No description available.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1973.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 196-218). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38837

Hamburg, University, Diss., 2004.

The compact structure of the eukaryotic genome dictates the accessibility to genes, and therefore adds an additional layer of regulation for gene expression. A specialized class of proteins called "chromatin remodelers" facilitates this process in the cell. The imitation switch (ISWI) subfamily of chromatin remodelers is a well studied class of proteins affecting gene expression. Its member ISW2 was recently shown to behave differently from other chromatin remodeling proteins. For instance, the ISW2 complex has been shown to be stimulated by ~5-6 fold in its ATPase activity when bound to a nucleosome rather than to a DNA molecule. Nucleosome remodeling by ISW2 has even been shown to depend on the N-terminal tail of histone H4 and therefore, the octamer of a nucleosome might be playing a significant role in nucleosome remodeling by the ISW2 complex. The aim in this investigation was to delineate the protein-protein interactions that the ISW2 complex establishes with the octamer upon binding to a nucleosome. Several histones with unique cysteines engineered at specific positions were refolded with other wild type histones to produce histone octamers with a single cysteine in one of the four histones. Based on previous reports from site-specific DNA photoaffinity cross-linking and hydroxyl-radical footprinting experiments, it was inferred that the SHL2, entry-exit position and the extranucleosomal linker DNA were contacted by the ISW2 complex on a nucleosome1. Considering these critical regions and taking into account the accessibility of residues in close proximity to these regions, five discrete positions were selected on the octamer surface for scanning the face of the nucleosome. The five sites were residues 19, 89 and 113 of histone H2A (H2A-19, H2A-89, H2A-113), residue 109 of histone H2B (H2B-109), and residue 80 of histone H3 (H3-80). Initially, octamers with cysteine at one position in one of the four histone proteins were reconstituted on a 0N70 DNA (where `N' represents the 147bp 601 DNA sequence, and the lengths of the linker DNA is represented by the numbers 0 and 70). Nucleosomes were modified with the protein-protein cross-linker- MAB (methanethiosulfonate-tetrafluorophenylazide-biotin) reagent. This reagent makes a disulfide bond with the cysteines in the octamer of a nucleosome. The MAB reagent had a distance of ~11.1Aº between its photoreactive tetrafluorophenylazide group and the disulfide forming methanethiosulfonate group. The ISW2 complex was bound to the modified mononucleosomes and cross-linked by irradiating with UV-light. Under reducing conditions the biotin moiety was transferred from the nucleosome to the ISW2 complex. The subunit of the remodeler that was photocross-linked at these positions on the nucleosome was blotted onto nitrocellulose and detected with streptavidin conjugated to horseradish peroxidase (HRP). The catalytic subunit-Isw2 of the ISW2 complex was cross-linked at all five positions but with the following order of intensity from most to least- H2A-89, H3-80, H2B-109, H2A-19, and H2A113. Mass spectrometry was used to decipher these residues, motifs or domains of the catalytic subunit- Isw2 that interacted with the octamer at each position. The ISW2 complex was digested with trypsin, and the biotinylated peptides were enriched using monomeric avidin affinity chromatography. The largest subunit of the ISW2 complex, Itc1, did not get cross-linked at any of the positions.

Expansive proliferation of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered cerebral cortex. NPC expansion places a heavy burden on proteins that regulate chromatin packaging and genome integrity, which is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. Accordingly, mutations in ATRX, a chromatin remodelling protein required for heterochromatin maintenance at telomeres and simple repeats, cause the ATR-X syndrome. Here, we demonstrate that proliferating ATRX-null cells accumulate DNA damage, while also exhibiting sensitivity to hydroxyurea-induced replication fork stalling. Specifically, PARP1 hyperactivation and replication-dependent double strand DNA breakage indicated replication fork protection defects, while DNA fiber assays confirmed that ATRX was required to protect replication forks from degradation. Interestingly, inhibition of the exonuclease MRE11 by the small molecule mirin could prevent degradation. Thus, ATRX is required to limit replication stress during NPC expansion.

