The fundamental unit of chromatin is the nucleosome, which consists of a core of eight proteins wrapped by DNA. This core is composed of four pairs of histone proteins: H2A, H2B, H3, and H4. The DNA wraps around the protein core ~1.7 times, facilitating compaction of DNA length in the cell. Further, the location of nucleosomes makes genomic elements encoded in the DNA, such as promoters or enhancers, accessible or inaccessible to RNA polymerase and transcription factors. Thus, where nucleosomes are located (or positioned), can play a major role in transcription or other cellular processes. Additionally, histone proteins are frequently post-translationally modified, and these modifications further play a role in cellular processes, and in some cases are even required for specific protein function. What positions nucleosomes, and the downstream results of positioning or post-translational modifications (PTMs) is a topic of prolific study. Nucleosome formation is not random. In vivo it is believed that chromatin remodelers are the primary determinant of where nucleosomes form, while in vitro the DNA itself is the primary determinant. Formation of nucleosomes in vitro is a potent tool to elucidate fundamentals of chromatin. Considering that in vitro nucleosome formation is dependent on free energy, morphology and base composition of the DNA influence the free energy of formation. We found that the ends of linear DNA fragments were much more likely to have in vitro nucleosomes form on them. While this has the potential to bias results, based on our observations we could not find any significant alteration of the overall underlying DNA sequence composition due to the end preference observed. Histone proteins frequently receive the PTMs of methylation or acetylation. Histone methylation is typically indicative of repressed genes, while histone acetylation is typically indicative of active genes. In vivo the addition and removal of methylation and acetylation is highly dynamic. We hypothesized that the histone PTMs of methylation and acetylation also played a role in where nucleosomes formed. Comparing both in vivo and in vitro datasets, we observed strikingly similar patterns of nucleosomes for several histone methylations and acetylations, suggesting that these PTMs do indeed direct nucleosome formation. Upon further investigation, the underlying DNA sequence preferences change when compared to unmodified nucleosomes. This suggests that the genome is encoded to position these marks in locations where they are likely to be needed.

There are about 20,000 genes in the human genome. The lowly nematode worm, C. elegans, has about the same number of genes. How could two organisms that are so different arise from a similar number of genes? The answer is epigenetics, or the factors that help control when and where genes are expressed. There are many layers that comprise the epigenetic control of genes. One of which is the structure or architecture of chromatin. Chromatin is a complex of DNA and proteins. Histone proteins with DNA wrapped around them form the fundamental component of chromatin, the nucleosome. Chromatin exists in two forms, euchromatin and heterochromatin. Euchromatin is made of loosely packed nucleosomes while in heterochromatin nucleosomes are tightly packed. Genome elements are not accessible in heterochromatin but are in euchromatin. In this way chromatin architecture provides a layer of control of genetic expression. Where nucleosome form is a function of several factors including the underlying DNA sequence, and binding competition between histones and other DNA binding proteins. Here we test the ability of various DNA sequences to position and repel histone proteins in C. elegans worms. We find that the 601sequence can position nucleosomes and that the PRS-322 sequence does repel nucleosomes in vivo. Assessing chromatin architecture requires sequences to be aligned to a reference genome, however, there are numerous programs with which to do this. Each program performs this task in a different way. These differences can have a large impact on the downstream analysis of the results. To this end, we have tested various alignment programs to assess how well they align reads to a reference genome. Here we have found that Bowtie2, BWA, and Chromap perform alignments accurately and we suggest using them. As an organism develops its genetic expression changes. This change in expression is often the result of temporally specific genomic elements such as enhancers. Understanding when enhancers are accessible during development can lead to a better understanding of the genetic control needed for development. Here we utilize data gathered at specific developmental stages in C. elegans to elucidate enhancer accessibility. In this work we have furthered the understanding of epigenetic control of expression by quantifying positioning and repelling sequences, testing read mapping programs for accuracy and identifying temporally specific enhancers in developing worms.

No description available.

