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1
Deniz, Ozgen. "Nucleosome Positioning in Budding Yeast = Posicionamiento de nucleosomas en Saccharomyces cerevisiae." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/145763.
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The nucleosome is the fundamental structural unit of DNA compaction in eukaryotic cells and is formed by the wrapping of 147 bp double stranded DNA around a histone octamer. Nucleosome organization plays a major role in controlling DNA accessibility to regulatory proteins, hence affecting cellular processes such as transcription, DNA replication and repair.
Our study focuses on genome-wide nucleosome positioning in S. cerevisiae to explore nucleosome determinants and plasticity throughout the cell cycle and their interplay with gene expression based on cell mRNA abundance.
We pursued the contribution of DNA physical properties on nucleosome organization around key regulatory regions such as TSSs and TTSs by analyzing genome-wide MNase-digestion profile of genomic DNA. We also implemented a systematic approach to standardize MNase-Seq experiments by minimizing the noise generated by extrinsic factors to enable an accurate analysis of the underlying principles of nucleosome positioning and dynamics. Moreover, we carried out a large-scale study of nucleosome plasticity throughout the cell cycle and its interplay with transcription based on a comparative analysis among nucleosome maps, gene expression data and MNase sensitivity assays. We then focused on nucleosome organization around DNA replication origins and its possible effect on origin activation. Finally, we sought to characterize centromeric nucleosome composition and its oscillation along cell cycle.
During the course of these studies, we found that key regulatory regions such as 5’ and 3’ nucleosome free regions (NFRs) contain unusual physical properties that are intrinsic to genomic DNA. We further demonstrated that DNA physical properties and transcription factors act synergistically to define NFRs, especially in genes with an open promoter structure. Once NFR is defined, the nucleosome positioning around TSSs can be predicted by a simple statistical model, supporting the energy barrier model for nucleosome positioning determination. However, we also observed that nucleosomes are quite dynamic at distal 5’ NFRs and do have distinct regulatory mechanisms.
Our comparative analysis of nucleosome organization along cell cycle revealed that chromatin exhibits a distinct configuration due to DNA replication-dependent organization at S phase, showing higher sensitivity to MNase and displaying fuzzier nucleosomes along the genome. Moreover, we observed different features at M phase, where chromatin compaction is the highest and displays a slightly different pattern than in G1 and G2 phases. Interestingly, these changes in chromatin organization are sudden and acute and only affect some regions of the genome, whereas the majority of genes present conserved nucleosome patterns along cell cycle. Our individual gene analysis disclosed that the largest changes take place in cell cycle-dependent genes, indicating the interplay between chromatin and transcription. Moreover, a distinct nucleosome organization at high and low transcription rates further supports this relationship.
The detailed analysis around replication origins shows that they display slightly wider NFRs at G1 phase due to pre-Replication complex binding. Once the replication origins are active, nucleosomes partially occupy NFRs up to a certain extent due to constitutive binding of ORC. Moreover, we provided further evidence that early firing origins tend to have more ordered nucleosome organization than late firing origins.
Finally we illustrated that centromeric nucleosomes display a perfect positioning, confirming their strong centromeric sequence-dependent recruitment to DNA. The characterization of histone composition under physiological cell conditions suggested that the octameric nucleosome assembly model is favored in centromeres. Yet, our analysis along cell cycle showed centromeric nucleosome dynamics, proposing that its composition might oscillate along cell cycle.
Taken together, our accurate study provides a dynamic picture of nucleosome positioning and its determinants; new insights into cell cycle-dependent chromatin organization on key regulatory regions and its interplay with gene expression; and adds a new dimension to the characterization of centromeric nucleosomes.Nuestro estudio se centra en el posicionamiento de nucleosomas a nivel genómico en levadura, con tal de explorar los factores determinantes de nucleosomas y su plasticidad a lo largo del ciclo celular, así como su relación con la expresión génica basándonos en la cantidad de mARN celular. Encontramos que las regiones libres de nucleosomas (NFRs en inglés) en 5’ y 3’ contienen propiedades físicas inusuales, las cuales son intrínsecas del ADN genómico. Además, demostramos que estas propiedades físicas actúan sinérgicamente con factores de transcripción para definir las NFRs. Una vez la NFR está definida, el posicionamiento de nucleosomas en torno al inicio de transcripción (TSS en inglés) puede predecirse con modelos estadísticos simples. No obstante, también observamos que los nucleosomas son bastante dinámicos en las regiones distales a 5’NFRs y poseen distintos mecanismos reguladores. Nuestro análisis comparativo acerca de la organización de los nucleosomas reveló que la cromatina de hecho exhibe una configuración distinta debido al reordenamiento dependiente de la replicación en fase S, mostrando una mayor sensibilidad de corte por el enzima MNase y un mayor número de nucleosomas deslocalizados a lo largo del genoma. Adicionalmente, observamos características particulares en fase M, donde la cromatina sufre un mayor grado de compactación. Notablemente, estos cambios en la organización de la cromatina son repentinos y agudos y sólo afectan a algunas regiones del genoma, mientras que la mayoría de genes presentan una conservación del patrón de nucleosomas a lo largo del ciclo celular. El análisis detallado en torno a los orígenes de replicación muestra una NFR más ancha en fase G1, debido a la unión del complejo pre-replicatorio. Una vez se activa el origen, los nucleosomas sólo ocupan parcialmente la NFR, debido a la unión constitutiva del complejo de origen de replicación (ORC en inglés). También proporcionamos evidencias de que los orígenes tempranos tienden a tener una organización nucleosomal más ordenada que los tardíos. Finalmente, ilustramos que los nucleosomas centroméricos poseen un posicionamiento idóneo y asimismo, un ensamblaje distinto. Sin embargo, nuestro análisis también mostró la dinámica de los nucleosomas centroméricos a lo largo del ciclo celular, indicando que de hecho su composición puede oscilar a lo largo del ciclo celular. En conjunto, nuestro detallado estudio proporciona una imagen dinámica del posicionamiento de nucleosomas y sus factores determinantes; nuevos indicios respecto a la organización de la cromatina en regiones reguladoras clave en base al ciclo celular y su conexión con la expresión génica; y finalmente, añade una nueva dimensión a la caracterización de los nucleosomas centroméricos.
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Wight, Andrew. "Adaptive NK Cell Memory and Nucleosome Interference: Two Tales of the Ly49 Receptor Family." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/37059.
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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.
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Koorsen, Gerrit. "The association of the secondary DNA-binding site of linker histone H5 in a nucleosome." Master's thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/4282.
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Bibliography: leaves 153-170.In order to understand the role of linker histones in the formation of the 30-run chromatin fibre as well as their role in transcriptional repression, it is essential to know their location on the nucleosome. In this study, we have modelled the location of the globular domain of chicken linker histone HS (GHS) on the nuc1eosome. The primary DNA binding site of GH5 was modelled by homology to the co-crystal structure of the E. coli CAP-DNA complex.
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FENG, YIHONG. "Controllable cell delivery and chromatin structure observation using DNA nanotechnology." Kyoto University, 2020. http://hdl.handle.net/2433/258987.
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Cook, April D. "Characterization of nucleosome occupancy in mammalian cells." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070019.
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Chromatin is a complex of genomic DNA, RNA, and associated proteins. Many of the processes that occur on chromatin regulate the accessibility of the genetic material of a cell. The nucleosome is the basic subunit of chromatin, composed of a histone octamer wrapped with approximately 150bp of DNA. Alterations to chromatin structure, including to nucleosomes and their location, underlie global transcriptional diversity. A striking example of this is the so-called "open" chromatin state in pluripotent cells, characterized by loosely bound chromatin proteins and rapid nucleosome turnover, that allows transcriptional flexibility for subsequent differentiation. In contrast, differentiated cells contain compacted chromatin that can selectively block access to DNA and subsequent transcription. Thus, characterizing the physical state of chromatin is important to understanding its regulatory state.
