Linker Histone Functions of HMO1- Implications for DNA repair

The DNA of eukaryotic cells does not exist in free linear strands; it is tightly packaged and wrapped around nuclear proteins in order to be accommodated it inside the nucleus. The basal repeating unit of chromatin, termed the nucleosome, provides the first level of compaction of DNA into the nucleus. Nucleosomes are interconnected by linker DNA and associated linker histones to form 30 nm fibers. The highly diverse linker histones are critical for compaction and stabilization of higher order chromatin structure by binding DNA entering and exiting the nucleosome. The lysine-rich C-terminal domain (CTD) of metazoan H1 is crucial for such stabilization. This study concerns the functions of Saccharomyces cerevisiae Hmo1p, an high mobility group (HMGB) family protein unique in containing a terminal lysine-rich domain and functions in stabilizing genomic DNA. My study suggests that Hmo1p shares with mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of Hmo1p or deletion of the Hmo1p CTD creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin remodeling events associated with DNA double strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of Hmo1p. Further, my data indicates that Hmo1p functions in the DNA damage response by directing lesions towards the error-free pathway. My results suggest that Hmo1p controls DNA end resection and favors the classical non- homologous end joining (NHEJ) over alternate end Joining (A-EJ) that is error-prone process. In all, my study identifies a novel linker histone function of Hmo1p in Saccharomyces cerevisiae with the ability to stabilize genomic DNA, and appears to go beyond conventional linker histone function

Induced Elastic Matrix Synthesis within 3-Dimensional Collagen Constructs

Elastin, a primary component of elastic arteries, maintains structural stability of the cyclically recoiling artery, and critically regulates vascular cell behavior. Accelerated degradation of elastic matrix, such as that seen in vascular pathologies like abdominal aortic aneurysms (AAA), can therefore severely compromise vessel homeostasis. Tissue engineering and in-situ matrix repair strategies evaluated so far are primarily limited in inducing adult vascular cells to replicate the complex elastic matrix assembly process, and restore lost matrix integrity. Previously, our lab established the elastogenic benefits of concurrent delivery of TGF-β1 and HA-oligomers (together termed elastogenic factors, EFs), within 2D cultures of rat aortic smooth muscle cells (SMCs). Since SMCs are known to switch to a synthetic, highly matrix producing phenotype, in a manner that cannot be replicated in vivo, we sought to develop a relevant 3D in vitro model system, where the benefits of EFs can be replicated. We chose a 3D collagen-gel model since the presence of a collagenous matrix is centric to replicating vascular tissue architecture and mechanics. Further, vascular cells, regardless of the choice of scaffolds, robustly synthesize collagen. Examining the impact of a pre-existing collagenous microenvironment on the ability of the SMCs to synthesize fibrous elastic matrix in response to provided EFs, is pertinent to its clinical translation. In the first set of studies, we examined a dose range of EFs on inducing rat SMCs-seeded within 3D collagen gel constructs. Relative to untreated control, all the three doses tested showed up to a 2-fold up-regulation in gene expression of the elastin crosslinking enzyme, lysyl oxidase, and increased the accumulation of matrix elastin up to 5-fold. The lowest dose combination of 0.1 ng/ml TGF-β1 and 0.2 µg/ml HA-o, was evaluated to be most elastogenic, and this was utilized in subsequent studies. Next, we evaluated the application of cyclic strains at varying frequencies in improving EF-induced elastic matrix output, and to obtain matrix and cell orientation in a manner similar to that required in vivo. Further, we tested this system on human SMCs seeded within tubular collagen-gel constructs, to examine if they respond to EF-treatment similar to rat SMCs. A bimodal trend in elastic matrix output was observed with increasing frequencies. Relative to static controls, constructs treated with EFs at 2.5% strains and 1.5 Hz were found to improve contractile SMC phenotype, up-regulate elastin gene expression up to 7-fold, and increase elastic matrix content by 5-fold. These parameters were therefore chosen for application in subsequent studies. The presence of high concentrations of matrix degrading proteases, such as MMPs-2 and -9, inherent to AAA wall, as well as within our 3D system, can compromise the accumulation and efficient assembly of newly synthesized elastic matrix components. In the next set of studies, we demonstrated that addition of Doxycycline (DOX), a non-specific MMP inhibitor, along with EFs, suppressed MMP-2 gene expression, within static and dynamic (2.5% strain at 1.5 Hz) tubular constructs, and markedly improved overall elastic matrix synthesis. Since the effects of EFs and DOX are highly dose-dependent, the successful in vivo translation of their benefits relies on their controlled and targeted delivery specifically to the site of disease. In the final set of studies, we tested the effects of TGF-β1 and DOX released from PLGA nanoparticles, incorporated within the cyclically stretched tubular 3D model optimized in previous studies. We were able to successfully demonstrate that such localized delivery was able to induce elastogenesis in a manner similar to exogenous delivery of the same factors. Overall, these results will be useful towards addressing a fundamental and widely absent aspect in vascular engineering, that of inducing adult vascular cells to replicate biological and structural mimics of native elastic fiber networks