Ly49 receptors are the canonical natural killer cell class-I major histocompatibility complex receptors expressed in mice. They have a well-defined role in natural killer cell self/non-self discrimination and in the developmental licensing of functional natural killer cells. In this thesis, I report two novel aspects of Ly49 receptor biology. First, I show that their expression may be regulated by specific nucleosome occupancy on AML-1 binding sites within the distal Ly49 promoter. This finding sheds light on a potential regulatory pathway that has thus far been unexplored in studies of the Ly49 receptor family, and highlights the Ly49 family as an ideal model system in which to study the impact of nucleosome occupancy in general. Second, I show that Ly49 receptors have a central and indispensable role in the emerging phenomenon known as adaptive natural killer cell memory. Natural killer cells have recently been observed displaying adaptive, long-lived, antigen specific memory responses comparable to T cell memory responses, but no explanatory mechanism has been discovered to describe how adaptive memory is possible in these ‘innate’ immune cells. Using Ly49-deficient mice, I show that the inhibitory, self-specific Ly49 receptors Ly49C and Ly49I are required for adaptive memory responses to chemical haptens or protein antigens. Moreover, I show that Ly49C/I binding capabilities are required during all stages of the memory response, as is antigen presentation in the context of class I major histocompatibility complex, again analogous to T cell memory responses. I present initial findings implicating these Ly49 receptors as key components of the antigen recognition process itself, and propose a mechanism based in evolutionarily ancient immunology to explain how this specificity could arise. Finally, I demonstrate that Ly49-dependent natural killer cell memory is capable of mediating powerful anti-cancer vaccination effects using an aggressive model of melanoma. Together, these findings in Ly49 family expression regulation and its functional role in adaptive NK cell responses open several new avenues of study in Ly49 receptor biology and natural killer cell immunology.

Nucleosomes, the primary unit of chromatin structure, are positioned either statistically or specifically. The statistical positioning denotes the arbitrary positioning of nucleosomes on DNA agreeing with the nucleosome’s broad coverage of the genome—however, there is evidence that nucleosomes are also positioned specifically at controlled positions. DNA sequences determine the specific nucleosome positions, and the presence or depletion of nucleosomes affects the availability of the DNA region to other proteins. The DNA sequences of H2A and H2A.Z nucleosomes in Drosophila were analysed in search of nucleosome positioning patterns. Dinucleotide patterns with 10 bp periodicity were identified from the DNA sequences of H2A nucleosomes. Compared with the yeast patterns, the Drosophila patterns had the same periodicity but different dinucleotides near the dyad, which was related to the different H3 structure between them. The nucleosome positioning patterns from the H2A.Z nucleosomes implied the specific histone-DNA interaction as a result of the deviations of the patterns where the different amino acids of H2A and H2A.Z interact with the DNA. The Ly49 gene cluster was selected as a model system to study the interplay between nucleosomes and transcription factors. Ly49 proteins, the surface receptors on NK cells, display variegated expression patterns, and the bidirectional promoter Pro-1 is known as a key determinant of the stochastic expression of each Ly49 gene. The systematic analysis of nucleosome positions based on the genome sequences in the Ly49 gene cluster revealed that the repressing Pro-1 reverse promoters are open, while the activating forward Pro-1 promoters were covered by nucleosomes. Furthermore, specific nucleosome positions covered transcription factor binding sites. The covered factor binding sites were further examined by their periodic appearances on the nucleosome-covered sequences, which revealed the accessibility to the sites. The sequence analysis predicted that the regulation by the transcription factor AML-1 would be sensitive to the nucleosome coverage; the prediction was confirmed by cell line experiments. The 10 bp periodic nucleosome positioning patterns interact with histones specifically. The long nucleosome positioning patterns coexist with the short sequence motifs for transcription factor binding sites adding another layer of the control to the transcriptional regulation.

No description available.