Digestion of chromatin with micrococcal nuclease (MNase) and subsequent sequencing of the protected DNA fragments produces a map of nucleosome occupancy. Traditional MNase mapping experiments capture a snapshot of nucleosome occupancy, providing information about nucleosomes that are accessible at the level of digestion used. We analyzed regions of difference in nucleosome occupancy between embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and differentiated cell types using traditional MNase-seq and found that differences in pluripotent and differentiated cells are punctate and correlate with regulatory regions important for pluripotency and development. Further, our analysis shows ESCs and iPSCs to be vastly more similar to each other in their chromatin structure than to the differentiated cells.
We then developed a new way of collecting and analyzing MNase-seq data that allows us to determine both nucleosome occupancy as well as the accessibility of DNA to regulatory factors. Our methodology discerns distinct physical states of chromatin and provides novel insights into the accessibility of regulatory regions. Additionally, we present a quantitative metric useful for characterizing local and global regions of the genome that should be useful in future cell type comparisons.
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Pohl, Andy 1979. "Nucleosome dynamics and analysis in breast cancer cells." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/328416.
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Genome-wide analysis of the nucleosome positioning and histone H1 isoform content of the T47D breast cancer cell line has found a number of observations, namely that with a gentle digestion of microccocal nuclease (MNase), a nucleosome is visible just upstream of the transcription start site, in the region known as the “nucleosome-free region” (NFR). H1 isoforms bind to chromatin mainly in a redundant manner, but H1.2 and H1.3 show some specificity while H1.5 increases its binding dramatically after a progesterone stimulus. In the course of these studies, a general-purpose software package was developed for the manipulation and analysis of bigWig files, a data format for storing continuous signal data assigned to genome coordinatesEn el meu estudi genòmic sobre el posicionament de nucleosomes i sobre elcontingut de les isoformes de la histona H1 en cèl•lules de càncer de mama T47D he dut a terme una sèrie d'observacions. Específicament he trobat que amb una digestió suau amb nucleasa micrococcal, es pot identificar un nucleosoma just abans del lloc d'inici de transcripció, en la regió coneguda com a "regió lliure de nucleosomes". També he vist que les diferents isoformes somàtiques de la histona H1 (H1.0-H1.5, H1x) s'uneixen a la cromatina de manera redundant, però que la H1.2 i la H1.3 presenten certa especificitat, mentre que la H1.5 mostra un augment de la unió generalitzat després d'estimular les cèl•lules amb progesterona. En el decurs de la meva recerca, he desenvolupat un programari general per la manipulació i l'anàlisi d'arxius amb format bigWig, un format per a l'emmagetzematge de dades de senyals continus al llarg de les coordenades del genoma.
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Gatta, R. "Chromatin configuration of CCAAT-containing cell cycle promoters." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/158416.
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The CCAAT box is a frequent promoter element bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone acetylations and methylations and the association of enzymatic activities. This thesis presents different novel findings. The first finding was that H3-H4 take part of core promoters under active conditions, with the expected cohort of “positive” modifications, while H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show also that NF-Y is important for H3K36me3 deposition and for Pol II elongation. The second finding was that H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Both functional and pharmacological inactivation led to state that KDM1 plays a positive role in transcription of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. Therefore, these preliminary data are the first in vivo indication of a crucial interplay between core histones and “deviant” histone-fold such as NF-Y, leading to fine tuning of histone methylations. The third finding was that NF-Y is not involved in histone acetyl-marks deposition as well as in histone methyl-marks: in fact, histone acetylation status of active cell cycle genes was only slightly perturbed after NF-Y removal. Moreover, this work proposed a special histone acetylation pattern typical of cell cycle gene cluster, characterized by H2BK120ac and H3K9,18,36ac deposited on H3 in repressive conditions. And finally, by in vivo analysis we showed GCN5 and PCAF involvement in the acetyl-marks deposition, and the probable NF-Y dependent recruitment of multisubunit complexes responsible of the chromatin remodelling, like STAGA and ATAC.
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Garza, Manero Sylvia Patricia. "The role of high mobility group of nucleosome binding proteins in stem cell biology and differentiation." Thesis, University of Glasgow, 2019. http://theses.gla.ac.uk/41111/.
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The high mobility group of nucleosome binding proteins (HMGNs) are chromatin architectural proteins that bind specifically to nucleosomes and influence chromatin structure and DNA-dependent functions. However, the mechanisms underlying these events remain largely unknown. HMGN1 and HMGN2 are highly expressed by embryonic stem cells and are downregulated as differentiation proceeds. Nevertheless, embryonic and adult neural stem cells retain elevated levels of these proteins. Chromatin plasticity is essential for the pluri- and multipotency of stem cells and it is achieved by maintaining an open and dynamic chromatin conformation. Conversely, developmental potential seems to be restricted by chromatin condensation. The present work shows that loss of HMGN1 or HMGN2 in pluripotent embryonal carcinoma cells leads to increased spontaneous neuronal differentiation, which is accompanied by a reduction in pluripotency markers and higher gene expression of lineage-specific transcription factors. Inhibition of signalling pathways relevant for neurogenesis does not re-establish the phenotype observed in Hmgn2-knockout cells. Withdrawal of the factors sustaining pluripotency in embryonal carcinoma cells results in higher induction of pro-neural factors in cells lacking HMGN1 or HMGN2. Neural stem cells derived from Hmgn-knockout cells also display higher gene expression of pro-neural transcription factors and increased spontaneous neuronal differentiation. Loss of HMGN2 disrupts the active histone modification landscape, and therefore affects the chromatin structure at local and global levels. The proposition is that the local changes directly influence the transcription rates of pluripotency and lineage-specific transcription factors, while the global changes may restrict chromatin plasticity. The present data support a hypothesis whereby HMGNs contribute to the chromatin plasticity of stem cells by promoting an active histone modification landscape and open chromatin conformation, which are essential for preserving the self-renewal and developmental potential of stem cells.
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Pünzeler, Sebastian [Verfasser], and Sandra [Akademischer Betreuer] Hake. "PWWP2A : a novel H2A.Z nucleosome interactor involved in cell cycle regulation / Sebastian Pünzeler. Betreuer: Sandra Hake." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1111505349/34.
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Unhavaithaya, Yingdee. "Conserved Nucleosome Remodeling/Histone Deacetylase Complex and Germ/Soma Distinction in C. elegans: A Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/239.
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A rapid cascade of regulatory events defines the differentiated fates of embryonic cells, however, once established, these differentiated fates and the underlying transcriptional programs can be remarkably stable. Here, we describe two proteins, MEP-1, a novel protein, and LET-418/Mi-2, both of which are required for the maintenance of somatic differentiation in C. elegans. MEP-1 was identified as an interactor of PIE-1, a germ-specific protein required for germ cell specification, while LET-418 is a protein homologous to Mi-2, a core component of the nuc1eosome remodeling/histone deacetylase (NuRD) complex. In animals lacking MEP-1 and LET-418, germline-specific genes become derepressed in somatic cells, and Polycomb group (PcG) and SET domain-related proteins promote this ectopic expression. We demonstrate that PIE-1 forms a complex with MEP-1, LET-418, and HDA-1. Furthermore, we show that the overexpression of PIE-1 can mimic the mep-1/let-418 phenotype, and that PIE-1 can inhibit the Histone deacetylase activity of the HDA-1 complex in COS cells. Our findings support a model in which PIE-1 transiently inhibits MEP-1 and associated factors to maintain the pluripotency of germ cells, while at later times MEP-1 and LET-418 remodel chromatin to establish new stage- or cell-type-specific differentiation potential.
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Mayes, Kimberly. "The Role of the Nucleosome Remodeling Factor NURF in Inhibiting T and Natural Killer Cell Mediated Antitumor Immunity by Suppressing Tumor Antigenicity and Natural Cytotoxicity Receptor Co-ligands." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4770.
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Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity.
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Amaya, Maria. "The Role of the Nucleosome Remodeling and Histone Deacetylase (NuRD) Complex in Fetal γ-Globin Expression." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/521.
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An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.
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Wright, Ashley Nicolle. "Analysis of Nucleosome Mobility, Fragility, and Recovery: From Embryonic Stem Cells to Invitrosomes." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/5285.