Nucleosomes were prepared from trout testis nuclei by micrococcal nuclease digestion. The reactivity toward N- [ethyl- ³Hlmaleimide (NEM) of the single sulfhydryl group of histone H3 in the nucleosomes was studied under a variety of conditions. Under conditions of low ionic strength, there is negligible reaction of nucleosomes with NEM, suggesting that the cysteinyl residue of H3 is buried. Complete denaturation of nucleosomes in 6 M guanidinium chloride leads to reaction of 2 moles of NEM per mole of nucleosomes, in agreement with the expected presence of 2 moles of H3 per particle. Exposure of nucleosomes to 2 M NaCI or 1 M MgCl₂ leads to exposure of the thiol group. At higher Mg⁺⁺ concentrations, the thiol group remains exposed, but in NaCI solutions, as the salt concentration is increased beyond 2 M, the thiol group returns to an inaccessible state. The reactivity of nucleosome thiol groups is relatively unaffected by urea to approximately 5 M. Between 5 and 8 M urea, a rapid increase in thiol reactivity indicates a cooperative unfolding of the nucleosome core. When added together, urea and salt act in a cooperative manner to expose the H3 sulfhydryl group. Mixtures of oligonucleosomes have also been studied under different conditions. They were found to behave in a similar fashion to monomers in 6 M guanidine, but their thiols react more slowly than those of monomers in high salt. Removal of the amino-terminal regions of the core histones by tryptic digestion has no noticeable effect on the accessibility of nucleosome thiol groups. It is concluded that the carboxy-terminal region of H3 containing Cys 110 is masked mainly by histone-histone interactions in the octameric core complex, and is located in a region which is relatively insensitive to the perturbations induced by trypsin or low concentrations of urea. Nucleosomes reconstituted in the presence of a sulfhydryl reducing agent were indistinguishable from native particles in their reactivity to NEM in low salt buffers, in 2 M NaCl and in 6 M guanidine hydrochloride. These studies indicate that the degree of exposure of H3 sulfhydryl groups in nucleosomes can be effectively monitored using NEM. The carboxy-terminal region of H3 containing Cys 110 seems to be located in a relatively stable region of the nucleosome core, perhaps at the interface between heterotypic tetramers. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

INO80, a member of the multi-subunit SWI2/SNF2 superfamily, is involved in transcription regulation, DNA repair and replication. Not much is known about its substrate specificity and remodeling mechanism or how it differs in comparison to SWI/SNF or ISWI. Site-directed mapping of histone-DNA contacts showed that INO80 generally remodels mononucleosomes by moving them to the center of DNA. The length of extranucleosomal DNA was found to play an important role in nucleosome binding as well as remodeling by INO80 much like ISW2 and ISW1a. INO80 preferentially binds to nucleosomes containing >20bp of extranucleosomal DNA. Similarly, INO80 remodeling of mononucleosomes with different lengths of extranucleosomal DNA showed that at least 33bp of extranucleosomal DNA on one side of the nucleosome was required for initiation of remodeling. These data suggest that INO80 behaves much like ISW2 and ISW1a complexes based on their requirement for extranucleosomal DNA. INO80 does not unravel or displace nucleosomes like SWI/SNF. There are several key aspects of how INO80 interacts with and remodels nucleosomes that are quite distinct from SWI/SNF, ISW2, and ISW1a. Previously SWI/SNF and ISW2 were shown to initiate nucleosome movement by translocating along nucleosomal DNA two helical turns from the dyad axis. Nucleosome movement by INO80 instead requires translocation by the complex along nucleosomal DNA near the entry/exit site at the dimer-tetramer interface. Sliding interference of INO80 by the presence of nicks indicated that torsional strain at the site of translocation is required for nucleosome mobilization by INO80. Hydroxyl radical footprinting of the INO80-nucleosome complex shows found that INO80 interactioninteracts with extranucleosomal DNA at, the entry-exit site and to lesser extent at the dyad axis, but it lacks the protection found indoes not contact 2 helical turns from the dyad like ISW2 and SWI/SNF at two helical turns from the dyad axis as determined by photoaffinity cross-linking studies. The catalytic subunit (Ino80) rather than being found associated 2 helical turns from the dyad, was bound to extranucleosomal DNA and nucleosomal DNA near the entry-exit site. Other subunits (Arp8p, Arp5p and Nhp10) were also found to be contacting both nucleosomal and extranucleosomal DNA. Site-specific histone cross-linking studies revealed that Ino80, Arp5 and Arp4 interact extensively with the histone dimer of the nucleosome in comparison to H3-H4 tetramer. Although N-terminal histone tails are often important for chromatin remodeling, INO80 shows no requirement of histone tails for its nucleosome binding and mobilizing activities. The deviation of INO80 from the canonical model of how ATP-dependent remodelers interact and mobilize nucleosome is apparently due to its unique role as a member of the remodeling complexes that promote the exchange of H2A/H2B dimer from core nucleosome particle.

Bivalent (poised or paused) chromatin comprises activating and repressing histone modifications at the same location. This combination of epigenetic marks at promoter or enhancer regions keeps genes expressed at low levels but poised for rapid activation. Typically, DNA at bivalent promoters is only lowly methylated in normal cells, but frequently shows elevated methylation levels in cancer samples. Here, we developed a universal classifier built from chromatin data that can identify cancer samples solely from hypermethylation of bivalent chromatin. Tested on over 7,000 DNA methylation data sets from several cancer types, it reaches an AUC of 0.92. Although higher levels of DNA methylation are often associated with transcriptional silencing, counter-intuitive positive statistical dependencies between DNA methylation and expression levels have been recently reported for two cancer types. Here, we re-analyze combined expression and DNA methylation data sets, comprising over 5,000 samples, and demonstrate that the conjunction of hypermethylation of bivalent chromatin and up-regulation of the corresponding genes is a general phenomenon in cancer. This up-regulation affects many developmental genes and transcription factors, including dozens of homeobox genes and other genes implicated in cancer. Thus, we reason that the disturbance of bivalent chromatin may be intimately linked to tumorigenesis.