<p>Transcription initiation is a key component in the regulation of gene expression. Recent high-throughput sequencing techniques have enhanced our understanding of mammalian transcription by revealing narrow and broad patterns of transcription start sites (TSSs). Transcription initiation is central to the determination of condition specificity, as distinct repertoires of transcription factors (TFs) that assist in the recruitment of the RNA polymerase II to the DNA are present under different conditions. However, our understanding of the presence and spatiotemporal architecture of the promoter patterns in the fruit fly remains in its infancy. Nucleosome organization and transcription initiation have been considered hallmarks of gene expression, but their cooperative regulation is also not yet understood.</p> <p>In this work, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs), and developed an improved paired-end sequencing strategy to explore the transcription initiation landscape of the D.melanogaster genome. We distinguished three initiation patterns: 'Peaked or Narrow Peak TSSs&#8219;, 'Broad Peak TSSs&#8219;, and 'Broad TSS cluster groups or Weak Peak TSSs&#8219;. The promoters of peaked TSSs contained the location specific sequence elements, and were bound by TATA Binding Protein (TBP), while the promoters of broad TSS cluster groups were associated with non-location-specific elements, and were bound by the TATA-box related Factor 2 (TRF2).</p> <p>Available ESTs and a tiling array time series enabled us to show that TSSs had distinct associations to conditions, and temporal patterns of embryonic activity differed across the majority of alternative promoters. Peaked promoters had an association to maternally inherited transcripts, and broad TSS cluster group promoters were more highly associated to zygotic utilization. The paired-end sequencing strategy identified a large number of 5' capped transcripts originating from coding exons that were unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications.</p> <p>We applied an innovative search program called FREE to embryo, head, and testes specific core promoter sequences and identified 123 motifs: 16 novel and 107 supported by other motif sources. Motifs in the embryo specific core promoters were found at location hotspots from the TSS. A family of oligos was discovered that matched the Pause Button motif that is associated with RNA pol II stalling.</p> <p>Lastly, we analyzed nucleosome organization, chromatin structure, and insulators across the three promoter patterns in the fruit fly and human genomes. The WP promoters showed higher associations with H2A.Z, DNase Hypersensitivity Sites (DHS), H3K4 methylations, and Class I insulators CTCF/BEAF32/CP190. Conversely, NP promoters had higher associations with polII and GAF binding. BP promoters exhibited a combination of features from both promoter patterns. Our study provides a comprehensive map of initiation sites and the conditions under which they are utilized in D. melanogaster. The presence of promoter specific histone replacements, chromatin modifications, and insulator elements support the existence of two divergent strategies of transcriptional regulation in higher eukaryotes. Together, these data illustrate the complex regulatory code of transcription initiation.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21946号 / 理博第4524号 / 新制||理||1650(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

<div><div><p>Important cellular processes such as migration, differentiation, and development often rely on precise timing. Yet, the molecular machinery that regulates timing is inherently noisy. How do cells achieve precise timing with noisy components? We investigate this question using a first-passage-time approach, for an event triggered by a molecule that crosses an abundance threshold. We investigate regulatory strategies that decrease the timing noise of molecular events. We look at several strategies which decrease the noise: i) Regulation performed by an accumulating activator, ii) Regulation dues to a degrading repressor, iii) Auto-regulation and the presence of feedback. We find that either activation or repression outperforms an unregulated strategy. The optimal regulation corresponds to a nonlinear increase in the amount of the target molecule over time, arises from a tradeoff between minimizing the timing noise of the regulator and that of the target molecule itself, and is robust to additional effects such as bursts and cell division. Our results are in quantitative agreement with the nonlinear increase and low noise of <i>mig-1</i> gene expression in migrating neuroblast cells during <i>Caenorhabditis elegans</i> development. These findings suggest that dynamic regulation may be a simple and powerful strategy for precise cellular timing.</p><p>Autoregulatory feedback increases noise. Yet, we find that in the presence of regulation by a second species, autoregulatory feedback decreases noise. To explain this finding, we develop a method to calculate the optimal regulation function that minimizes the timing noise. Our method reveals that the combination of feedback and regulation minimizes noise by maximizing the number of molecular events that must happen in sequence before a threshold is crossed. We compute the optimal timing precision for all two-node networks with regulation and feedback, derive a generic lower bound on timing noise, and compare our results with the neuroblast migration during <i>C. elegans</i> development, as well as two mutants. We finds that indeed our model is aligned with the experimental findings.</p></div></div><div><p>Furthermore, we apply our framework of temporal regulation to explain how the stopping point of the migrating cells in <i>C. elegans</i> depends on the body size. Considering temporal regulation, we find the termination point of the cell for various larval sizes. We discuss three possible mechanisms: i) No compensation; here the migration velocity is constant across the mutants of <i>C. elegans</i>, and this results in the migration distance to be constant but the relative position to be different across various sizes; ii) Total compensation; here the velocity is compensated with body size, hence resulting in the same relative position of cells across mutants; and iii) Partial compensation; here the velocity of migration is correlated with body size to some degree, resulting in a non-linear relationship between termination point and body size. We find that our partial compensation model is consistent with experimental observations of cell termination.</p><p>Finally, we look at the detection of traveling waves by single-celled organisms. Cells must use temporal and spatial information to sense the direction of traveling waves, as seen in cAMP detection by the <i>amoeba </i><i>Dictyostelium</i>. If a cell only uses spatial information to sense the direction of the wave then the cell will move forward when the wave hits the front of the cell, and move backward when the wave hits the back of the cell, resulting in neutral movement. Cells must use temporal information along with spatial information to effectively move towards the source. Here we develop a mechanism by which cells are able to integrate the spatial and temporal information through a system of inhibitors. We find the optimal time to release the inhibitors for maximizing the precision of directional sensing.</p></div>

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016