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Studies of the nucleosome core particle structure in Phsarum polycephalumStone, G. R. January 1985 (has links)
No description available.
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A study of chromatin heterogeneity using immunochemical methodsBlanchard, A. D. January 1987 (has links)
No description available.
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DNA damage and repair in chromatin during carcinogenesisRyan, A. J. January 1987 (has links)
No description available.
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Phosphorylation and distribution of High-Mobility Group protein HMGN1 in the context of Immediate-Early (IE) gene inductionPogna, Edgar Allan January 2012 (has links)
Eukaryotic genomes are highly organized and packaged into chromatin, a complex structure formed of proteins and DNA, in which the basic repeating unit is the nucleosome. Chromatin can be arranged in condensed or relaxed structures influencing accessibility of proteins that regulate transcription, replication, recombination and repair. One class of transiently chromatin-associated proteins is the High-Mobility Group (HMG) protein family. HMG proteins are subdivided into three subgroups: HMGA, HMGB and HMGN. HMGN1, the subject of this study, is a prominent member of the HMGN (High-Mobility Group Nucleosome-binding) protein family, the only HMG proteins that specifically binds to the nucleosomes. HMGNs are maintained in dynamic balance between nucleosome-associated and nucleosome-free pools. Regulation of chromatin involves several enzymatic activities that modify specific residues on chromatin proteins, which may influence these interactions. While associated with nucleosomes, HMGNs can interfere with some modifications of histone tails. Modification of HMGN1 on specific residues and post-translational modification (PTM) of histones are concomitantly regulated by the complex signalling networks associated with the induction of immediate early (IE) genes. Induction of IE genes is associated with phosphorylation of HMGN1 which has been suggested to increase the rate of dissociation of HMGN1 from the nucleosome, thus allowing access and modification of histone tails. My research has been focused on characterizing HMGN1 isoforms present in different cellular compartments and at different time-points during IE gene induction with various stimuli, including epidermal growth factor (EGF), anisomycin (An) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, I investigated the localization of HMGN1 within the nucleus and at specific IE gene loci, especially at sites where post-translationally modified histones are localised. In my analysis only the phosphorylation at serine 6 of HMGN1 shows a correlation with gene induction. Analysis of DNA sequences from chromatin immunoprecipitation (ChIP) has shown that HMGN1 is present at equal levels in active and inactive genes. It appears that HMGN1 localization on DNA is not dictated by a particular preference for any gene elements such as promoters, exons, introns or gene termination sequences.
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Potential roles for chromatin structures in the differential regulation of the 5s rRNA genesHowe, LeAnn Judith 22 June 2017 (has links)
In 1871, a unique substance was isolated from the white blood cells of pus. This substance, which later became known as chromatin, was shown to be a nucleoprotein complex which encompasses the majority of genomic DNA in all eukaryotes. Although chromatin was once viewed as primarily a structural component of the nucleus, it is now accepted that it also plays an important role in the modulation of transcription of individual genes. In this study, the 5S rRNA genes in Xenopus laevis were used as a system to investigate potential roles for chromatin structures in transcription regulation. X. laevis produces two major classes of 5S rRNA: the somatic type is present in most cells whereas the oocyte type is produced only during oogenesis and the early stages of embryogenesis. These two gene families share a very similar coding region and employ identical transcription machinery, leading researchers to believe that it is how these genes are packed into chromatin which is responsible for the differential developmental regulation.
Initially, this study focused on the binding constraints placed on the RNA polymerase III basal transcription factor, transcription factor IIIA (TFIIIA), by a histone octamer. Five overlapping fragments of the X. laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes and it was shown that each fragment positions a histone octamer at unique translational sites. Using these nucleosomes it was demonstrated that nucleosome translational positioning is the major determinant of the binding of TFIIIA to the 5S rRNA genes.
The relationship between core histone acetylation and transcription of the X. laevis 5S rRNA genes was also investigated. By immunopreciptitating chromatin fragments from a X. laevis kidney cell line with an antibody specific for hyperacetylated histone H4, it was shown that the oocyte 5S rRNA genes are packaged with hypoacetylated histone H4 when transcriptionally repressed.This taken together with the results of others, suggests a link between histone acetylation and RNA polymerase III transcription. However this study
was unable to shed light on the basis for this relationship as it was found that histone acetylation did not affect the binding of TFIIIA to nucleosomal DNA.
In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. It was shown that despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin and TFIIIA from Xenopus laevis.
