Biochemical and Functional Analysis of Homeoprotein Nkx3.1

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dc.contributor.advisor Jonathan Horowitz, Committee Chair en_US
dc.contributor.advisor Robert Smart, Committee Member en_US
dc.contributor.advisor James Mahaffey, Committee Member en_US
dc.contributor.advisor Gregg Dean, Committee Member en_US
dc.contributor.author Simmons, Steven O'Neal en_US
dc.date.accessioned 2010-04-02T19:00:35Z
dc.date.available 2010-04-02T19:00:35Z
dc.date.issued 2006-06-08 en_US
dc.identifier.other etd-06022006-094339 en_US
dc.identifier.uri http://www.lib.ncsu.edu/resolver/1840.16/4758
dc.description.abstract Nkx3.1 is a homeodomain-containing transcription factor that is expressed early in the development of the prostate gland and is likely to play an important role in the differentiation of prostatic epithelia. Loss of Nkx3.1 protein expression is often an early event in prostate tumorigenesis, and the abundance of Nkx3.1-negative epithelial cells increases with disease progression. In a number of systems, homeodomain proteins collaborate with zinc-'finger'-containing transcription factors to bind and regulate target genes. Herein I report that Nkx3.1 collaborates with Sp-family members in the regulation of prostate specific antigen (PSA) in prostate-derived cells. Nkx3.1 forms protein complexes with Sp proteins dependent on their respective DNA-binding domains and an amino-terminal segment of Nkx3.1 and Nkx3.1 negatively regulates Sp-mediated transcription via Trichostatin A-sensitive and —insensitive mechanisms through a distal portion of the PSA promoter. Nkx3.1 DNA-binding activity is not required for trans-repression of Sp-driven PSA activity. I conclude that Nkx3.1 negatively regulates Sp-mediated transcription via the tethering of histone deacetylases and other co-repressors and/or inhibiting the association of Sp proteins with co-activators. Additionally, I outline my efforts to characterize the subcellular localization of Nkx3.1. I report that Nkx3.1 is a nuclear protein and contains at least one nuclear localization signal (NLS), likely within the carboxy-terminal 20 amino acids of the homeodomain. I also show that Nkx3.1 associates with the nuclear matrix likely via a nuclear matrix targeting sequence (NMTS) that may be coincident with the NLS within Nkx3.1 homeodomain. I show further that a functionally intact Nkx3.1 homeodomain is not required for nuclear localization but is required for association with the nuclear matrix. In addition to these results I show that Nkx3.1 is associated with mitotic chromatin throughout most, if not all, of mitosis and that a functionally intact Nkx3.1 homeodomain is sufficient for inclusion within mitotic chromosomes. Finally, I report the expression of Nkx3.1 in insect and mammalian cells, including LNCaP prostate epithelial cells does not lead to the detection of Nkx3.1 DNA-binding activity in cell extracts. My inability to detect Nkx3.1 protein/DNA binding activity does not appear to be due to an inhibitory phosphorylation event or to inhibitory protein-protein interactions. I also report my efforts to identify Nkx3.1 target genes using a genome-wide approach. This genome-wide screen for putative Nkx3.1 target genes yielded 42 clones containing novel, human genomic DNA. Ten of these clones harbored unique genomic fragments, while the remaining 32 clones carried sequences that were isolated repeatedly and could be subdivided into three sequence classes. Many of the recovered sequences mapped to locations that are within or near known genes and most carried one or more consensus Nkx3.1 DNA-binding sites. Further work must be performed to corroborate that the results from this genome-wide screen represent in vivo Nkx3.1 targets. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject Sp4 en_US
dc.subject ChIP en_US
dc.subject prostate cancer en_US
dc.subject PSA en_US
dc.subject nuclear matrix en_US
dc.subject Sp4 en_US
dc.subject Sp2 en_US
dc.subject Sp1 en_US
dc.subject Nkx3.1 en_US
dc.subject PIN en_US
dc.subject Sp5 en_US
dc.title Biochemical and Functional Analysis of Homeoprotein Nkx3.1 en_US
dc.degree.name PhD en_US
dc.degree.level dissertation en_US
dc.degree.discipline Toxicology en_US


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