DNA in eukaryotes is packaged into nucleosomes, which present steric impediments to many of the factors and enzymes that act on DNA, including DNA repair enzymes. Within the nucleosome, DNA remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway. Other DNA repair pathways, including Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Recombination (HR), and Non-homologous End-Joining (NHEJ) are all thought to require nucleosome remodeling or disruption. In contrast, it was reported that the first step of BER does not require or induce nucleosome disruption. For example, the human DNA glycosylase hNTH1 (human Endonuclease III) was discovered to excise thymine glycol lesions from nucleosomes without nucleosome disruption, and could excise optimally oriented lesions with an efficiency approaching that seen for naked DNA (Prasad, Wallace, and Pederson 2007). To determine if the properties of hNTH1 are shared by other human DNA glycosylases, we compared hNTH1 with NEIL1, a human DNA glycoylase that also excises thymine glycol from DNA, with respect to their activities on nucleosome substrates. We found that the cellular concentrations and apparent kcat/KM ratios for hNTH1 and NEIL1 are similar. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. After adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities towards nucleosome substrates. We next wanted to examine the effects of nucleosomes on enzymes that catalyze the remaining steps in BER. We therefore assembled the entire four-step BER reaction with model, lesion-containing nucleosomes. The rates of substrate processing during the first three steps in BER, catalyzed by a DNA glycosylase, AP endonuclease, and DNA Polymerase Pol), varied with the helical orientation of the substrate relative to the underlying histone octamer. In contrast, the rate of action by DNA Ligase III- (in association with XRCC1) was independent of lesion orientation. These results are consistent with structural studies of BER enzymes and the previously proposed DNA unwrapping model for how BER enzymes gain access to lesions in nucleosomes (Prasad, Wallace, and Pederson 2007). During these investigations, we also discovered a synergistic interaction between Pol and Ligase III- complexed with XRCC1 that enhances the repair of lesions in nucleosomes. Together, our results support the hypothesis that DNA glycosylases have evolved to function in specific cellular environments (e.g. NEIL1 may function exclusively during DNA replication), but also possess DNA binding motifs and mechanisms of substrate recognition that impart a similar intrinsic activity on nucleosomes. In addition to hNTH1 and NEIL1, we have discovered that lesion orientation is also an important factor to the activities of APE and Pol and that the complete BER reaction can occur without requiring or inducing nucleosome disruption. Finally, protein-protein interactions between XRCC1 and Pol may be important for the efficient in vivo repair of lesions in nucleosomes.

The nucleosome remodeling factor (NURF) is an evolutionary conserved ATP-dependent chromatin remodeling factor. It was first isolated from Drosophila as a complex with enzymatic activity that once recruited to nucleosome, it slides the nucleosome to provide accessibility for transcription factors. Since then, numerous works from animal models and cell lines show the role of NURF as a regulator of gene expression. NURF interacts with H3K4me3 and sequence specific transcription factors that recruit the complex to promoter regions. Whether this is the only mechanism by which NURF regulates gene expression is not known. However, other ATP-dependent chromatin remodeling complexes are known to regulate gene expression independent from transcription initiation. In order to explore the role of NURF in regulating gene expression, we utilized two genome wide approaches to map NURF binding and NURF dependent changes in chromatin structure using ChIP-Seq and FAIRE-Seq, respectively. From these analyses, we discovered that NURF broadly localizes in the genome with preferences to gene bodies and 3’ends of genes. Also, we found that NURF maintains open chromatin regions at upstream, intron and downstream of genes. These novel findings shed light on new roles for NURF complex within genes, in addition to its classical role at promoter regions. Furthermore, we discovered the function of a previously uncharacterized domain in the NURF specific subunit BPTF. We show that the N-terminal the plant homeodomain (PHD) of BPTF directly interacts with THOC4, a protein associated with RNA-pol 2. Also, we show using ChIP analyses that this interaction recruits BPTF to gene bodies. Next, we investigated functional consequences for NURF recruitment to gene bodies using Cyclin D1 (Ccnd1) gene as a model. These analyses revealed that NURF is required for normal mRNA processing and loss of NURF induces intron retention, which results in unstable transcripts. Finally, we show that the defect in mRNA processing is not specific to the Ccnd1 gene, as we observe similar defects in four other BPTF dependent genes. Together, our work uncovered new role of mammalian NURF complex in regulating gene expression through mRNA processing.

