Thesis (Ph.D. in Biochemistry & Molecular Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 169-185). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

The dynamic nature of eukaryotic chromatin enables the packaging of large amounts of genetic material in a small space. At the same time, it provides controlled access to genomic DNA for a variety of nuclear processes for example, transcription and DNA repair. The transition between open and closed chromatin states is largely governed by ATP-dependent chromatin remodeling complexes, which operate on nucleosomes in concert, to modulate chromatin structure and composition. Exchange of the canonical and variant forms of histones in nucleosomes, and altering the spacing between consecutive nucleosomes, are two major ways which regulate chromatin-based processes and chromatin higher-order organization. The evolutionarily conserved INO80 and ISW1a complexes mediate these two aspects of nucleosome remodeling, respectively. Despite sharing conserved domain architecture of the core remodeling machinery, chromatin remodelers differ significantly in their modes of interaction with nucleosomes, and how they alter histone-DNA contacts. In this study, we have used a site-specific photocrosslinking approach coupled with peptide mapping to determine the interactions of subunits and domains of the S. cerevisiae INO80 and ISW1a complexes with nucleosomes. We find that specific interactions of remodelers with different regions of the nucleosome largely dictate their specialized functions and mechanisms. The ATP-dependent helicase-like (ATPase) domains of remodelers belonging to the ISWI and SWI/SNF families translocate along DNA close to the center of nucleosomes in order to mobilize, space or disassemble nucleosomes. In contrast, we observed that INO80 has a strikingly distinct mechanism, which is different even from its paralog SWR1. INO80 mobilizes nucleosomes as well as catalyzes the exchange of histone variant H2A.Z for the canonical histone H2A, while SWR1 mediates the reverse exchange of H2A for H2A.Z, without being able to mobilize nucleosomes. We have found that INO80, in order to promote H2A-H2B dimer exchange, translocates along DNA at the H2A-H2B interface close to the edge of nucleosomes and persistently displace DNA from H2A-H2B. Blocking either DNA translocation or the accumulation of DNA torsions close to the edge of the nucleosome interferes with this dimer exchange by INO80. SWR1 and other SWI/SNF and ISWI remodeling complexes translocate along DNA at the H3-H4 interface and do not persistently displace DNA from the histone octamer as does INO80. This study shows for the first time an ATP-dependent chromatin remodeler that invades nucleosomes at the DNA entry site instead of the center − a more logical approach for the displacement of H2A-H2B. We also investigated nucleosomal DNA interactions of other INO80 subunits and domains to understand the architecture of INO80 bound to nucleosomes. We found that the HSA (helicase-SANT-associated) domain of Ino80 along with actin-related protein (Arp) subunits Arp8 and Arp4 bind to the extranucleosomal DNA and is potentially involved in a coupling mechanism with the ATPase domain to regulate its activity. We also mapped the DNA binding regions of Arp8 and Arp4, which might be involved in recruiting INO80 to genomic sites. The ISWI remodeler ISW1a regulates the distance (spacing) between nucleosomes in an array by simultaneously interacting with two nucleosomes and directionally remodels one of them. We mapped DNA interactions of ISW1a subunits in mono- and di-nucleosomes. Our results show that the catalytic Isw1 subunit specifically interacts with the region of DNA translocation and DNA entry site of the asymmetrically positioned nucleosome in a di-nucleosome, which is preferentially mobilized. In contrast, the Ioc3 subunit interacts extensively with the linker DNA as well as the extranucleosomal DNA of the un-remodeled nucleosome. This bias in nucleosomal DNA interactions of ISW1a enables directional remodeling, which reveals the molecular basis of nucleosome spacing. We have identified a novel domain within the non-catalytic Ioc3 subunit of ISW1a that regulates nucleosome spacing. We found that when this domain is deleted, the catalytic Isw1 subunit loses its specificity and interacts with both the nucleosomes of a di-nucleosome substrate. This is consistent with the domain-deleted ISW1a mobilizing both nucleosomes efficiently, leading to the loss of its nucleosome spacing activity. In summary, this dissertation explores how different remodeling complexes have customized and regulated modes of nucleosome interaction in order to accomplish specialized remodeling outcomes. INO80 places its ATPase domain for translocation at the H2A-H2B dimer interface and persistently displaces DNA from its surface to promote H2A.Z exchange. Nucleosome spacing by ISW1a requires the catalytic Isw1 subunit to engage with and reposition one out of two consecutive nucleosomes in an array, while the Ioc3 subunit likely monitors the distance between them.

Chromatin diminution is defined as the elimination of DNA during the differentiation of early embryonic cells into pre-somatic cells. While it was first observed in the nematode Parascaris equorum, it also been identified in other parasitic nematodes, hagfish and copepods. In the copepod Mesocyclops edax, up to 90% of genomic DNA is eliminated during chromatin diminution. It was previously shown that the eliminated DNA contained highly repetitive heterochromatic sequences. Here, we digested pre- and post-diminution DNA with BamHI and produced small libraries of clones from each. Analyses revealed no decrease in low copy numbered sequences, such as transposable elements. Rather, both libraries are found to be surprisingly similar in all aspects analysed. Further comparison also demonstrated similarity of our libraries with the DNA sequences eliminated from Cyclops kolensis. Consequently, we suggest that M. edax eliminates portions of euchromatic DNA, in addition to the previously characterized satellite sequences.

