Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 18 (1958) no. 3, p. 766. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 33-35).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Universiẗat, Diss., 2003--Heidelberg.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Tierärztl. Hochsch., Diss., 2004--Hannover.

Universiẗat, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

SWI/SNF, the founding member of ATP dependent chromatin remodelers and its paralog RSC in yeast perform similar yet distinct functions inside the cell. In vitro these complexes use ATP dependent DNA translocation to either mobilize or disassemble nucleosomes. However, how these complexes interact with nucleosomes and the mechanism by which chromatin remodeling is achieved is not fully understood. Further, it is not understood how they perform disparate roles in vivo despite their similar biochemical activities. To understand the fundamental differences between these complexes the substrate specificity of RSC and SWI/SNF and their interaction with different parts of the nucleosome were investigated. SWI/SNF and RSC exhibited almost identical nucleosome binding affinities (~7 nm) with a minimal requirement of 20 bp of extranucleosomal DNA for efficient binding. Hydroxical-radical footprinting of RSC-nucleosome complex showed that RSC, unlike SWI/SNF, interacts extensively with approximately 50 bp of extranucleosomal DNA near the nucleosome entry site. RSC also interacts, but not as strongly as SWI/SNF, with almost one gyre of nucleosomal DNA (SHL-2 to SHL-6) on the same side of the extranucleosomal DNA. Analogous to the previously observed SWI/SNF-footprint the second gyre of nucleosomal DNA had no protection and in fact enhanced cleavage was seen starting from 3-4 helical turns from the dyad axis up to the exit site where DNA leaves the nucleosome. The asymmetry of the DNA footprint pattern confirmed binding of RSC in one preferred orientation guided by the extranucleosomal DNA at one end of the nucleosome like ISW2 and also like SWI/SNF but only when recruited by transcription factors. DNA crosslinking revealed that most of the SWI/SNF contacts are with a small region spanning the DNA translocation start site near SHL2 and does not extend to the rest of the footprint. Further, the SWI/SNF contacts are primarily through its catalytic subunit Snf2 which is found to intercalate between the DNA gyre and the histone octamer at SHL2. Consistent with its DNA footprint, RSC however makes extensive contacts with both nucleosomal and extranucleosomal DNA through five major subunits Sth1, Rsc2, Rsc3, Rsc30 and Rsc4. Excepting the catalytic subunit Sth1 which is highly homologous to Snf2, the remaining four are unique to RSC. Sth1 contacts a much broader region in the nucleosomal DNA than Snf2 with the primary contact being at SHL2 where it wedges between the DNA and the histone octamer surface. The accessory subunits Rsc2, Rsc3 and Rsc30 mostly contribute to the extranucleosomal DNA contacts of RSC. These subunits also make a second major contact near the dyad, with those made by Rsc3 and Rsc30 being the strongest. The histone N-terminal tails that emanate out of the nucleosome structure are implicated in the regulation of chromatin remodeling, in general, and in the activation of several SWI/SNF dependent genes, in particular. Remodeling kinetics studies with tailless nucleosomes revealed that the histone H4 tail is required for nucleosome mobilization, H2A/H2B dimer displacement and nucleosome disassembly by both RSC and SWI/SNF. Further, the H4 tail modulates RSC and SWI/SNF remodeling without affecting ATP hydrolysis or nucleosome binding. These data suggest a similarity between SWI/SNF and ISWI class of chromatin remodelers based on their dependence on the H4 tail. Owing to the presence of acetyl-lysine binding bromodomains in these complexes and to a greater extent in RSC the differences in their remodeling activities, if any, were expected to be accentuated by histone acetylation. Studies with H3 and H4 tail acetylated nucleosomes provided evidence for two pathways that work synergistically to recruit SWI/SNF and RSC to chromatin. While one of the pathways involves transcription activators, the other pathway of SWI/SNF recruitment is dependent on covalent acetylation of histone H3 tail. Bromodomain mediated recognition of these acetyl marks not only facilitates SWI/SNF recruitment but also stimulates their catalytic activity to mobilize nucleosomes. Importantly, extensive conformational changes occur in SWI/SNF in response to H3 tail acetylation. Chromatin remodeling by SWI/SNF and RSC is also regulated to different degrees by H3 tail acetylation depending on the number of bromodomains. The higher responsiveness of RSC to H3 tail acetylation than SWI/SNF can provide additional regulatory mechanisms for RSC which might ultimately account for their different functional roles inside the cell. When these same acetyl marks are within the H3 globular core and reside near the dyad axis of symmetry they are found to act in synergy with RSC and SWI/SNF to facilitate nucleosome movement as well as nucleosome disassembly. Unlike H3 tail acetylation, the remodeling enhancement by H3 core acetylation occurs via an acetyl lysine-bromodomain recognition independent mechanism. Further, supporting this recognition-independent mechanism H3 core acetylation does not affect the recruitment of these complexes. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.

