Histone variants in archaea

Eukaryotic histone variants are involved in a wide range of processes and play a key role in altering nucleosome dynamics to shape the architecture of chromatin. The importance of individual variants has been studied extensively in many eukaryotes. In comparison, we know relatively little about histones in archaea. Despite sequence variation and evidence for potential functional differences between histone paralogs in the same species, whether archaea have histone variants, and therefore the potential for complex histone-based chromatin, has not been comprehensively explored. In this work, I apply structural and sequence-based approaches and present evidence that histone variants exist in archaea. In silico modelling suggests that, similarly to some eukaryotic variants, paralogs in archaea can be identified by unique structural properties. In particular, I describe one such variant, a “capstone”, that can drastically alter histone-based chromatin by limiting oligomerisation. Other paralogs have less extreme structural properties but are shared between species which separated hundreds of millions of years ago, on par with some eukaryotic histone variants. Although there are shared features between the two, histones in archaea have appear to have explored a different sequence space to eukaryotic histones, evolving separately and in parallel.Open Acces

Actin-Related Proteins Regulate The Structure And Function Of The Rsc Chromatin-Remodeling Complex In An Rtt102-Dependent Manner

Chromatin remodeling complexes (remodelers) alter chromatin structure to regulate access to DNA. Remodelers belong to four families, but each are assembled around a catalytic subunit, featuring a conserved, DNA-dependent ATPase domain flanked by family-specific domains. These can intra-molecularly interact with the ATPase domain to regulate its function and can also recruit auxiliary subunits for additional regulation. Sth1, the catalytic subunit of the yeast SWI/SNF-family remodeler RSC, recruits a heterodimer of actin-related proteins (Arps) 7 and 9 through an N-terminal helicase/SANT-associated (HSA) domain. An additional auxiliary subunit, Rtt102, co-purifies with Arp7/9. In this dissertation, I sought to understand how Arp7/9 binding to the HSA domain of Sth1 regulates the structure and function of the central ATPase domain, and how this regulation is modulated by Rtt102. Using Isothermal Titration Calorimetry (ITC), Small angle X-ray scattering (SAXS) and X-ray crystallography. I discovered that Rtt102 binds with nanomolar affinity to stabilize a compact conformation of the Arp7/9 heterodimer, which is required for full binding to the HSA domain and formation of a stable complex with the ATPase domain. The crystal structure of the Rtt102-Arp7/9 complex reveals that ATP binds to Arp7 to help stabilize the compact conformation of Rtt102-Arp7/9 required for tight association with the HSA domain. To correlate these findings to remodeler function, I designed a novel biochemical assay to test for the effects of Arp7/9-binding on intramolecular interactions within Sth1. This approach revealed that two conserved sequences, Protrusion-1 (P1) within the ATPase domain and a region adjacent to the HSA domain known as the post-HSA (pHSA) domain, interact directly with each other. Binding of Arp7/9 to the HSA domain weakens this interaction in an Rtt102-dependent manner. Fluorescence anisotropy experiments further showed that the weakening of P1-pHSA interaction by Rtt102-Arp7/9 reduces the affinity of the ATPase domain for DNA. Taken together, Rtt102 thus emerges as an important factor that stabilizes Arp7/9, allowing it to modulate regulatory intra-molecular interactions within Sth1 and thereby control DNA binding to the ATPase domain. As such, this work establishes a novel molecular mechanism for regulation of the RSC remodeler

Computational Investigations of Biomolecular Motions and Interactions in Genomic Maintenance and Regulation

The most critical biochemistry in an organism supports the central dogma of molecular biology: transcription of DNA to RNA and translation of RNA to peptide sequence. Proteins are then responsible for catalyzing, regulating and ensuring the fidelity of transcription and translation. At the heart of these processes lie selective biomolecular interactions and specific dynamics that are necessary for complex formation and catalytic activity. Through advanced biophysical and computational methods, it has become possible to probe these macromolecular dynamics and interactions at the molecular and atomic levels to tease out their underlying physical bases. To the end of a more thorough understanding of these physical bases, we have performed studies to probe the motions and interactions intrinsic to the function of biomolecular complexes: modeling the dual-base flipping strategy of alkylpurine glycosylase D, dynamically tracing evolution and epistasis in the 3-ketosteroid family of nuclear receptors, discovering the allosteric and conformational aspects of transcription regulation in liver receptor homologue 1, leveraging specific contacts in tyrosyl-DNA phosphodiesterase 2 for the development of novel inhibitor scaffolds, and detailing the experimentally observed connection between solvation and sequence-specific binding affinity in PU.1-DNA complexes at the atomic level. While each study seeks to solve system-specific problems, the collection outlines a general and broadly applicable description of the biophysical motivations of biochemical processes