The molecular target of epigenetic signaling is chromatin. Histones are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. As PTMs occur at multiple positions within histones, the degree to which these modifications might influence each other remains one of the major challenges of chromatin biology. Although major discoveries in understanding the complex repertoire of histone modifications were achieved using reductionist experimental systems with synthetic histone peptides, they do not explain the role of putative PTM cross-talks in a chromatin context. However, generating chromatin substrates of defined modification status has proved to be a technically challenging task. In this thesis, I first demonstrate our work on establishing a novel approach to produce libraries of modified nucleosomes. We employed protein trans-splicing and sortase-mediated ligation strategies to incorporate chemical modifications on histone tails of ‘ligation-ready’ nucleosomes. Subsequently, the ‘designer’ nucleosome libraries were used for testing the binding of heterochromatin protein 1 (HP1) and elucidated the previously uncharacterized crosstalk of H3K9me2 and S28ph marks. Further investigations explained the mechanism of this crosstalk and highlighted the importance of developing chemical biology tools for elucidating complex chromatin signaling. Second, I describe our reconstitution systems for the assembly of semisynthetic recombinant chromatin carrying methylation marks on DNA and distinct modifications on histones, e.g. H3K9me3. I aimed to understand the mechanisms of the interplay between chromatin and one of the DNA maintenance methylation factors, UHRF1. I showed that UHRF1 strongly interacts with nucleosomes containing linker DNA. However, it exerts only residual enzymatic activity in this context. Based on functional H3 ubiquitylation assays in vitro, I found that hemi-methylated nucleosomes stimulate enzymatic activity of UHRF1, suggesting that the protein’s chromatin targeting and activation are a two-step process. The positioning of hemi-methylated CpG on nucleosome regulates UHRF1 target selectivity. Further, mutational analysis revealed that the PHD domain of the factor is indispensable for H3 binding and that its SRA domain is required for catalytic activation. Overall, our work adds a new layer of positional complexity to the me½CpG-dependent regulation of UHRF1 and expands the current model of DNA methylation maintenance.

Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...

Call number: LD2668 .T4 1979 F57 / Master of Science

Aspects of higher order chromatin structure such as replication timing, lamina association and Hi-C inter-locus interactions have been recently studied in several human and mouse cell types and it has been suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and little is known about the characteristics of any divergent loci defined. Here we generate an orthologous dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between different mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the majority of the orthologous genome. This broad similarity is observed in spite of the substantial time since the species diverged, differences in experimental procedures among the datasets examined, and the presence of cell type specific structures at many loci. However, we also identify hundreds of regions showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. Divergent regions are enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in mammalian evolution. They are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions may underlie divergent regulation. Divergent regions show unusual shifts in compositional bias, sequence divergence and are unevenly distributed across both genomes. We investigate the mechanisms of divergence in higher order structure by examining the influence of sequence divergence and also many features of primary level chromatin, such as histone modification and DNA methylation patterns. Using multiple regression, we identify the dominant factors that appear to have shaped the physical structure of the mammalian genome. These data suggest that, though relatively rare, divergence in higher order chromatin structure has played important roles during evolution.

Background: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin structure dictates gene expression, and therefore function, further investigation of this phenomenon may increase our understanding of therapeutic responses. I hypothesised that nuclear morphological changes in cancer in response to DNA-damage by chemotherapy are mediated by histone deacetylases (de Ruijter, van Gennip et al.). Methods: Ovarian cancer cell lines PEO1/PEO4 (platinum sensitive/resistant) were selected as in vitro models, and primary ovarian cancer xenografts OV1002 and HOX424 as in vivo models. Expression levels of HDACs, heterochromatin protein 1 (HP1), and DNA damage response (DDR) proteins were profiled by Western blot analysis after treatment with cisplatin. Immunofluorescence imaging was undertaken using confocal microscopy, and nuclear texture and γH2AX foci were measured in Image J. Cell cycle and apoptosis were detected by flow cytometry. Thirty eight different ovarian cancer biopsies and 175 xenograft samples were assessed for HDAC and HP1 expression in response to chemotherapy by quantitative immunofluorescence. HDAC2 expression was modulated by interfering RNAs (siRNA). Results: I demonstrated nuclear morphological changes in clinical tumours, xenografts, and cell lines in response to platinum chemotherapy by robust measurement of nuclear texture. Expression of HDAC2 increased in PEO1 cells treated with cisplatin at 24h, and this was accompanied by high expression of HP1s. Expression of components of both HDACs and DDR pathways (pBRCA1, γH2AX, pATM, pATR) showed time dependent changes after cisplatin treatment. Knockdown of HDAC2 reduced the expression of HP1, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DDR induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, I observed reversible alteration of chromatin patterns during cisplatin treatment to some degree. In clinical ovarian cancer specimens, expression of HDAC4, HDAC8 and HP1γ significantly increased after chemotherapy in sensitive patients, with enhanced heterogeneity in chromatin pattern. HDAC2, HDAC8, and HP1 expression were also increased after carboplatin treatment in carboplatin-sensitive xenografts. Conclusion: These results demonstrate alterations in nuclear morphology after chemotherapy, and implicate HDACs in having a role in higher order chromatin changes and in cellular DNA damage responses in ovarian cancer both in vitro and in vivo.