Finally, it has long been proposed that selective binding of histone H1 is, in part, responsible for the differential developmental regulation of the oocyte and somatic 5S rRNA genes in Xenopus laevis. In this study it was shown that histone H1 bound both oocyte and somatic genes equally after reconstitution into mononucleosomes or oligonucleosome arrays. Furthermore it was shown that the binding of histone H1 selectively repressed only oocyte gene transcription, and that a RNA polymerase III selectively repressed only oocyte gene transcription, and that a RNA polymerase III transcription complex was able to initiate transcription of nucleosomal somatic templates regardless of whether histone H1 was present. These results support a model in which the differential regulation of the 5S rRNA genes is not due simply to the prevention of histone HI binding by transcription complexes on the somatic genes, but rather a difference in the interaction of histone HI with the somatic and oocyte genes. / Graduate
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Epigenome control by chromatin modifiers: roles for histone H3 lysine modifiers in the regulation of repetitive elementsGrady, Patrick James Robert January 2015 (has links)
Thesis advisor: Hugh P. Cam / Chromatin is the site of numerous structural features that contribute to the regulation of the genome. Although numerous posttranslational modifications to the histone proteins that make up chromatin have been identified, it remains unclear whether and to what extent these modifications might regulate transposons and other repetitive sequences. One such modification is methylation of histone H3 lysine 4 (H3K4me), which is catalyzed by Set1 and its associated complex Set1C/COMPASS. Although H3K4me is associated with actively transcribed regions in euchromatin, an emerging body of evidence suggests that Set1-mediated transcriptional control is often repressive. This thesis work describes expanded functions for Set1C/COMPASS as a regulatory module with roles throughout the genome. We identify novel locus-dependent repressive functions for Set1 at repetitive genomic regions. Interestingly, Set1 has multiple repressive modes that are dependent and independent of H3K4me. Additionally, we show that Set1 controls the nuclear organization of Tf2 retrotransposons by antagonizing H3K4 acetylation. We describe how the roles of Set1 in the nuclear organization and transcriptional repression of Tf2 cooperate to restrict Tf2 transposition. Finally, we identify an H3K4-dependent role in countering the reduced dosage of histone H3 genes to help maintain genome stability and silencing of Tf2s and pericentromeric heterochromatin. Our study considerably expands the regulatory repertoire of an important histone modifier and highlights the multifaceted function by a highly conserved chromatin-modifying complex with diverse roles in genome control. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Quantitative proteomics of human chromatinFurlan, Cristina January 2013 (has links)
The work presented in this thesis aims at unravelling human chromatin composition by quantitative proteomics to outline the functional and structural changes occurring during the life of human cells. Chromatin is the structure formed by proteins and RNAs interacting with the genetic material. At present, chromatin is not well defined. It is not easy to investigate either the composition of its constituent proteins or how this arrangement changes. We set out to analyse the chromatin composition changes occurring during the cell cycle. Our procedure couples a SILAC mass spectrometry-based approach with a newly developed biochemical chromatin purification method, which involves fixation of proteins to DNA. By testing two different fixation times (5 and 10 minutes) and three phases of the cell cycle (G1/S, G2, M), we quantified ~3000 proteins providing a broad picture of the global changes on chromatin protein composition. Surprisingly, chromatin seems to be occupied by many unexpected proteins (40%) that appear to be increased during mitosis. We hypothesized that these unexpected proteins come into contact with DNA during mitosis when the nuclear envelope breaks down and the highly negatively charged DNA can be found in proximity to extra nuclear proteins. We used Pulse-SILAC technique that allows to distinguish newly synthesized proteins to test this possibility. By comparing in a single cell cycle and during G0 arrest the incorporation of new proteins into chromatin with their synthesis in the cytoplasm and in the whole cell, we could not find a different behaviour for the unexpected proteins as result of mitosis. Despite the efforts in tracking down the origin of these unexpected proteins, it is still uncertain whether their presence on chromatin is the result of a biological process or, in part, a drawback of the purification methods adopted. However, proving their genuine presence on chromatin will be important to elucidate how chromatin functions.
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Contemporary Genetic Tools for in Vivo Investigations of H3K27 Demethylases in Zebrafish CardiogenesisAkerberg, Alexander 21 November 2016 (has links)
Dynamic histone modification has emerged as a robust and versatile regulator of gene expression in eukaryotic cells. One such modification, the trimethylation of lysine 27 on histone H3 (H3K27me3) is facilitated by the Polycomb repressive complex 2 (PRC2) and contributes to the localized repression of transcription. Subsequently, lysine specific demethylase Kdm6b (Jmjd3) can relieve the repressive H3K27me3 mark, allowing for transcriptional activation. In vitro studies have suggested a role for Kdm6b during mesodermal and cardiovascular differentiation in mammalian embryonic stem cells; however, this relationship has yet to be characterized in vivo. I utilized the advantages of the zebrafish model to investigate the in vivo roles of Kdm6b-family demethylases during development using a reverse genetic approach.
I carried out two independent loss-of-function studies to analyze the role of Kdm6b-family demethylases during embryonic development in zebrafish. By comparing genetic loss-of-function and morpholino-mediated knockdown approaches, I found that morpholino–mediated knockdown of kdm6bb transcript produces off-target effects and does not portray an accurate representation of in vivo function. I then show that, while not required for early cardiogenesis, histone demethylases kdm6ba and kdm6bb function redundantly to promote late stage proliferation during heart ventricle trabeculation. These data reveal a previously unknown functional relationship and support the hypothesis that Kdm6b-family demethylases function primarily during later stages of development. Additionally, my description of morpholino-induced off-target effects supports the need to use extreme caution when interpreting morphant phenotypes.