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Several factors direct the placement of specific nucleosomes, which in turn have the ability to regulate DNA accessibility. These factors include, but are not limited to, nucleotide sequence preference, nucleotide modifications, the type of histones present within the nucleosome, and the presence of additional transcription factor or chromatin remodelers. A combination of these and other factors are responsible for tightly controlled efficient transcription within the eukaryotic cell. In order to contribute to the understanding of these complicated processes, three separate hypothesis-driven investigations were carried out. First, we looked into nucleosome positioning and phasing within closely related cells lines. Second, we examined domain level nucleosome occupancy on various portions of the chromosome. Finally, we generated a novel method that significantly reduces data loss in in vitro nucleosome reconstitution experiments caused by nucleosome fragment-end bias. All three of our investigations yielded separate results. First, by examining positions and phasing patterns within similar cell types we find common patterns and minor differences within similar cell types. The presence of minor differences in nucleosome positions may cause unique expression patterns. Secondly, we found that decreased domain level nucleosome occupancy at the chromosome arms is not caused by the presence of a class of nucleosomes, termed fragile nucleosomes. Finally, we found that when our nucleosome recovery method is applied conservatively to our dataset, it is possible to recover 80% of the lost nucleosome reconstitution data.
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Tacheva, Silvia K. "Post-translational Modifications of Newly Synthesized Histones H3 and the Role of H3 K56 Acetylation on Chromatin Assembly in Mammalian Cells." Thesis, Boston College, 2010. http://hdl.handle.net/2345/1745.
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Thesis advisor: Anthony T. AnnunziatoThe project I am presenting aimed to: 1. Elucidate the pattern of post- translational modification on the different variants of newly synthesized histones H3 in mammalian cells; 2. Reveal whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone into chromatin in mammalian cells; and 3. Determine whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone specifically in replicating chromatin in mammalian cells. The experiments to answer these questions were performed using HEK293 cells with inducible expression of FLAG-histones, enabling us to control the synthesis of new histones of interest and to detect and analyze their presence and relative levels in the cells. The results suggest that the acetylation of lysine 56 on histone H3 may play a positive role in the incorporation of the histone into new chromatin, and lack of acetylation may be reducing the efficiency of incorporation compared to acetylated histones
Thesis (MS) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Font, Mateu Jofre 1977. "Dynamics of progesterone receptor interactors in breast cancer cells upon hormone exposure." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/511363.
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El receptor de la progesterona és un regulador clau per la proliferació de les cèl·lules de càncer de mama dependents d’hormona. El mecanisme d’acció del PR ha tingut un paper important en la resolució del mecanisme molecular d’activació de la transcripció. No obstant això, no hi ha hagut un estudi a fons de les seves interaccions en resposta a hormona. En aquest treball s'han identificat per RIME (immunoprecipitació ràpida per l'espectrometria de masses de proteïnes endògenes) 315 interactors d’alta confiança del PR en cèl·lules de càncer de mama exposades a la potent agonista de la progesterona R5020 durant 0, 1, 5, 15, 30 i 60 minuts. Hem identificat 20 interactors coneguts del PR i 295 de nous. Els interactors del PR trobats formen 4 grups dinàmics; El grup basal, 66 proteïnes presents en nivells similars en tots els temps; grup 1, 41 proteïnes que disminueixen la seva interacció després de l'hormona; grup 2, 115 proteïnes que augmenten la seva interacció ràpidament després de l'hormona; i el grup 3, 91 proteïnes que tenen un augment de la seva interacció constant amb el temps. Els interactors del PR formen complexes funcionals que intervenen en la regulació transcripcional, remodelació de la cromatina, el processament del ARNm, reparació de l’ADN danyat, la degradació proteosomal, proteïnes estabilitzadores i proteïnes de l’estructura nuclear. L'exposició de cèl·lules a l’antagonista parcial de la progesterona RU486 manté la majoria dels interactors del PR, però perd els relacionats amb la regulació de la transcripció. Aquest estudi estableix les bases per a l'anàlisi de les noves funcions dels receptors de progesterona en cèl·lules de càncer de mama.Progesterone receptor is a key regulatory element in hormone-dependent breast cancer cells proliferation. The mechanism of action of PR has played an important role in solving the molecular mechanism of transcription regulation. However, it has not been a thorough study of its interactors in response to hormone. In this work we have identified by RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) 315 high confidence PR interactors in breast cancer cells exposed to the potent progesterone agonist R5020 for 0, 1, 5, 15, 30 and 60 minutes. We have identified 20 known PR interactors and 295 new ones. The found PR interactors form 4 dynamic clusters; Basal cluster, 66 proteins present at similar level at all time points; Cluster 1, 41 proteins decreasing their interaction after hormone; cluster 2, 115 proteins increasing their interaction rapidly after hormone; and cluster 3, 93 proteins increasing their interaction steadily over time. PR interactors form functional complexes involved in transcriptional regulation, chromatin remodelling, mRNA processing, DNA damage repair, proteosomal degradation, protein stability and nuclear structural proteins. Exposure of cells to progesterone partial antagonist RU486 maintain the majority of PR interactors, but loses the interactors related to transcription regulation. This study set the bases for analyses of new functions of progesterone receptor in breast cancer cells.
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Hoch, Duane A. "Thermodynamic studies of the Escherichia coli factor for inversion stimulation and the eukaryotic nucleosome core particle." Online access for everyone, 2006. http://www.dissertations.wsu.edu/Dissertations/Fall2006/D_Hoch_112006.pdf.
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Qiu, Runan [Verfasser]. "Effects of nucleoside analogues on protein expression in cells of the SerW3 cell line / Runan Qiu." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1062536800/34.
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Liang, Lu. "Nucleoside transport in sheep reticulocytes : evidence for an intracellular transporter pool." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60695.
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We tested the existence of an intracellular nucleoside transporter pool in sheep reticulocytes. The number of cell surface NBMPR binding sites in sheep reticulocytes is 5.1 $ pm$ 0.5 (S.D.) fmol/10$ sp{6}$ cells (n = 9), compared to 6.9 $ pm$ 0.4 (S.D.) fmol/10$ sp{6}$ cells (n = 9) for total binding. Resealed ghosts made from sheep reticulocytes show no fewer NBMPR binding sites than cell surface, but somewhat fewer than total binding sites. Furthermore, the number of NBMPR binding sites is the same in ghosts $ pm$ saponin, suggesting that after cell lysis and resealing, NBMPR is accessible in the intracellular matrix. The resealed ghosts also show increased permeability to pCMBS (an inhibitor of -SH groups on the cytoplasmic side of the nucleoside transporter in sheep reticulocytes). NBMPR binding sites exhibit a preferential loss compared to those on the cell surface during in vitro maturation of sheep reticulocytes. Species differences in NBMPR binding on cell surface and total were also examined.
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Pettitt, Andrew R. "Cell survival, P53 dysfunction and nucleoside action in chronic lymphocytic leukaemia." Thesis, University of Liverpool, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366481.
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Britton, Fiona Catherine. "The molecular and biochemical characterisation of thymidine kinase 1 from normal and transformed mammary cell lines." Thesis, University of Ulster, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268554.
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Borgland, Stephanie L. "Investigations of interactions between nucleoside transporters and adenosine receptors in three cell models." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ32902.pdf.
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Clarke, Anna B. "Mitochondrial toxicity of nucleoside reverse transcriptase inhibitors in a rat phaeochromocytoma cell line." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430314.
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Shaw, Margaret Mary. "Differential effects of guanosine nucleoside analogue inhibitors of herpes simplex virus on cell death." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621019.
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Harastani, Mohamad. "Image analysis methods development for in vitro and in situ cryo-electron tomography studies of conformational variability of biomolecular complexes : Case of nucleosome structural and dynamics studies." Electronic Thesis or Diss., Sorbonne université, 2022. http://www.theses.fr/2022SORUS283.
