Multicellular organisms are composed of a number of different specialised cells that all carry the same genetic material but are highly divergent in their functions and characteristics. This diversity is only allowed because sets of specific genes are expressed in one type of cells and silent in others. A precise control mechanism is required to fine-tune gene regulation and relies on chromatin structure and regulatory proteins. One of the largest families of DNA-binding factors that influence this in human and mouse is the KRAB zinc finger protein (KZFP) family. KZFPs are thought to have rapidly evolved alongside transposable elements and be mediators of transcriptional repression. The few KZFPs that have been characterised so far have been shown to be involved in a wide range of regulatory and biological processes; hence it is hard to make functional generalisations. During my PhD, I studied one member of the KZFP family in mouse, ZFP263, with the aim of understanding its mechanism of action in mouse embryonic stem cells (mESCs) and its role in mice. My work has shown that ZFP263 is an ancient protein highly conserved in mammals and under purifying selection. One of its two functional domains however is divergent from the consensus sequence found in most KZFPs and suggests that ZFP263 might have lost the ability to recruit repressive chromatin states. My research identified the targets of ZFP263 binding in mESCs and showed that it does not bind and silence transposable elements. Indeed it targets unique regions of the genome, mostly within transcribed regions of genes. These genes show a wide range of expression levels and are involved in several key biological processes. Surprisingly, binding sites are not associated with the canonical KZFP co-factor but mostly co-localize with active histone marks. My findings lead me to hypothesise that ZFP263 has evolved to target active epigenetic states to unique regions that are positive regulators of transcription, in contrast to the more canonical model of KZFP function. To test this hypothesis, I have generated targeted mutations at Zfp263 in mice using CRISPR-Cas9 and my preliminary results suggest that Zfp263 mutants have growth defects indicating a role for this protein in mouse development. My findings indicate that ZFP263 is a unique KZFP with non-canonical properties and provide novel insights into the evolution and functions of KZFPs in mammals.

"October, 2004" / Bibliography: leaves 118-154. / xii, 154 leaves : ill. (some col.), plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Microbiology and Immunology, 2005

The main focus of this thesis is to study the physical characteristics of the N-terminus of human XAF1 protein, a 17kD protein named NTA1, by biophysical methods. Structural studies of the N-terminus of XAF1 serves as a base for the studies of the structure and function relationship of the N-terminus, and the same maybe true for the full length XAF1. Bioinformatics analysis shows that NTA1 shares high sequence identity with the TRAF-type zinc finger domain-containing protein 1 (TRAFD1) and FLN29. Protein structure prediction has been performed on NTA1 by the I-TASSER web server. The prediction result suggests that NTA1 is a structure that consists of α-helices which are joined by flexible linkers. The loose structure shown by I-TASSER is expected to have high solvent accessibility. This coincides with the deuterium exchange data. In addition, by the CD approach, NTA1 was estimated to contain high α-helix content. This result is consistent with the bioinformatics prediction and the secondary structure obtained from the chemical shift index method as well. The physical characterizations of NTA1 showed that NTA1 is a loosely packed protein; and the five zinc ions are bound in the protein structure. Based on the chemical shifts of β-carbons, the Cysteine residues Cys8, Cys11, Cys34, Cys37, Cys50, Cys59, Cys62, Cys86, Cys89 and Cys115 showed a significantly downfield shift, they are probably involved in the zinc ions coordination. The dynamic property of NTA1 was investigated by NMR techniques. Backbone dynamics of NTA1 reveal that NTA1 does not have a typical spherical structure, it is anisotropic. Residues corresponding to the zinc finger regions in the predicted structure show large R2/R1 and S2 values, while regions shown to be flexible linkers in the 3D structure prediction show small R2/R1 and S2 values. Thus, the protein structure homology modeling data are supported by the backbone dynamics data. / published_or_final_version / Chemistry / Master / Master of Philosophy

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Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Chemistry. Includes bibliographical references. Also available online via the Internet.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 166-180). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, December 1999. / Includes bibliographical references. Also available on the Internet.

Genetic competence is a developmental process in bacteria that allows natural transformation. Competent Gram positive bacteria such as Bacillus subtilis carry a cytosolic helicase which is required for efficient transformation. In this work ComFA is confirmed as a DEAD-box helicase. I also describe a new accessory motif in ComFA that contributes to transformation independently of the helicase activity in ComFA. The newly discovered metal-binding motif consists of four cysteines which are required for transformation and zinc binding. While the zinc finger is required for full function, it is not required for DNA binding. As DEAD-box family helicases are generally non-processive, it appears that at least part of the rapid DNA uptake process is mediated by a non-processive helicase. Active uptake using the ComFA helicase motor may be required to maintain the integrity of the incoming DNA to allow subsequent recombination.

No description available.