Toward a molecular understanding of yeast silent chromatin : roles for H4K16 acetylation and the Sir3 C-terminus

Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression. In budding yeast, silent chromatin is characterized by the loading of the Silent Information Regulatory (Sir) proteins (Sir2, Sir3 and Sir4) onto unmodified nucleosomes. This requires the deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions between Sir proteins forming the Sir2-3-4 complex. During my PhD thesis I sought to advance our understanding of these phenomena from a molecular perspective. Previous studies of Sir-chromatin interactions made use of histone peptides and recombinant Sir protein fragments. This gave us an idea of possible interactions, but could not elucidate the role of histone modifications in the assembly of silent chromatin. This required that we examine nucleosomal arrays exposed to full length Sir proteins or the holo Sir complex. In Chapter 2, I made use of an in vitro reconstitution system, that allows the loading of Sir proteins (Sir3, Sir2-4 or Sir2-3-4) onto arrays of regularly spaced nucleosomes, to examine the impact of specific histone modifications (methylation of H3K79, acetylation of H3K56 and H4K16) on Sir protein binding and linker DNA accessibility. The “active” H4K16ac mark is thought to limit the loading of the Sir proteins to silent domain thus favoring the formation of silent regions indirectly by increasing Sir concentration locally. Strikingly, I found that the Sir2-4 subcomplex, unlike Sir3, has a slight higher affinity for H4K16ac-containing chromatin in vitro, consistent with H4K16ac being a substrate for Sir2. In addition the NAD-dependent deacetylation of H4K16ac promotes the binding of the holo Sir complex to chromatin beyond generating hypoacetylated histone tails. We conclude that the Sir2-dependent turnover of the “active” H4K16ac mark directly helps to seed repression. The tight association of the holo Sir complex within silent domains relies on the ability of Sir3 to bind unmodified nucleosomes. In addition, Sir3 dimerization is thought to reinforce and propagate silent domains. However, no Sir3 mutants that fail to dimerize were characterized to date. It was unclear which domain of Sir3 mediates dimerization in vivo. In Chapter 3, we present the X-ray crystal structure of the Sir3 extreme C-terminus (aa 840-978), which folds into a variant winged helix-turn-helix (Sir3 wH) and forms a stable homodimer through a large hydrophobic interface. Loss of wH homodimerization impairs holo Sir3 dimerization in vitro showing that the Sir3 wH module is key to Sir3-Sir3 interaction. Homodimerization mediated by the wH domain can be fully recapitulated by an unrelated bacterial homodimerization domain and is essential for stable association of the Sir2-3-4 complex with chromatin and the formation of silent chromatin in vivo

Dynamics and Heterogeneity of Gene Expression and Epigenetic Regulation at the Single-Cell Level

The ability of cells to establish and remember their gene expression states is a cornerstone of multicellular life. This thesis explores how gene expression states are regulated dynamically, and how these regulations differ in individual cells even under the same conditions. These properties underlie cellular state decisions and often determine the balance between different cell types in a multicellular system, but are typically inaccessible to conventional techniques that rely on static snapshots and population averaging. We address these issues in two separate contexts, one natural and one synthetic, using time-lapse imaging and other single-cell techniques. In the first context, we use embryonic stem cells (ES), which were shown to exist in a mixed population of at least two cellular states with distinct differentiation propensities, as a model to study natural dynamics of cellular states. These cells display rare, stochastic, and spontaneous transitions between the two states, as well as more frequent fluctuations in gene expression levels within each state. Our system enables us to further investigate how these dynamics are modulated under a cell signaling environment that enhances pluripotency, and the role DNA methylation plays in maintaining these states. In the second context, we investigate how chromatin regulators (CRs), part of a complex system that enables cells to modulate gene expression and epigenetic memory, operate dynamically in individual cells. We build a synthetic platform to measure the isolated effect of recruitment and de-recruitment of four individual CRs. In contrast to conventional transcription factor control, all CRs tested regulate gene expression in all-or-none events, controlling the probability of stochastic transitions between fully active and silent states rather than the strength of gene expression. The qualitative and quantitative responses of a cell population are determined by the set of event rates associated with each CR, as well as the duration of CR recruitment. These results provide a framework for understanding and engineering chromatin-based cellular states and their dynamics. </p

