Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Two novel and highly related C₂H₂ zinc finger proteins (CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and hematopoietic cell development. However, the precise cellular function(s) and the contribution of these proteins to neoplastic processes and hematopoietic cell development remain unknown. The goal of the studies described herein was to elucidate the molecular mechanisms underlying the transcriptional repression mediated by these proteins to understand their biological properties, and ultimately, their cellular function(s). CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive manner, suggesting that this repression mechanism(s) may not involve TSA-sensitive histone deacetylation catalyzed by member(s) of class I and II HDACs. One possible mechanism is that CTIP proteins may exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was found to interact with CTIP proteins both in vitro and in mammalian cells, and was recruited to the promoter template in a CTIP-dependent manner. The proline-rich regions of CTIP proteins and the sirtuin homology domain of SIRT1 were found to be essential for mediating CTIPs•SIRT1 interactions. Moreover, column chromatography revealed that SIRT1 and CTIP2 were components of a large complex in Jurkat cell nuclear extracts. Based on the findings that SIRT1 associates with CTIP proteins in mammalian cells, SIRT1 may underlie the transcriptional repression activity of CTIP proteins. The following results support the hypothesis that SIRT1 may underlie the mechanism(s) of CTIP-mediated transcriptional repression. First, CTIP-mediated transcriptional repression was inhibited, at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive HDACs. Second, the decrease in levels of acetylated histones H3 and/or H4 at the promoter region of a reporter gene was observed upon overexpression of CTIP proteins, and this effect was inhibited, at least partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the promoter template of a reporter gene in mammalian cells upon overexpression of CTIP proteins. Fourth, SIRT1 enhanced the transcriptional repression mediated by CTIP proteins and this enhancement required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected cells. In summary, results described herein strongly suggest that CTIP-mediated transcriptional repression involves the recruitment of SIRT1 to the template, at which the TSA-insensitive, but nicotinamide-sensitive histone deacetylase catalyzes deacetylation of promoter-associated histones H3 and/or H4. These results contribute additional understanding to the molecular mechanisms underlying transcriptional activity of CTIP proteins, which might be helpful for identification and characterization of the target genes under the control of CTIP proteins in cells of hematopoietic system and/or the central nervous system. / Graduation date: 2004

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Not available / text

Wilms' tumor suppressor protein (WTI) is a key regulatory factor involved in controlling the development and normal physiology of the genitourinary tract. Mutations within WT1 result in multiple syndromes affecting the kidney and gonads with the most severe effects being Wilms' tumor, a pediatric kidney cancer. The WTI protein is composed of two distinct functional domains; the amino terminus is a proline and glutamine rich regulatory domain, while the carboxyl terminus is a DNA binding domain which contains four C2H2 zinc fingers. Although the zinc finger motif is small in size, proteins containing zinc fingers are extremely diverse in their functions. The functional diversity of WT1 is exemplified through its interactions with a wide range of ligands, such as DNA, RNA and proteins. The interaction between WT1 and DNA has been well characterized, while the interactions with RNA and proteins still require intensive investigation. Recent studies have identified a diverse group of WT1 protein partners but the characterization of the protein-protein interactions has been limited and inconclusive. Therefore, the experiments conducted in this study focused on investigating the mechanism of interaction between WTI with the protein ligands Ciao 1, p53, par4, and U2AF65. To identify which WTI zinc finger(s) are critical in protein binding, a series of finger swap and deletion mutant proteins were created using site directed mutagenic PCR. The effects the mutant proteins had on the protein interactions were analyzed qualitatively using GST pulldown assays. Two different approaches were used for the GST pulldown assays. The first approach utilized bacterially expressed and purified proteins. None of the mutant WTI proteins exhibited a decrease in protein binding in these assays. Numerous pulldown trials involving various zinc fmger proteins revealed non-specific protein-protein interactions were occurring. The second approach employed in vitro translated 35S-labelled proteins. The results from these assays demonstrate a clear role for WT lzf3, and a possible role for WTI zf4 in the WT 1-par4 interaction. The replacement of WT 1 zinc fingers 3 and 4 with those from YY1 caused a distinct reduction in binding to par4 which was exclusive for the WT1-par4 interaction. YY1 is a transcription factor from yeast that contains four C2H2 type zinc fingers. A decrease in binding between the chimeric proteins WTI :YY1 and the protein partners Ciao 1 and U2AF65 was also observed, although to a much lesser extent. This difference in binding ability may indicate that the interactions between WT1 and its protein ligands involve different zinc fingers.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Sydney, 2009. / Title from title screen (viewed February 10, 2009). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Degree awarded 2009; thesis submitted 2008. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--Georgia State University, 2008. / Title from file title page. Markus W. Germann, committee chair; Kathryn B. Grant , W. David Wilson, committee members. Electronic text (147 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Oct. 6, 2008. Includes bibliographical references.