Consequences of local and global chromatin mechanics to adaption and genome stability in the budding yeast Saccharomyces cerevisiae

Le génome de la levure de boulanger Saccharomyces cerevisiae a évolué à partir d'un ancêtre chez lequel une profonde décompaction du génome s'est produite à la suite de la perte de la méthylation de la lysine 9 de l'histone H3, il y a environ 300 millions d'années. Il a été proposé que cette décompaction du génome a entraîné une capacité accrue des levures à évoluer par des mécanismes impliquant des taux de recombinaison méiotique et de mutation exceptionnellement élevés. La capacité à évoluer accrue qui en résulte pourrait avoir permis des adaptations uniques, qui en ont fait un eucaryote modèle idéal et un outil biotechnologique. Dans cette thèse, je présenterai deux exemples de la façon dont les adaptations locales et globales du génome se reflètent dans les changements des propriétés mécaniques de la chromatine qui, à leur tour, indiquent un phénomène de séparation de phase causée par les modifications post-traductionnelles des histones et des changements dans les taux d'échange des histones. Dans un premier manuscrit, je présente des preuves d'un mécanisme par lequel la relocalisation du locus INO1, gène actif répondant à la déplétion en inositol, du nucléoplasme vers l'enveloppe nucléaire, augmente la vitesse d'adaptation et la robustesse métabolique aux ressources fluctuantes, en augmentant le transport des ARNm vers le cytosol et leur traduction. La répartition d'INO1 vers l'enveloppe nucléaire est déterminée par une augmentation locale des taux d'échange d'histones, ce qui entraîne sa séparation de phase du nucléoplasme en une phase de faible densité plus proche de la périphérie nucléaire. J'ai quantifié les propriétés mécaniques de la chromatine du locus du gène dans les états réprimé et actif en analysant le déplacement de 128 sites LacO fusionnés au gène liant LacI-GFP en calculant diffèrent paramètres tel que la constante de ressort effective et le rayons de confinement du locus. De plus, j'ai mesuré l'amplitude et le taux d'expansion en fonction du temps du réseau LacO et j'ai observé une diminution significative du locus à l'état actif, ce qui est cohérent avec le comportement de ressort entropique de la chromatine décompactée. J'ai montré que les séquences d'éléments en cis dans le promoteur du locus, essentielles à la séparation de phase, sont des sites de liaison pour les complexes de remodelage de la chromatine effectuant l'acétylation des histones. Ces modifications de la chromatine entraînent une augmentation des taux d'échanges des sous-unités des complexes d'histones, et une séparation de phase locale de la chromatine. Enfin, je présente l’analyse de simulations in silico qui montrent que la séparation de phase locale de la chromatine peut être prédite à partir d'un modèle de formation/disruption des interactions multivalentes protéine-protéine et protéine-ADN qui entraîne une diminution de la dynamique de l'ADN. Ces résultats suggèrent un mécanisme général permettant de contrôler la formation rapide des domaines de la chromatine, bien que les processus spécifiques contribuant à la diminution de la dynamique de l'ADN restent à étudier. Dans un second manuscrit, je décris comment nous avons induit la « retro-évolution » de la levure en réintroduisant la méthylation de la lysine 9 de l'histone H3 par l'expression de deux gènes de la levure Schizosaccaromyces pombe Spswi6 et Spclr4. Le mutant résultant présente une augmentation de la compaction de la chromatine, ce qui entraîne une réduction remarquable des taux de mutation et de recombinaison. Ces résultats suggèrent que la perte de la méthylation de la lysine 9 de l'histone H3 pourrait avoir augmenté la capacité à l'évoluer. La stabilité inhabituelle du génome conférée par ces mutations pourrait être utile pour l'ingénierie métabolique de S. cerevisiae, dans laquelle il est difficile de maintenir des gènes exogènes intégrés pour les applications de nombreux processus biotechnologiques courants tels que la production de vin, de bière, de pain et de biocarburants. Ces résultats soulignent l'influence des propriétés physiques d'un génome sur son architecture et sa fonction globales.The genome of the budding yeast Saccharomyces cerevisiae evolved from an ancestor in which a profound genome decompaction occurred as the result of the loss of histone H3 lysine 9 methylation, approximately 300 million years ago. This decompaction may have resulted in an increased capacity of yeasts to evolve by mechanisms that include unusually high meiotic recombination and mutation rates. Resultant increased evolvability may have enabled unique adaptations, which have made it an ideal model eukaryote and biotechnological tool. In this thesis I will present two examples of how local and global genome adaptations are reflected in changes in the mechanical properties of chromatin. In a first manuscript, I present evidence for a mechanism by which partitioning of the active inositol depletion-responsive gene locus INO1 from nucleoplasm to the nuclear envelope increases the speed of adaptation and metabolic robustness to fluctuating resources, by increasing mRNA transport to the cytosol and their translation. Partitioning of INO1 to the nuclear envelope is driven by a local increase in histone exchange rates, resulting in its phase separation from the nucleoplasm into a low-density phase closer to the nuclear periphery. I quantified the mechanical properties of the gene locus chromatin in repressed and active states by monitoring mean-squared displacement of an array of 128 LacO sites fused to the gene binding LacI-GFP and calculating effective spring constants and radii of confinement of the array. Furthermore, I measured amplitude and rate of time-dependent expansion of the LacO array, and observed a significant decrease for the active-state locus which is consistent with entropic spring behavior of decompacted chromatin. I showed that cis element sequences in the promoter and upstream of the locus that are essential to phase separation are binding sites for chromatin remodeling complexes that perform histone acetylation among other modifications that result in increased histone complex exchange rates, and consequent local chromatin phase separation. Finally, I present analytical simulations that show that local phase separation of chromatin can be predicted from a model of formation/disruption of multivalent protein-protein and protein-DNA interactions that results in decreased DNA dynamics. These results suggest a general mechanism to control rapid formation of chromatin domains, although the specific processes contributing to the decreased DNA dynamics remain to be investigated. In a second manuscript, I describe how we retro-evolutionarily engineered yeast by reintroducing histone H3 lysine 9 methylation through the expression of two genes from the yeast Schizosaccaromyces pombe Spswi6 and Spclr4. This mutant shows an increase in compaction, resulting in remarkable reduced mutation and recombination rates. These results suggest that loss of histone H3 lysine 9 methylation may have increased evolvability. The unusual genome stability imparted by these mutations could be of value to metabolically engineering S. cerevisiae, in which it is difficult to maintain integrated exogenous genes for applications for many common biotechnological processes such as wine, beer, bread, and biofuels production. These results highlight the influence of the physical properties of a genome on its overall architecture and function