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La tomographie électronique cryogénique (cryo-TE) permet de visualiser des complexes biomoléculaires in situ. Les données 3D de biomolécules produites à l'aide de cryo-ET sont bruitées, souffrent d'anisotropies spatiales et sont difficiles à analyser individuellement. Les biomolécules sont flexibles et l'analyse de leur variabilité conformationnelle est nécessaire pour comprendre leurs mécanismes fonctionnels. Les méthodes standards de traitement de données de cryo-ET moyennent plusieurs copies de biomolécules individuelles pour obtenir des structures à des résolutions plus élevées et considèrent que la variabilité conformationnelle biomoléculaire est discrète plutôt que continue en utilisant la classification. Cette thèse présente les deux premières méthodes de traitement de données cryo-ET pour l'analyse de la variabilité conformationnelle continue biomoléculaire, HEMNMA-3D et TomoFlow. HEMNMA-3D analyse des données expérimentales avec les directions de mouvement simulées par l'Analyse de Modes Normaux et permet la découverte d'une large gamme de mouvements biomoléculaires. TomoFlow extrait des mouvements à partir des données en utilisant la technique de vision par ordinateur de Flux Optique. Je montre le potentiel de ces deux méthodes sur des données cryo-ET expérimentales de variabilité conformationnelle des nucléosomes dans les cellules. Les deux méthodes montrent des résultats cohérents, faisant la lumière sur la variabilité conformationnelle des nucléosomes dans les cellulesCryogenic electron tomography (cryo-ET) allows visualizing biomolecular complexes in situ. 3D data of biomolecules produced using cryo-ET are noisy, suffer from spacial anisotropies, and are difficult to analyze individually. Biomolecules are flexible, and analyzing their conformational variability is necessary to understand their functional mechanisms. Standard cryo-ET data processing methods average multiple copies of individual biomolecules to obtain structures at higher resolutions and consider that biomolecular conformational variability is discrete rather than continuous using the classification. This thesis presents the first two cryo-ET data processing methods for analyzing biomolecular continuous conformational variability, HEMNMA-3D and TomoFlow. HEMNMA-3D analyzes experimental data with the motion directions simulated by Normal Mode Analysis and allows the discovery of a large range of biomolecular motions. TomoFlow extracts motions from the data using the computer vision technique of Optical Flow. I show the potential of these two methods on experimental cryo-ET data of nucleosome conformational variability in cells. The two methods show coherent results, shedding light on the conformational variability of nucleosomes in cells
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Wallerman, Ola. "Genome-Wide Studies of Transcriptional Regulation in Mammalian Cells." Doctoral thesis, Uppsala universitet, Medicinsk genetik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-132882.
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The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.
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Doherty, Andrew J. "Nucleoside uptake in rabbit outer cortical basolateral membrane vesicles and a variety of cultured renal cell lines." Thesis, University of Kent, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280309.
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Banjer, Hamsa Jameel. "Role of the human Concentrative Nucleoside Transporter 1 (hCNT1) in oncogenesis." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586350.
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Transportome alterations have been associated with oncogenesis. The loss of human Concentrative Nucleoside Transporter 1 (hCNT1) during carcinogenesis seems to be a relatively common event in tumors and might by itself contributing to oncogenesis. In fact, hCNT1 can play a dual role in cell biology being a nucleoside and nucleoside-derived drug transporter but also an important player in the modulation of a variety of cellular functions. Accordingly, we have previously reported that hCNT1 function is able to induce physiological changes that are relevant to tumor biology in a transport independent-manner. These observations argued in favor of hCNT1 being a transceptor protein. Thus, in this thesis we focused our study on the role of hCNT1 in cell physiology beyond the mere nucleoside salvage. We found that hCNT1 is able to induce antiproliferative effects in different tumor backgrounds, and its restitution in Hepatocellular carcinoma (HCC) derived cell lines alters several different signaling cascades that are important to many physiological functions of the cell, including cell survival and cell migration. Moreover, hCNT1-induced cell death seems to be triggered by an increase in intracellular Ca2+ levels, which might be related to the interaction of the Ca2+-binding protein S100A11 with hCNT1.
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Usher, Sarah Louise. "Nucleosome positioning in Arabidopsis." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/3212/.
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The aim of this project was to test hypotheses relating to nucleosome positioning in Arabidopsis to provide a basis for better understanding of epigenetic transcriptional regulation in plants. Prior to this study, virtually no information existed regarding nucleosome positioning in plants. Eukaryote chromosomes consist of chromatin, composed of nucleosomes separated by linker DNA of variable lengths. Nucleosomes consist of 147 bp of DNA wrapped 1.7 times around a histone octamer. Whilst no consensus nucleosome positioning DNA sequence exists, sequence preferences influence positioning, and contribute to the complex epigenetic processes which act to control transcriptional activity. These details of the underlying mechanisms are known to differ between the plant and animal kingdoms. High-throughput sequencing technologies were utilised to generate large datasets of mono- and di-nucleosome sequences from wild-type Arabidopsis. These enabled genome-wide analysis and inference of plant-specific patterns of nucleosome positioning and sequence properties. Further data were generated from a methyltransferase antisense (MET1) which is depleted in methylated CG epigenetic marks. The internal distributions of dinucleotides within Arabidopsis nucleosomes were similar to those observed in non-plant eukaryotes. A unique periodicity in the distribution of linker lengths was detected in Arabidopsis wild type chromatin. In contrast, the MET1 antisense line displayed the expected periodicity, indicating systematic differences in chromatin organisation. There was a significant increase in nucleosome occupancy within exons compared with introns. However, this difference was less marked in the MET1 antisense. Specific patterns of nucleosome phasing were observed around transcription start sites. Linker lengths within rRNA gene clusters associated with nucleolar organiser regions (NORs) differed depending on chromosome of origin, suggesting differences in higher order chromatin structure between the NORs. Comparison of the nucleosome position and DNA methylation within the rRNA gene cluster revealed interesting differences between the two regions, which may reflect interactions affecting chromatin structure and transcriptional regulation.
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Wiesendanger, Barbara. "I. The proportion of non-nucleosomal, transcribable and nucleosomal, non-transcribable ribosomal RNA gene copies is cell type-specific : II. Replication fork barriers in Xenopus laevis and Xenopus borealis ribosomal DNA /." Zürich, 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10801.
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Chi, Xiuling. "BIOSYNTHETIC PATHWAY OF THE AMINORIBOSYL COMPONENT OF LIPOPEPTIDYL NUCLEOSIDE ANTIBIOTICS." UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/23.
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Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis
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Javaid, Sarah. "Nucleosome Remodeling by hMSH2-hMSH6." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1293732523.
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Venkataraman, Shanmugasundaram. "Histone acetylation and nucleosome dynamics." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23234.
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In this report, I will describe purification of core histone octamers from chicken blood, HeLa nuclei and yeast cells, along with preparation of DNA fragments containing the 208 bp 5S rDNA gene and the adult beta (bA)-globin gene promoter. In vitro experiments studying the effect of histone acetylation on the positioning and mobility of nucleosomes on the sea urchin 5S rDNA gene and the chicken bA-globin gene promoter will be described. The former provides a well studied nucleosome positioning and mobility model system, while the latter is a developmentally regulated gene, with globin gene switching through the early stages of the lifetime of the chicken, and a proposed involvement of positioned nucleosomes in its regulation. The aim was to determine the difference between hypoacetylated and hyperacetylated core histones in terms of their influence upon nucleosome positioning and mobility. In earlier studies, it was noted that there was a difference in relative positioning intensities between the two forms (ie. hypoacetylated core histones preferentially positioned at certain sites, while hyperacetylated core histones positioned at the same sites but with different relative affinities). Therefore, acetylation affects where a nucleosomes is able to position. I have carried on this work to further characterize nucleosome positioning and to study the implications of histone acetylation on nucleosome mobility. I have found subtle differences in the thermodynamics and kinetics of hyperacetylated nucleosomes compared to hypeoacetylated nucleosomes: hyperacetylated nucleosomes appear to have a lower threshold in both these parameters when studied using the 208 pb rDNA fragment. Experiments involving two other types of core histones, trypsinized chicken core histone octamers and chicken core histone tetramers will also be described, which will be placed into the context of the results found with the other types of core histones. Finally, I will describe the effect of reconstituting hyperacetylated core histones with methylated DNA, long known to be a mediator of transcriptional repression, in the form of the chicken bA-globin gene promoter.