The genome is more than a linear sequence of bases; its spatial organisation is a key part of its function. In humans, three billion base pairs, or approximately two metres of DNA are packaged into a nucleus a few micrometres in diameter. The genome must also be organised so that it can be replicated and partitioned into daughter cells, and so that regulatory elements are positioned to affect their targets. Until recently, little was known about the organisation of the genome at the scale of single genes. The packaging of DNA onto nucleosomes, and the segregation of chromosomes into chromosome territories was well understood, but the development of chromatin conformation capture (3C) techniques has enabled the first thorough study of intermediate scales. These methods provide information about the distances between pairs of genomic loci, which gives indirect information about their positions. By applying these techniques to single cells, it has become possible to calculate a structure from the observed distance restraints. Through the prior constraints placed on the model, such as the existence of a continuous backbone, these structures provide additional information about the conformation of DNA. To overcome the limitations of 3C, it is useful to integrate additional sources of information. I present several methods for the validation and improvement of Hi-C structures by adding data from microscopy, and for characterising dyes used in single-molecule light microscopy. It is found that single-cell Hi-C structures agree with fluorescence microscopy when observing the distance of genes from the edge of the nucleus, and that centromeres are not a suitable label for direct validation. Adding absolute positional restraints from images is shown to be useful in better determining chromatin structure in synthetic tests. Finally, the presence of a FRET acceptor near a fluorescent protein is shown to improve its photophysical properties.

Trimethylation of histone H3 lysine 27 (H3K27me3) marks facultative heterochromatin, containing silent genes. My research investigated factors that influence the distribution of H3K27me3 in the filamentous fungus Neurospora crassa. The H3K27 methyltransferase complex, PRC2, is well conserved in eukaryotes and consists of four core members: E(Z), EED, SUZ12 and P55. I showed that three of the PRC2 subunits (SET-7, the homolog of E(Z), EED and SUZ12) are required for H3K27me3 in Neurospora, while NPF, the homolog of P55, is only required for a subset of H3K27me3 domains. H3K27me3 is organized into large, gene-rich domains in Neurospora and normally does not overlap with constitutive heterochromatin, which is marked by both H3K9me3 and DNA methylation and bound by heterochromatin protein 1 (HP1). I discovered that loss of HP1 binding results in a genome-wide relocalization of H3K27me3. Specifically, it is lost from many of its normal domains while it becomes associated with much of the genome that is constitutive heterochromatin. This contrasts plant and mouse studies in which the loss of DNA methylation relocalizes H3K27me3. The DCDC complex is the H3K9-specific methyltransferase consisting of DIM-5, DIM-7, DIM-9, CUL4 and DIM-8. Separate deletions of DCDC subunits, with the exception of dim-7, relocalized H3K27me3 to constitutive heterochromatin, presumably due to the loss of HP1 binding. The deletion of dim-7 resulted in the loss of all H3K27me3, suggesting a novel role for dim-7. To look for a recruitment signal for PRC2, I moved large fragments contained within an H3K27me3 domain to loci devoid of H3K27me3, his-3 and csr-1. None of the fragments induced H3K27me3, demonstrating that a recruitment signal is not present within every fragment of H3K27me3-marked DNA. Large chromosomal rearrangements had profound effects on H3K27me3 domains, resulting in the loss of some H3K27me3 domains and the formation of others. In Drosophila and mammals, a subset of PRC2 complexes contains the histone deacetylase, Rpd3. A close homolog of Rpd3 in Neurospora, HDA-3, did not appear to be a member of PRC2 in Neurospora. This dissertation includes both previously published and unpublished co-authored material.

NF-κB signalling induces transcriptional upregulation of a wide array of genes in response to inflammatory signalling caused by, for example, TNFα cytokine. In addition to inducing the expression of factors which mediate an intracellular response, such stimuli also cause the expression of further signalling factors, including TNFα itself, to propagate and refine an initial stimulus. However, while such positive feedback signalling can be seen to be beneficial in amplifying potentially small initial stimuli, excessive production can cause hyper-inflammatory responses; an occurrence linked to several autoimmune diseases. Therefore, correct regulation – in regards to both too little and too much TNFα signal production – is essential for a balanced immune response. In this thesis I have focussed on the effects of the IκB protein family member BCL-3 on TNFΑ transcription: demonstrating NF-κB dependent induction of both TNFΑ and BCL3 genes and a subsequent negative role for BCL-3 in regulating TNFΑ transcription in the human fibrosarcoma HT1080 cell line – forming an Incoherent Feed Forward Loop (I-FFL) motif. Notably, I have shown a differential rate of induction of TNFΑ (rapid) and BCL3 (delayed) transcript levels; demonstrating that while the TNFΑ gene has a pre-stimulus RNA polymerase II bound and poised for a rapid response, the BCL3 promoter requires histone modification and chromatin remodelling for binding of NF-κB and RNA polymerase II. Extensive characterisation of the temporal sequence of events constituting BCL3 promoter remodelling, mRNA plus protein levels and NF-κB nuclear localisation through live cell microscopy allowed the construction of a mathematical model which has been tested to ensure it can accurately recreate biological behaviour. This model has been utilised to show that the delayed production of inhibitory BCL-3 produces distinct TNFΑ transcript dynamics: (i.) initially allowing a high magnitude response but coupled to later strong repression of TNFΑ expression and (ii.) producing a non-monotonic response to pulsed stimuli. This behaviour cannot be quantitatively recreated with models in which BCL3 transcription is induced simultaneously with TNFΑ and proposed physiological benefits are outlined. Based on this work, time delays in I-FFLs are proposed as a novel mechanism to produce varied output dynamics. Future research tools have also been developed in this work - including generation of an expression vector to visualise BCL-3 protein in live cells (utilising a BAC recombinant engineering approach) - plus further research questions and predictions regarding TNFα signalling have been raised by additional modelling work.