of Brt1, gfp is transcribed. Also consistent with our findings, an increase in both the transcription of trp1 and badA and the formation of a biofilm in mutants of the brt1 gene was observed. Furthermore, to determine the role of the Trp in regulating badA, an electrophoretic mobility shift assay was carried out. The data confirms that Trp1 binds the promoter region of badA gene to regulate gene expression. In summary, the brt1/trp1 regulon affects badA transcription and biofilm formation in B. henselae. Understanding the mechanism and condition(s) by which the brt/trp regulatory system regulates badA is a plausible approach to the development of treatments that target the formation of biofilm-related diseases and persistent bacteremia in humans.

The PhD thesis is focused on a transcriptomics-based comparison of barley cultivars differing with respect to their low temperature acclimation capacity, with a particular focus on genes transcribed in the leaf and crown. The crown was of interest because of its importance for the winter survival of the plant. To involve both the first and the second phase of hardening, the test plants were exposed first to +3řC for 21 days, followed by - 3řC for one day. Freezing damage was assessed by measuring electrolyte leakage (Papers 2 and 3), using a modified version of a protocol developed by Prášil and Zámečník (1998). The same protocol was adapted to evaluate crown regrowth (Paper 2); for this purpose, the plants were cooled, then replanted and cut above the crown, and their survival rate calculated over the following week. Each RNA sample was queried by hybridization to an Affymetrix 22 K Barley1 GeneChip Genome Array (Close et al. 2004). The data were statistically analysed with the help of the software packages R, MAS 5.0 (Ihaka & Gentleman 1996) (Papers 2 and 3), Gene Spring GX 7.3 (Agilent Technologies, Santa Clara CA) and MapMan (Thimm et al. 2004; Usadel et al. 2005) (Paper 2), the "Self-Organizing Maps" algorithm (Kohonen et al. 1996) (Paper 3) and MIPS FunCat (Ruepp et al. 2004) (Paper 2). Paper...

The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

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.

The Piwi interacting RNA pathway (piRNA) transcriptionally and post-transcriptionally silences transposons in the germline to maintain host genome integrity and faithful transmission of the genetic materials. In Drosophilaovaries, maternally loaded piRNAs kick-start piRNA biogenesis and convert precursor transcripts into piRNAs to replenish the piRNA pool during oogenesis. piRNA clusters are the genomic source of piRNA precursors, which are determined by the HP1 homolog Rhino and accessary factors. Rhino specifically binds to piRNA cluster chromatin. I was intrigued by how Rhino localizes to piRNA clusters to specify piRNA precursors. TREX is a conserved mRNA biogenesis complex composed of UAP56 and the THO complex. Identification of UAP56 as a cluster transcript-processing factor established the link between piRNA biogenesis and the general mRNA processing machinery. In my thesis, I investigated the functions of UAP56 and THO in piRNA cluster transcript processing. I characterized an RNP specific to cluster transcripts, defined by binding with both factors, which is distinct from RNP of bulk mRNA transcripts, and found that assembly of these RNPs depends on Rhino. These findings imply that piRNA precursors are specified co-transcriptionally. Additionally, I found that TREX mutants lead to a loss of Rhino binding specificity. I propose that Rhino and TREX co-transcriptionally scan for cluster and transposon sequences to establish loci that produce piRNA precursors. Surprisingly, I also discovered a piRNA-independent function for TREX in transposon silencing. I showed that TREX mutants lead to transcriptionally activation of a number of transposon families without affecting their piRNA biogenesis and piRNA mediated repressive histone modifications. I propose that TREX could mediate a conserved transposon silencing mechanism.