Ces dernières années ont vu se multiplier les études portant sur le positionnment du nucléosome. Ce phénomène est en effet essentiel pour comprendre correctement les processus liés à l'expression génique. Ces travaux soulignent notamment la multiplicité des facteurs qui interviennent dans le positionnement en vivo, toutefois le rôle de la séquence d'ADN elle même est bien reconnu que ce soit sous formes de séquences préférées ou "excluantes". Afin d'identifier les propriétés structurales et dynamiques des séquences qui possèdent une forte capacité à positionner le nucléosome, nous avons procédé à une série d'études structurales sur les oligomères d'ADN libres correspondant à la séquence de l'une des plus connues d'entre elles, la séquence 601. Nous nous sommes plus particulièrement intéressés à une séquence de 39 paires de bases correspondant à la moitié d'un tour complet autour du cœur d'histone. L'utilisation des méthodes de RMN permet de déterminer les propriétés d'oligomères en solution. Néanmoins la difficulté d'attribuer les résonances phosphore sur des séquences oligonucléotides longues nous a conduit à diviser la séquence d'intérêt en quatre parties égales (12bp) sur lesquelles les attribution penvent être réalisées. L'analyse des 72 déplacements chimiques du phosphore (&#948-P) et des 249 distances séquentielles inter-protons recueillies sur les oligomères montre que les mouvements de l'ADN à l'échelle de temps de la nanosecondes, des groupements phosphates, des sucres et des bases sont fortement couplés et directement reliés à la séquence dinucléotidique. Les données expérimentales recueillies dans ce travail sont en accord avec les prédictions de l'échelle TRX (Twist,Roll,X-disp) qui quantifie les mouvement à l'échelle de la nanoseconde de l'ADN-B. L'annotation de l'échelle TRX de la séquence 601 montre une alternance périodique de régions rigides et flexibles le long de la séquence, cette périodicité est de 10 pairs de bases. De façon frappante, cette alternance se superpose aux variations sinusoïdales des largeurs des petits sillons observées sur les structures cristallographiques des nucléosomes. L'élément TTAAA, a fait l'objet d'une attention particulière dans ce travail. Il est soupçonné d'être crucial dans le contexte de la formation des nucléosomes, cette séquence présente en effet quelques particularités remarquables, tels que la présence de plusieurs enchaînements dinucléotidiques riche en BI (dP décalé vers les hauts champs et distances inter-nucléotidiques courtes)- ceux-ci se trouvent associés à un petit sillon très étroit. Les résultats relatifs aux couplages dipolaires résiduels (RDC), qui reflètent l'angle de déviation entre le vecteur carbone-proton et l'axe l'ADN ont montré que les différences entre les couplages dipolaires résiduels de deux résidus successifs &#916-(RDC), sont également corrélées aux &#948-P. Ce résultat confirme définitivement que la structure et la dynamique des groupes phosphates, les positions relatives des bases et les conformations sucres de l'ADN-B sont fortement couplés. En outre, nos résultats permettent de commencer à déchiffrer le processus influençant la formation des nucléosomes, ils montrent que l'échelle TRX est un outil puissant de prédiction des conformations et la dynamique des ADN. / Nowadays, nucleosome positioning is being studied intensively due to the critical role in the regulation and transcription by modulating the accessibility of specific targeted sites for proteins in eukaryotic cell. As demonstrated by recent studies that positioning in vivo could be modulated by various factors, among which the role of DNA sequence is repeatedly underlined due to the form of preferred or excluding sequences. In an attempt to identify the structural and dynamic properties of sequences exhibiting strong ability to precisely position the nucleosome, we carried out a series of structural studies on free DNA oligomers corresponding to the core of this sequence. The synthetic 601 sequence was selected as a proper candidate for the experiment and the oligomers cover 39bp of the 5’ half of the sequence. The utilization of NMR technology allows the observation of the oligomer properties in solution. However, it’s difficult to assign phosphorus resonances signals to long DNA oligonucleotides, which could be realized to divide the sequence of interested part into four equivalents (12 bp). Analysis of a large set of NMR data on the four oligomers (72 dP (phosphorus chemical shift) and 249 sequential inter-proton distances) indicate the strong coupling between nanoseconde timescale motions of phosphate group and those involving sugar and bases. In the four dodecamers, the nanosecond timescale dynamics is dominated by the dinucleotide sequence, modulated at the tetranucleotide level. Importantly, the experimental data collected here validate our previously published NMR-based TRX scale that quantifies the nanosecond timescale intrinsic malleability of the ten complementary dinucleotides in B-DNA. The TRX annotation of the whole 601 sequence shows a 10 base-pair periodic alternation of stiff and flexible regions that can be exactly superimposed to the sinusoidal variations of minor groove width observed on the crystallographic structures of nucleosome. The TTAAA element was the subject of particular attention in this work. It is suspected to be crucial in the nucleosome formation context, exhibiting remarkable features, such as the constitution of a succession of BI-profiles (high-shifted dP and short internucleotide distances) associated to a narrow minor groove. Furthermore, the results related to the residual dipolar couplings (RDC), which reflect the deflection angle between carbon-proton vectors and the long axis of DNA have shown that the differences between the residual dipolar couplings of two successive residues, &#916-(RDC), are also correlated with &#61540-P. This result definitively confirms that the structure and the dynamic of phosphate group, bases and sugars in B-DNA are highly coupled. In addition, our results start to decipher the indirect readout process underlying the nucleosome formation. Further, they establish that the TRX scale is a powerful, predictive tool for better understanding DNA-protein interactions, based on flexibility criteria. It represents a new step towards a simplified determination of properties of DNA by NMR.

No description available.