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Mikdar, Mahmoud. "Role of nucleotide metabolite transporters in erythropoiesis and red blood cell functions." Thesis, Université de Paris (2019-....), 2020. http://www.theses.fr/2020UNIP7099.
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De récents travaux ont montré que les voies métaboliques sont fortement impliquées dans la régulation de l’engagement et la différentiation des cellules souches hématopoïétiques (CSHs) vers les différentes lignées hématopoïétiques. Les nucléosides sont des métabolites et précurseurs indispensables pour la biosynthèse des nucléotides, et dont la disponibilité intracellulaire dépend de leur transport à travers la membrane plasmique. Cependant, le rôle des transporteurs de nucléosides et de nucléotides dans l’engagement érythroïde des CSHs reste mal défini. Au cours de ma thèse, l’analyse fonctionnelle des cellules sanguines de patients déficitaires de ENT1 ou ABCC4 a démontré pour la première fois que le transport des nucléosides (ENT1) et nucléotides (ABCC4) est essentiel pour maintenir (i) le métabolisme des nucléotides cycliques et désoxynucléotide, (ii) une morphologie et une déformabilité normales des globules rouges, (iii) la phosphorylation des protéines de la membrane érythrocytaire ainsi que l’organisation du cytosquelette, (iv) la physiologie plaquettaire et (v) l’érythropoïèse ex vivo. De plus, nos expériences mécanistiques ont démontré que ENT1 joue un rôle crucial dans l’engagement des CSHs vers la lignée érythroïde en contrôlant le métabolisme des nucléotides cycliques et l’activation des facteurs de transcriptions érythroïdes. Ce travail a également montré qu’une mutation compensatoire dans le gène ABCC4, retrouvée chez deux patients ENT1null, diminuait les altérations érythroïdes chez ces patients, ce qui confirme le rôle de ces deux transporteurs dans la régulation intracellulaire de l’AMP cyclique. De plus, ce résultat a été confirmé par un modèle souris Ent1-/- puisque le traitement de ces souris avec un inhibiteur de ABCC4 augmente l’engagement érythroïde des CSHs, l’érythropoïèse et l’hématocrite. En outre, une étude protéomique et génomique sur des sujets PEL-négatif a montré qu’une large délétion dans le gène ABCC4 est responsable de ce groupe sanguin. L’inactivation du gène ABCC4 dans une lignée cellulaire par CRISPR-Cas9 a confirmé que l’antigène de groupe sanguin PEL est portée par la protéine ABCC4. Contrairement à ENT1, l’absence totale de ABCC4 est associée à une altération de la fonction plaquettaire sans anomalie de la lignée érythroïde et dont le mécanisme de compensation n’implique pas ENT1. En conclusion, mon travail de thèse dévoile un nouveau mécanisme moléculaire régulant l’érythropoïèse, et démontre le rôle important du métabolisme des nucléotides dans l’engagement des CSHs et dans la biologie érythroïde. Nos résultats ouvrent de nouvelles perspectives pour le développement de stratégies thérapeutiques pour le traitement de l’anémie basées sur la disponibilité intracellulaire des nucléosides et des nucléotidesRecently, metabolic pathways have emerged as critical components in the regulation of hematopoietic stem cell (HSC) renewal, as well as in lineage commitment and differentiation. Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside and nucleotide transporters in the differentiation of HSCs to the erythroid lineage remains undefined. In the present work, we demonstrate for the first time that nucleoside (ENT1) and nucleotide (ABCC4) transport is essential for the (i) metabolism of cyclic nucleotides and deoxynucleotides (ii) erythrocyte morphology and deformability, (iii) erythrocyte membrane protein phosphorylation and skeleton organization, (iv) platelet function, and (v) ex vivo erythropoiesis. Interestingly, functional and mechanistic experiments showed that the equilibrative nucleoside transporter 1 (ENT1) controls the nucleotide metabolism and the activation of erythroid transcription factors. This finding explains the role of ENT1 in maintaining the optimal erythroid commitment and differentiation of HSCs. On the other hand, although the downregulation of the ABC nucleotide transporter ABCC4 attenuates the erythroid disorders in ENT1null patients, its total loss results in abnormal platelet function without erythroid disorders. Importantly, we demonstrate that a large deletion in ABCC4 gene is associated with the PEL-negative null blood phenotype. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Targeting ENT1 and ABCC4 transporters either by knockout or pharmacological inhibition in mice lead to a marked change in blood cells counts. Specifically, the genetic deletion of Ent1 in mice results in a decreased RBC production and macrocytosis. While mice treated with ABCC4 inhibitor increased the erythroid commitment of HSCs, and enhanced erythropoiesis as demonstrated by the increase of circulating erythrocytes and reticulocytes. Overall, our findings reveal a new molecular mechanism regulating erythropoiesis and highlight the important role of nucleotide metabolism in the lineage commitment of HSCs and erythroid biology. Our findings open new avenues for the development of novel therapeutic strategies for the treatment of anemia
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Goswami, Anwesha. "ELUCIDATING THE MECHANISM OF LIPL: A NON-HEME FE(II), α -KETOGLUTARATE: URIDINE-5’-MONOPHOSPHATE DIOXYGENASE." UKnowledge, 2015. http://uknowledge.uky.edu/pharmacy_etds/45.
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Several nucleoside natural product antibiotics from Streptomyces sp. and actinomycetes have recently been shown to target bacterial peptidoglycan cell wall biosynthesis by inhibiting the bacterial translocase I (MraY). The biosynthetic gene clusters for A-90289, liposidomycins and caprazamycins revealed a protein with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD). This enzyme (LipL) is a mononuclear, non-heme, Fe(II) dependent α-keto glutarate (α-KG) :uridine monophosphate (UMP) dioxygenase responsible for the net dephosphorylation and two electron oxidation of UMP to uridine-5’-aldehyde. The postulated reaction coordinates involving the activation of the C-5’ center in UMP and the corresponding formation of uridine-5’-aldehyde are modeled on extensive spectroscopic and structural characterizations of TauD. In this dissertation, the postulated radical mechanism for LipL involving the formation of an unstable hydroxylated intermediate is investigated via the characterization of a key product obtained from the reaction of LipL (and its homolog Cpr19) with a synthetically modified surrogate substrate where the bridging phosphoester oxygen in UMP is replaced with a 5’ C-P bond. We further validate our hypothesis by analyzing the reactions of both LipL and Cpr19 with specifically 2H1 – labeled UMP substrate and confirming the expected products via mass spectrometry. In addition, we explore substrate promiscuity of the enzymes and utilize a set of site specific mutants of Cpr19 as means of gaining better insight into the active site residues. Predictive models for Cpr19 and LipL structures are developed by the combination of experimental results and chemical logic.
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Bharath, M. M. Srinivas. "Towards The Understanding Of The Structural Biology Of Histone H1." Thesis, Indian Institute of Science, 2000. https://etd.iisc.ac.in/handle/2005/167.
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In the eukaryotic nucleus, an immense length of DNA is compactly packaged to generate an ordered three-dimensional hierarchical structure called chromatin (van Holde, 1988; Wolffe, A.P, 1998). This organization forms a template for various DNA transaction processes like replication, transcription, recombination etc. The different stages of organization of the chromatin finally results in the 10,000-fold compaction observed in the metaphase chromosome. The problem of how the fibres of chromatin are folded has interested biologists and biochemists for decades. It has long been recognized that the Histones play a major part in this folding. However, the distinctly different roles of the Histones H2A, H2B, H3 and H4 on one hand and the lysine rich Histones such as Histone H1 and its cognates on the other, were not understood until after the discovery of the nucleosomes in the early 1970s. Some of the early insights into the structure of chromatin came through the digestion of nuclear chromatin with calcium-dependent endonucleases like micrococcal nuclease. A repeating kinetic intermediate of about 200 bp of DNA with Histones was obtained (Simpson, 1978). Based on repeating pattern of micrococcal nuclease digested chromatin and structural studies, Kornberg (1974) proposed that chromatin is composed of a flexible chain of repeating units of 100 A0 diameter. These units were termed as "nucleosomes" (Oudet et al, 1975). It then became clear that the Histones H2A, H2B, H3 and H4 were constituents of the nucleosome core particle whereas the lysine rich Histone H1 was somehow associated with the linker DNA between core particles. Hence, the formers are called core Histones and the latter as linker Histones. On further digestion of nucleosome, a nucleosome core was obtained in which wrapping of 146 bp of DNA about the Histone octamer to form the core particle provided the first level of folding. Electron microscopy and X-ray diffraction techniques suggested that this particle is a disk, 57 A0 thick and 110 A0 in diameter, and that the DNA is wound around the Histone core (Finch et al, 1977), But this cannot account for the many thousand-fold condensation of the DNA in the eukaryotic nucleus. The "string of beads" structure observed obviously could not satisfy the compaction requirement. It soon became evident that there exists some level of higher order folding of the chromatin fiber. In a classical paper, Finch and Klug (1976), showed that the extended nucleosomal filaments condense into irregular fibers of about 30 nm diameter in the presence of low concentrations of Mg 2+. Based on the data from earlier structural studies, these authors proposed a solenoid model in which nucleosomes were wrapped into a regular helix with a pitch of about 11nm. Later, it was observed that the formation of well defined fibers requires the presence of lysine rich Histones such as Histone H1.