Agricultural impacts on plant beneficial pseudomonads

The soil microbiome is a dynamic and complex environment that offers numerous ecosystem services. Beneficial Pseudomonas spp. are agriculturally relevant bacteria with a plethora of plant growth promoting (PGP) traits, making them desirable targets for microbial inoculant development. Microbial inoculants have typically failed to produce reliable results, which can be attributed to the introduction of microbes into ecologically unsuitable environments. Its therefore important to better understand factors that can alter Pseudomonas spp. community structure and functioning. Crop domestication and land management have both played important roles in the development of agriculture over the last 10,000 years, however they have been associated with negative impacts on the soil microbiome. Here, the impacts of these agricultural components on soil pseudomonads was investigated. The study of 17 domesticated and ancestral wheat genotypes cultivated in a grassland soil revealed no clear difference in pseudomonad community structure within rhizosphere or bulk soil. The Highfield experiment at Rothamsted Research tests the impact of land management and revealed various impacts to soil properties, wheat physiology and total microbial abundance across grassland, arable and bare fallow managed soils. However, pseudomonad abundance was not found to significantly differ in bulk soil and rhizosphere communities. Additional studies looking at the more closely associated root compartment of wheat grown in soils from distinct land uses, revealed differences in abundance and phylogeny of cultivated pseudomonads. A range of PGP genetic and functional potentials including siderophore production, anti-fungal activity and phosphate solubilisation differed in isolates according to land use. The presence of the 1-Aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS) was of particular interest, due to its potential to reduce levels of stress ethylene in plants by degrading its precursor ACC. Intriguingly, acdS gene abundance, phylogeny and functional activity appeared to differ in pseudomonads associated with the different land uses. The rhizosphere and root compartments of wheat had a higher acdS gene abundance, particularly in the bare fallow soil which is known to have degraded soil properties. This suggests factors associated with wheat grown in different land managements were driving the selection of ACC deaminase producing pseudomonads. In vitro attempts to promote wheat growth under salt stress by applying ACC deaminase-containing isolates was not successful. Overall this thesis evidences the functional potential of pseudomonads for use in microbial inoculants, whilst providing an insight into the complexity of soil-plant-microbe interactions

Quantitative dissection of RNA structure formation reveals a cooperative and modular folding and assembly landscape

Structured RNAs are pervasive in biology with ubiquitous roles in gene expression and regulation. RNAs must fold from a linear chain of nucleotide sequence to attain three-dimensional structure. RNA folding can be described as modular and hierarchical with tiers of structure that form independently: secondary structure forms first and defines the helices followed by formation of tertiary structure. The separation between secondary and tertiary structure is not absolute. Many biological RNAs couple secondary structure changes to RNA tertiary structure formation and link these changes to downstream functional consequences. To predict how these biological RNAs fold requires a deep understanding of the structural intermediates, folding pathways, and mechanisms of cooperativity that promote folding. To test the modularity and predictability of secondary and tertiary RNA folding and assembly, we have investigated the folding and assembly of the P5abc subdomain from the Tetrahymena thermophila Group I intron ribozyme. P5abc folds cooperatively in isolation, binding Mg²⁺ ions and adopting tertiary structure. Mg²⁺ binding is linked to a shift in the secondary structure of seventeen nucleotides and prior work concluded that there is a high degree of cooperativity for this seemingly concerted transition. With the already established principles of RNA modularity in mind, we develop a reconstitution hypothesis to test if cooperative secondary and tertiary folding and assembly of P5abc can be understood from the component pieces. By using rational mutagenesis, we find that higher order folding of P5abc is modular, and we elucidate the physical origins of cooperativity (Chapter 2). With our knowledge of isolated P5abc folding, we demonstrate that the local folding transition within P5abc controls the rate and pathway of assembly with the P5abc-deleted ribozyme core (E[superscript ΔP5abc]), further highlighting the modularity of RNA structure (Chapter 3). Lastly, we show that the kinetics of assembly can be attributed to specific tertiary contacts that form in the assembly transition state such that the rate of a particular folding pathway is dictated by the properties of an individual tertiary contact (Chapter 4). The modularity of RNA structure makes it a reasonable molecule for the origins of life and an adaptable tool for bioengineering applications.Biochemistr

A Unique Role for Nonmuscle Myosin Heavy Chain IIA in Regulation of Epithelial Apical Junctions

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis