Zugl.: Göttingen, Univ., Diss., 2009

Biological nanomaterials are defined as materials with sizes within the nanoscale range of 1 - 100 nm. The fundamental functionalities and biocompatibility of these materials can be tailored for biotechnology applications. In 1983, Ned Seeman successfully developed the first customized DNA nanostructures, Holliday junctions. Since then, the field has continued to expand rapidly and various 2D and 3D nanostructures has been designed. Although the high predictability of DNA base-pairing is essential for the design of complex DNA nanostructures, it greatly limits its functional versatility; therefore, proteins are conjugated with DNA nanostructures to compensate for that. DNA origami-protein hybrid nanostructures were introduced in 2012. However, the structural units based on DNA origami-protein hybrid nanostructures are still limited, and the majority are constructed by covalent or sequence-specific non-covalent interactions. Here we utilize the inherent, non-sequence-specific interaction between DNA and histones to present sequence-independent self-assembled DNA origami-protein hybrid nanostructures. We demonstrated using various molecular biology and imaging techniques that ssDNAs and histone proteins self-assemble into structurally well-defined complexes. We successfully assembled DNA origami–histone hybrid nanostructures using two different shapes of DNA origami: rectangular (PF-3), and rectangular with central aperture (PF-2) nanostructures. We observed precise localization of nucleosome-like histone-ssDNA nanostructures at the edge (PF-3) or the center (PF-2) of the DNA origami. In addition, we demonstrated that this DNA origami-histone interaction results in the assembly of larger DNA origami complexes, including a head-to-head type dimer and a cross-shape complex. Our results suggest the successful self-assembly of the DNA origami–histone hybrid nanostructures provide a principal structural unit for constructing higher-order nanostructures. Given the reversible nature of the geometry-based noncovalent interaction between the DNA origami and the nucleosome-like histone-ssDNA nanostructures, the self-assembly/disassembly of DNA-histones hybrid nanostructures may open new opportunities to construct stimuli-responsive DNA-protein hybrid nanostructures.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21028号 / 医科博第89号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 浅野 雅秀, 教授 玉木 敬二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.