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Bharath, M. M. Srinivas. "Towards The Understanding Of The Structural Biology Of Histone H1." Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/167.
Full textAbstract:
In the eukaryotic nucleus, an immense length of DNA is compactly packaged to generate an ordered three-dimensional hierarchical structure called chromatin (van Holde, 1988; Wolffe, A.P, 1998). This organization forms a template for various DNA transaction processes like replication, transcription, recombination etc. The different stages of organization of the chromatin finally results in the 10,000-fold compaction observed in the metaphase chromosome. The problem of how the fibres of chromatin are folded has interested biologists and biochemists for decades. It has long been recognized that the Histones play a major part in this folding. However, the distinctly different roles of the Histones H2A, H2B, H3 and H4 on one hand and the lysine rich Histones such as Histone H1 and its cognates on the other, were not understood until after the discovery of the nucleosomes in the early 1970s. Some of the early insights into the structure of chromatin came through the digestion of nuclear chromatin with calcium-dependent endonucleases like micrococcal nuclease. A repeating kinetic intermediate of about 200 bp of DNA with Histones was obtained (Simpson, 1978). Based on repeating pattern of micrococcal nuclease digested chromatin and structural studies, Kornberg (1974) proposed that chromatin is composed of a flexible chain of repeating units of 100 A0 diameter. These units were termed as "nucleosomes" (Oudet et al, 1975). It then became clear that the Histones H2A, H2B, H3 and H4 were constituents of the nucleosome core particle whereas the lysine rich Histone H1 was somehow associated with the linker DNA between core particles. Hence, the formers are called core Histones and the latter as linker Histones. On further digestion of nucleosome, a nucleosome core was obtained in which wrapping of 146 bp of DNA about the Histone octamer to form the core particle provided the first level of folding. Electron microscopy and X-ray diffraction techniques suggested that this particle is a disk, 57 A0 thick and 110 A0 in diameter, and that the DNA is wound around the Histone core (Finch et al, 1977), But this cannot account for the many thousand-fold condensation of the DNA in the eukaryotic nucleus. The "string of beads" structure observed obviously could not satisfy the compaction requirement. It soon became evident that there exists some level of higher order folding of the chromatin fiber. In a classical paper, Finch and Klug (1976), showed that the extended nucleosomal filaments condense into irregular fibers of about 30 nm diameter in the presence of low concentrations of Mg 2+. Based on the data from earlier structural studies, these authors proposed a solenoid model in which nucleosomes were wrapped into a regular helix with a pitch of about 11nm. Later, it was observed that the formation of well defined fibers requires the presence of lysine rich Histones such as Histone H1.
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Huh, Joon Hoi. "A biochemical and biophysical study of nucleosome assembly by the Saccharomyces cerevisiae Nucleosome Assembly Protein 1." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3211726.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed June 14, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 119-126).
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Fraser, Ross Macdonald. "Computational analysis of nucleosome positioning datasets." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/29110.
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Monomer extension (ME) is an established in vitro experimental technique which maps the positions adopted by reconstituted core histone octamers on a defined DNA sequence. It provides quantitative positioning information, at high resolution, over long continuous stretches of DNA sequence. This technique has been employed to map several genes: globin genes (8 kbp), the beta-lactoglobulin gene (10 kbp) and various imprinting genes (4 kbp). This study explores and analyses this unique dataset, utilising computational and stochastic techniques, to gain insight into the potential influence of nucleosomal positioning on the structure and function of chromatin. The first section of this thesis expands upon prior analyses, explores general features of the dataset using common bioinformatics tools, and attempts to relate the quantitative positioning information from ME to data from other commonly used competitive reconstitution protocols. Finally, evidence of a correlation between the in vitro ME dataset and in vivo nucleosome positions for the beta-lactoglobulin gene region is presented. The second section presents the development of a novel method for the analysis of ME maps using Monte Carlo simulation methods. The goal was to use the ME datasets to simulate a higher order chromatin fibre, taking advantage of the long-range and quantitative nature of the ME datasets. The Monte Carlo simulations have allowed new insights to be gleaned from the datasets. Analysis of the beta-lactoglobulin positioning map indicates the potential for discrete disruption of nucleosomal organisation, at specific physiological nucleosome densities, over regions found to have unusual chromatin structure in vivo. This suggests a correspondence between the quantitative histone octamer positioning information in vitro and the positioning of nucleosomes in vivo. Taken together, these studies lend weight to the hypothesis that necleosome positioning information encoded within DNA plays a fundamental role in directing chromatin structure in vivo.
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Klinker, Henrike. "ATP-dependent nucleosome sliding by ISWI." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-180992.
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Oberbeckmann, Elisa [Verfasser], and Philipp [Akademischer Betreuer] Korber. "Absolute nucleosome occupancy and reconstitution of nucleosome positioning mechanisms for Saccharomyces cerevisiae / Elisa Oberbeckmann ; Betreuer: Philipp Korber." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1223369897/34.
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Dorigo, Benedetta. "Studies of nucleosome array structure and dynamics /." Zürich, 2004. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15710.
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Sayad-Rahim, Azin. "Motif discovery algorithms incorporating nucleosome positioning information." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86682.
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Transcription factor binding sites are essential components of the machinery that controls gene expression. In the absence of experimental data, computational approaches are used to predict binding sites based on promoter DNA sequence. However transcription factor binding depends not just on sequence but also the packaging of the DNA molecule. Nucleosomes, as the smallest unit of DNA packaging, affect transcription factor binding by obstructing protein-DNA interactions.We use an empirically-derived relationship between binding sites and nucleosome positioning to augment an existing computational approach to predicting transcription factor binding sites. We demonstrate that the inclusion of experimentally-derived nucleosome positioning data improves the prediction capabilities of the basic computational approach using a large dataset of experimentally confirmed transcription factor binding sites.
Les sites de liaison de facteurs de transcription sont des composants essentiels du méchanisme de contrôle de l'expression génique. En l'absence de données expérimentales, les approches informatiques sont utilisées pour prédire les sites de liaison basée sur la séquence d'ADN promoteur. Toutefois la liaison de facteurs de transcription dépend non seulement de la séquence mais également de l'emballage biologique de la molécule d'ADN. Les nucléosomes, en tant qu'unité d'emballage de base de l'ADN, ont un effet marqué sur le positionnement des sites de liaison de facteurs de transcription.
Nous dérivons une relation empirique entre les sites de liaison et le positionnement des nucléosomes pour améliorer un algorithme de prédiction de sites de liaison. Nous démontrons que l'inclusion de données de positionnement de nucléosome améliore la performance de l'algorithme de base en utilisant un ensemble de données de sites de liaison confirmé expérimentalement.
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Hu, Zhenhua. "Nucleosome positioning dynamics in evolution and disease." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25399.
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Nucleosome positioning is involved in a variety of cellular processes, and it provides a likely substrate for species evolution and may play roles in human disease. However, many fundamental aspects of nucleosome positioning remain controversial, such as the relative importance of underlying sequence features, genomic neighbourhood and trans-acting factors. In this thesis, I have focused on analyses of the divergence and conservation of nucleosome positioning, associated substitution spectra, and the interplay between them. I have investigated the extent to which nucleosome positioning patterns change following the duplication of a DNA sequence and its insertion into a new genomic region within the same species, by assessing the relative nucleosome positioning between paralogous regions in both the human (using in vitro and in vivo datasets) and yeast (in vivo) genomes. I observed that the positioning of paralogous nucleosomes is generally well conserved and detected a strong rotational preference where nucleosome positioning has diverged. I have also found, in all datasets, that DNA sequence features appear to be more important than local chromosomal environments in nucleosome positioning evolution, while controlling for trans-acting factors that can potentially confound inter-species comparisons. I have also examined the relationships between chromatin structure and DNA sequence variation, with a particular focus on the spectra of (germline and somatic) substitutions seen in human diseases. Both somatic and germline substitutions are found to be enriched at sequences coinciding with nucleosome cores. In addition, transitions appear to be enriched in germline relative to somatic substitutions at nucleosome core regions. This difference in transition to transversion ratio is also seen at transcription start sites (TSSs) genome wide. However, the contrasts seen between somatic and germline mutational spectra do not appear to be attributable to alterations in nucleosome positioning between cell types. Examination of multiple human nucleosome positioning datasets shows conserved positioning across TSSs and strongly conserved global phasing between 4 cancer cell lines and 7 non-cancer cell lines. This suggests that the particular mutational profiles seen for somatic and germline cells occur upon a common landscape of conserved chromatin structure. I extended my studies of mutational spectra by analysing genome sequencing data from various tissues in a cohort of individuals to identify human somatic mutations. This allowed an assessment of the relationship between age and mutation accumulation and a search for inherited genetic variants linked to high somatic mutation rates. A list of candidate germline variants that potentially predispose to increased somatic mutation rates was the outcome. Together these analyses contribute to an integrated view of genome evolution, encompassing the divergence of DNA sequence and chromatin structure, and explorations of how they may interact in human disease.
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CAROT, VALERIE, and P. COHEN. "Surenroulement de l'adn et dynamique du nucleosome." Paris 6, 1994. http://www.theses.fr/1994PA066782.
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Chez les eucaryotes, l'adn interagit avec les histones et les proteines non-histones pour former une superstructure nucleoproteique: la chromatine. Une des fonctions les plus evidentes de la chromatine est de permettre la condensation de l'adn dans le noyau. Plusieurs niveaux de compaction allant de l'adn au chromosome en metaphase ont ainsi ete distingues. Son role ne peut cependant etre restreint a cette seule fonction car les processus biologiques fondamentaux que sont la replication, la transcription, la recombinaison et la reparation s'operent au sein de la chromatine. La comprehension exacte de ces mecanismes requiert donc une connaissance approfondie de la structure et de la superstructure de la chromatine. Nous presentons dans l'introduction un apercu des connaissances actuelles sur la structure de la double helice. Cette structure conditionne en effet son interaction avec differentes proteines et en particulier les histones, avec lesquelles elle forme le nucleosome. La structure des nucleosomes sera presentee dans le second chapitre. Nous verrons par la suite comment ceux-ci peuvent influencer l'activite transcriptionnelle d'un gene. Nous avons montre que le surenroulement, en plus de ses effets deja connus sur la dynamique de l'adn et son interaction avec certaines proteines impliquees dans la transcription peut jouer egalement un role dans son interaction avec d'autres proteines, les histones, et particulierement au niveau d'une sequence strategique telle qu'un promoteur. Nos resultats montrent que le tetramere (h3-h4)2 est une structure douee d'une grande flexibilite conformationnelle. En effet, le tetramere surenroule preferentiellement l'adn en une superhelice gauche mais peut aussi, sous l'influence des fluctuations thermiques, surenrouler l'adn en une superhelice droite. Cet enroulement droit suggere un changement de structure du tetramere negatif au tetramere positif. La flexibilite conformationnelle du tetramere pourrait etre utilisee dans l'ensemble des processus impliquant l'adn dans la chromatine, et pourrait en particulier constituer la fameuse gyrase eucaryote
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Xie, Yunwei. "nucleosome, transcription and transcription regulation in Archaea." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1127830717.
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Aguilar, Gurrieri Carmen. "Etudes structurales sur l'assemblage du nucléosome." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV017/document.
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Au sein du noyau, l'ADN est organise en chromatine dont l'unité de base est le nucléosome. La structure de la chromatine est très dynamique, ce qui est nécessaire pour la plupart des opérations qui se produisent dans l'ADN telles que la réplication, la transcription, la réparation et la recombinaison. Le nucléosome est constitué de deux dimères H2A/H2B et deux dimères H3/H4 associés avec 147 paires de bases d'ADN. La protéine Nap1 est un chaperon d'histone H2A/H2B impliquée dans l'assemblage et démontage des nucléosomes. Nap1 protège les interactions non spécifiques entre l'ADN chargé négativement et les dimères H2A/H2B chargés positivement, afin de permettre la formation de la structure ordonnée des nucléosomes. Lors de l'assemblage des nucléosomes, les dimères d'histones H3/H4 sont déposés en premier lieu, suivi par le dépôt de dimères H2A/H2B. Lors du démontage du nucléosome, les dimères H2A/H2B sont retirés avant le retrait des dimères H3/H4. La determination de la structure du complexe Nap1-H2A/H2B pourra permettre une meilleure compréhension du processus d'assemblage du nucléosome. Dans cette étude, nous voulons comprendre comment le chaperon Nap1 cible spécifiquement les dimères d'histones H2A/H2B pour l'assemblage des nucléosomes. Notre objectif est de caractériser la structure et la fonction du complexe de Nap1-H2A/H2B. Ainsi nous nous sommes tout d'abord intéresse à la stoechiometrie de ce complexe. Nous avons trouvé qu'un dimère de Nap1 s'associe à un dimère H2A/H2B (Nap1_2-H2A/H2B). D'autre part, l'analyse par spectrométrie de masse non-dénaturante a montré que ce complexe de base peut s'oligomériser et contenir jusqu'à 6 copies de Nap1_2-H2A/H2B. L'analyse de ce complexe par spectrométrie de masse non-dénaturant a montré que ce complexe peu oligomériser dans un grand complexe contenant jusqu'à 6 copies de Nap1_2-H2A/H2B. Nous avons également obtenu la première structure cristalline à basse résolution de ce complexe. L'analyse du même complexe par microscopie électronique à coloration négative a révélé la présence en solution du même oligomère que dans l'unité asymétrique du cristal, qui contient aussi 6 copies de Nap1_2-H2A/H2B. Ainsi, nous avons pu mettre en évidence de nouvelles interfaces d'interaction entre les différents composants de ce complexe qui nous permettent de mieux comprendre le processus d'assemblage des nucléosomes. Le remodelage de la chromatine permet l'expression des gènes eucaryotes. Ce remodelage nécessite des enzymes telles que des histone acétyltransférases (HAT) et les chaperons d'histones. Les HATs acétylent les chaînes latérales des lysines. Il a été proposé que les HATs et les histones chaperons agissent en synergie pour moduler la structure de la chromatine pendant la transcription. La HAT p300 a été proposé d'interagir avec l'histone chaperon Nap1. Nous avons entrepris de caractériser cette interaction. Malheureusement, nos expériences n'ont pas pu détecter d'interaction directe entre ces protéinesAssembly of chromatin is an essential process that concerns most DNA transactions in eukaryotic cells. The basic repeating unit of chromatin are nucleosomes, macromolecular complexes that consist of a histone octamer that organizes 147 bp of DNA in two superhelical turns. Although, the structures of nucleosomes are known in detail, their assembly is poorly understood. In vivo, nucleosome assembly is orchestrated by ATP-dependent remodelling enzymes, histone-modifying enzymes and a number of at least partially redundant histone chaperones. Histone chaperons are a structurally diverse class of proteins that direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange. The currently accepted model is that nucleosome assembly is a sequential process that begins with the interaction of H3/H4 with DNA to form a (H3/H4)2 tetramer-DNA complex. The addition of two H2A/H2B dimers completes a canonical nucleosome. High-resolution structures of histone chaperons in complex with H3/H4 histones have resulted in detailed insights into the process of nucleosome assembly. However, our understanding of the mechanism of nucleosome assembly has been hampered by the as yet limited number of co-crystal structures of histone–chaperone complexes. In particular it remains unclear how histone chaperons mediate H2A/H2B deposition to complete nucleosome assembly. In this work, we have investigated the role of the H2A/H2B chaperon Nap1 (Nucleosome assembly protein 1) in nucleosome assembly. We have determined the crystal structure of the complex between Nap1 and H2A/H2B and analysed the assembly by various biophysical methods. The structure shows that a Nap1 dimer binds to one copy of H2A/H2B (Nap1_2-H2A/H2B). A large ~550 kDa macromolecular assembly containing 6 copies of the Nap12-H2A/H2B complex is seen in the asymmetric crystallographic unit. We confirmed by both non-denaturing mass spectroscopy and negative stain electron microscopy studies that this assembly is the predominant form of the Nap1_2-H2A/H2B complex in solution. We further investigated the potential interplay between p300-mediated histone acetylation and nucleosome assembly. Together, the structure and associated functional analysis provide a detailed mechanism for the Nap1 chaperon activity, its role in H2A/H2B deposition and in nucleosome assembly
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Cabrespines, Alban. "Etude du role des anticorps anti-nucleosome dans la pathogenicite et l'immunogenicite du nucleosome dans le lupus erythemateux dissemine." Paris 6, 1997. http://www.theses.fr/1997PA066034.