Due to the embryonic lethality exhibited by kdm6b-deficient embryos and the limited tools available for spatiotemporal transgene control in zebrafish, I was unable to investigate demethylase function within adult animals. I attempted to circumvent these limitations by creating an inducible gene expression system that uses tissue-specific transgenes that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. I showed that these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. I then demonstrated how this technology could be used to define developmental windows of gene function by spatiotemporally controlling expression of constitutively active Notch1 in embryos.
This dissertation contains previously published co-authored material.
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New approaches to the investigation of the interactions between enhancers and promoters during haemopoiesisDavies, James January 2017 (has links)
Next generation sequencing based methods allow us to identify active genes and potential regulatory elements rapidly across the genome, but an outstanding challenge is to unravel which regulatory elements control which genes. This is problematic because regulatory elements control genes over huge distances (over 1 million base pairs) and they have an unpredictable distribution around the genes they influence. In order to enhance transcription the protein complexes at distal regulatory elements make physical contact with the promoter. These interactions can be detected using Chromosome Conformation Capture (3C) technology and the position of regulatory elements can be deduced from these data. However, these methods are either low throughput or low resolution and they are prone to bias. In this 3C techniques were specifically developed to determine the interactions between regulatory elements and gene promoters promoter. The focus has been the development of Next Generation Capture-C, which allows many genetic loci and samples to be analysed simultaneously, with greater sensitivity and accuracy than has previously been possible. High resolution data can be produced from as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. Nanostring technology has also been developed for analysis of 3C libraries, as this allows interaction profiles to be determined without PCR or sequencing bias. The Nanostring data show remarkable correlation with the interaction profiles generated by NG Capture-C.
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Chromatin dynamics at the Sonic Hedgehog locus : a study using limb derived Sonic Hedgehog inducible cell lines to investigate chromatin architectureDouglas, Adam Thomas January 2017 (has links)
Enhancers are cis-regulatory sequences which promote the expression of target genes in a spatial and temporal fashion. They can be located within genes or between them and can act at distances of over 1 Mb. There are several different mechanisms by which enhancers regulate gene expression. Some, such as those regulating the Hox genes, are located close to each other in the genome in a structure referred to as a regulatory archipelago. These come together and act in combination to regulate gene expression, with different enhancer combinations resulting in different patterns of expression. On the other hand, enhancers can act individually, with designated enhancers responsible for regulating the expression of the same gene in different tissues or at different stages of development. Indeed, this is the case for the Sonic Hedgehog gene (Shh) where several different enhancers located within a gene sparse region referred to as a gene desert, act separately leading to Shh expression in areas such as the brain, the lungs, the notochord and neural tube and the limbs. Within the developing mouse embryo, Shh is expressed over roughly a two day period from E10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA). Ectopic expression in anterior regions has been observed in some common congenital diseases which affect the limbs, sometimes resulting in the formation of extra digits. The reason for this mis-expression is largely due to defects in the Shh limb enhancer commonly referred to as the Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highly conserved sequence create additional protein binding sites thus activating the enhancer in the wrong locations. The associated diseases are known collectively as the ZRS associated syndromes and can range from the less severe phenotype of preaxial polydactyly type II (characterised by an extra digit near the thumb) to the more severe Werner Mesomelic Syndrome (WMS), where patients present with a clear displacement of their tibia. The mechanism by which the ZRS functions is yet to be fully elucidated, with current studies producing conflicting data. What is known, is that the region encapsulating the Shh gene is highly compact, with both the gene and its enhancers located in a highly conserved Toplogical Associated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domain are likely created by the binding of the protein CTCF to specified binding sites located at the either end of the locus. This restricts the ability of the enhancers to regulate the expression of genes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression, the Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5 limb bud. Gene expression in these cells is highly reflective of the posterior limb bud, with the key exception being Shh, which is not expressed. However, using different drug treatments or biological manipulations Shh can be activated thereby making this the perfect system to analyse the mechanisms leading to Shh activation. In this investigation the cell lines were used to determine how the position of the ZRS changes upon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with either fosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locus is indeed highly compact in both Shh expressing and non-expressing cells. However, no differences were observed in terms of the distance between the ZRS and Shh between these two conditions in our cell lines. Next, both carbon copy chromosome conformation capture (5C) and circular chromosome conformation capture (4C) were used to look at changes to the Shh locus in different conditions. This confirmed Hi-C experiments and other recent publications suggesting that Shh is located within a TAD, the position of which is highly conserved between different conditions and cell lines. Furthermore, treatments activating the Shh gene resulted in significant deviations to the chromatin interactions within the locus suggesting a repositioning of structures when the gene is active. It is believed that the use of Shh inducible limb derived cell lines will prove extremely useful in future scientific endeavours to study the mechanisms of mammalian limb development. These provide a quick and easy means of accessing large numbers of Shh expressing cells, a feature which is increasingly important in an era where large cell numbers are needed for conducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.
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