Dissecting cis and trans Determinants of Nucleosome Positioning: A Dissertation

Eukaryotic DNA is packaged in chromatin, whose repeating subunit, the nucleosome, consists of an octamer of histone proteins wrapped by about 147bp of DNA. This packaging affects the accessibility of DNA and hence any process that occurs on DNA, such as replication, repair, and transcription. An early observation from genome-wide nucleosome mapping in yeast was that genes had a surprisingly characteristic structure, which has motivated studies to understand what determines this architecture. Both sequence and trans acting factors are known to influence chromatin packaging, but the relative contributions of cis and trans determinants of nucleosome positioning is debated. Here we present data using genetic approaches to examine the contributions of cis and trans acting factors on nucleosome positioning in budding yeast. We developed the use of yeast artificial chromosomes to exploit quantitative differences in the chromatin structures of different yeast species. This allows us to place approximately 150kb of sequence from any species into the S.cerevisiae cellular environment and compare the nucleosome positions on this same sequence in different environments to discover what features are variant and hence regulated by trans acting factors. This method allowed us to conclusively show that the great preponderance of nucleosomes are positioned by trans acting factors. We observe the maintenance of nucleosome depletion over some promoter sequences, but partial fill-in of NDRs in some of the YAC v promoters indicates that even this feature is regulated to varying extents by trans acting factors. We are able to extend our use of evolutionary divergence in order to search for specific trans regulators whose effects vary between the species. We find that a subset of transcription factors can compete with histones to help generate some NDRs, with clear effects documented in a cbf1 deletion mutant. In addition, we find that Chd1p acts as a potential “molecular ruler” involved in defining the nucleosome repeat length differences between S.cerevisiae and K.lactis. The mechanism of this measurement is unclear as the alteration in activity is partially attributable to the N-terminal portion of the protein, for which there is no structural data. Our observations of a specialized chromatin structure at de novo transcriptional units along with results from nucleosome mapping in the absence of active transcription indicate that transcription plays a role in engineering genic nucleosome architecture. This work strongly supports the role of trans acting factors in setting up a dynamic, regulated chromatin structure that allows for robustness and fine-tuning of gene expression