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Le lupus erythemateux dissemine (led) est une maladie autoimmune non specifique d'organe caracterisee par la production d'autoanticorps anti-adn et anti-histone parmi lesquels certains ont un role pathogene. L'etude de la cible de ces autoanticorps montre qu'ils reconnaissent l'adn et les histones dans une structure physiologique : le nucleosome. Nous avons cherche a caracteriser le role potentiel du nucleosome, en tant qu'immunogene et antigene cible, dans le developpement du led. Nous montrons la presence d'anticorps anti-nucleosome restreints reconnaissant des determinants conformationnels generes par l'association de l'adn et des histones au sein du nucleosome. Leur apparition precede, chez les souris lupiques mrl-mp +/+ et lpr/lpr, celle des anticorps anti-adn double brin et anti-histone, et leur presence dans les reins leur confere un role potentiellement pathogene. Nous avons caracterise le nucleosome issu de l'apoptose cellulaire. Il presente un profil degrade, et est heterogene lorsqu'il est complexe par differents anticorps anti-nucleosome, suggerant que la specificite fine des anticorps determine sa structure. De plus, nous montrons l'existence de recepteurs specifiques du nucleosome a la surface de differents modeles cellulaires. Ces recepteurs lient et internalisent les nucleosomes (libres ou complexes par des anticorps anti-nucleosome), qui sont ensuite achemines dans des compartiments intracellulaires riches en molecules de classe ii du cmh. Les cellules presentatrices d'antigenes incubees en presence de nucleosomes ou de complexes immuns sont capables d'activer des cellules t cd4#+ issues de souris mrl-mp+/+. Dans l'ensemble, ces donnees mettent en avant le role du nucleosome et des complexes nucleosome/anticorps anti-nucleosome dans le declenchement et/ou l'amplification de la reponse autoimmune, ainsi que dans la pathogenicite de la maladie.
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Iannone, Camilla 1984. "Links between chromatin structure and regulation of alternative pre-mRNA splicing in mammalian cells." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/301439.
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Intron removal is a necessary step for expression of most genes in higher eukaryotes, and alternative splice selection is a highly regulated mechanism that endows a single gene with the possibility to codify for multiple transcripts. Pre-mRNA splicing occurs largely co-transcriptionally, and its outcome is influenced by transcription elongation and chromatin structure. In this thesis we have used two different approaches to study novel links between chromatin structure and alternative splicing regulation.
In the first approach, we identified genome-wide progesterone-regulated cassette exons and compared them with nucleosome density profiles, with the aim of finding correlations between changes in nucleosome positioning and changes in alternative splicing. We find that, even if all exons harbor a well-positioned exonic nucleosome, four different classes of nucleosome density profiles can be identified around alternative exons, which strongly correlate with the DNA sequence GC content. Transitions between these profiles occur upon hormone stimulation and can be correlated with alternative splicing changes, although changes in nucleosome profiles are also observed in non-regulated exons. In particular hormone-induced exon inclusion is more frequently linked to changes in nucleosome density than hormone-induced skipped exons, which tend to have low nucleosome density profiles even before hormone treatment. Peaks of nucleosome density before alternative exons tend to correlate with exon inclusion.
In the second approach, we took advantage of ENCODE data of chromatin epigenetic signatures and RNA-Seq in multiple cell lines to evaluate functional enrichment of histone modifications over alternative exons. We find that three histone modifications (H3K4me3, H3K27ac and H3K9ac) co-occur in a subset of exons when they are highly included. These features are sufficient to predict differential inclusion levels in other cell lines. Moreover, they are enriched in exons characterized by the presence of DNase hypersensitive sites, promoter signatures and RNA Pol II accumulation. These observations suggest a functional role for 3-dimensional genome structure in the regulation of alternative splicing.La eliminación de intrones es un paso necesario para expresar la mayoría de los genes en eucariotas superiores. La selección alternativa del corte y empalme (pre-mRNA splicing) es un mecanismo altamente regulado que dota a un solo gen con la posibilidad de codificar para múltiples transcritos. El splicing del pre-mRNA se produce en gran parte de manera cotranscripcional y por esto resultado está influenciado por la elongación de la transcripción y la estructura de la cromatina. En esta tesis se han utilizado dos enfoques diferentes para estudiar nuevos vínculos entre la estructura de la cromatina y la regulación del splicing alternativo. En el primer enfoque hemos identificado, a nivel de todo el genoma, exons internos regulados por progesterona y los hemos comparados con los perfiles de densidad de nucleosomas, con el objetivo de encontrar correlaciones entre los cambios en el posicionamiento de nucleosomas y en el splicing alternativo. Hemos encontrado que, aunque todos los exones albergan un nucleosoma bien posicionado, se pueden identificar cuatro clases diferentes de perfiles de densidad de nucleosomas alrededor de exones alternativos, que se correlacionan fuertemente con el contenido G+C en la secuencia del ADN. Las transiciones entre estos perfiles se producen tras la estimulación con la hormona y se pueden correlacionar con los cambios en splicing alternativo, aunque se observan también cambios en los perfiles de nucleosomas en exones no regulados. La inclusión de exones inducida por la hormona está relacionada más a menudo con cambios en la densidad de nucleosomas que la exclusión. Estos exones excluidos tienden a tener perfiles de baja densidad nucleosomal incluso antes del tratamiento hormonal. Los picos de densidad de nucleosomas antes de exones alternativos tienden a correlacionarse con la exclusión de exones. En el segundo enfoque nos aprovechamos de los datos de ENCODE de marcas epigenéticas de la cromatina y de RNA-Seq en múltiples líneas celulares, para evaluar el enriquecimiento funcional de las modificaciones de histonas en exones alternativos. Encontramos que tres modificaciones de las histonas (H3K4me3, H3K27ac y H3K9ac) co-ocurren en un subconjunto de exones mas incluidos. Estas características son suficientes para predecir los niveles de inclusión diferenciales de estos exones entre líneas celulares. Además, estos exones se caracterizan también por la presencia de sitios hipersensibles a la DNasa, de marcas de promotores y la acumulación de ARN Pol II . Estas observaciones sugieren un papel funcional para la estructura en 3 dimensiones del genoma en la regulación del splicing alternativo.
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Hartley, Paul D. "Mechanisms that specify promoter nucleosome location and identity." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390046.
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Thanakiatkrai, Phuvadol. "Degradation, quantification and the theory of nucleosome protection." Thesis, University of Strathclyde, 2011. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=15335.
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