Centromere Identity and the Nature of the Cenp-A-Containing Nucleosome

The centromere is an essential chromosomal locus that serves as the site of kinetochore formation, ensuring accurate chromosome segregation during mitosis and meiosis. While most centromeres form on repetitive DNA, the underlying DNA sequence is neither necessary nor sufficient to support centromere function, suggesting that this locus is epigenetically defined. The histone H3 variant centromere protein A (CENP-A) replaces H3 in nucleosomes at the centromere and is the best candidate to provide this epigenetic mark. This thesis aims to understand the features of the CENP-A nucleosome that impart its ability to mark and stabilize functional centromeres. In the first part of the thesis, our work provides an in-depth study on the structure of CENP-A-containing nucleosomes and shows that CENP-A nucleosomes adopt an unconventional conformation in solution that results in both an altered histone core and wrap of DNA. Upon binding of the nonhistone protein CENP-C, the histone core and path of DNA wrapping it revert back to a canonical shape, but DNA termini flexibility becomes enhanced. These structural transitions imparted by CENP-C have important functional consequences, as endogenous centromeres lacking CENP-C lose CENP-A and have increased mitotic defects. In the second part of the thesis, I discuss the role that DNA sequence plays in centromere function. While seemingly indispensable, both the quality of DNA sequence and the quantity of DNA repeats are important for maintaining centromeres, and I outline our work that aims to understand both of these phenomena. Taken together, these works greatly increase our understanding of the intrinsic and extrinsic components of the CENP-A nucleosome and its role in centromere identity

Punctuated Evolution Within a Eurythermic Genus (Mesenchytraeus) of Segmented Worms: Genetic Modification of the Glacier Ice Worm F1F0 ATP Synthase

Segmented worms (Annelida) are among the most successful animal inhabitants of extreme environments worldwide. An unusual group of Mesenchytraeus worms endemic to the Pacific Northwest of North America occupy geographically proximal ecozones ranging from low elevation temperate rainforests to high altitude glaciers. Along this altitudinal transect, Mesenchytraeus representatives from disparate habitat types were collected and subjected to deep mitochondrial and nuclear phylogenetic analyses. Evidence presented here employing modern bioinformatic analyses (i.e., maximum likelihood, Bayesian inference, multi-species coalescent) supports a Mesenchytraeus “explosion” in the upper Miocene (5-10 million years ago) that gave rise to ice, snow and terrestrial worms, derived from a common aquatic ancestor. Among these ecologically-disparate but genetically-close worms, those maintaining the highest intracellular ATP levels reside permanently on glacier ice (i.e., M. solifugus). A comparative molecular analysis of 11 core structural subunits of the F1Fo-ATP synthase revealed extraordinary conservation across species, with a few notable exceptions. Most strikingly, the ice worm mitochondrial-encoded ATP6 (a) subunit – the ATP throttle known to regulate proton flux, hence ATP synthesis – encoded a highly basic, 15 amino acid carboxy-terminal extension likely to have been acquired by lateral gene transfer from an ancestral prokaryote. This insertion is supported by transcriptome raw read reconstruction and independent PCR amplifications from three geographically-distinct ice worm populations, and represents a rare example of a mitochondrial-based gene transfer event. The position and biochemical properties of the extension domain suggest a role in ATP synthase dimerization and/or proton shuttling, both of which would predictably enhance ATP production