Biochemical and Functional Analysis of Homeoprotein Nkx3.1

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Title: Biochemical and Functional Analysis of Homeoprotein Nkx3.1
Author: Simmons, Steven O'Neal
Advisors: Jonathan Horowitz, Committee Chair
Robert Smart, Committee Member
James Mahaffey, Committee Member
Gregg Dean, Committee Member
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.
Date: 2006-06-08
Degree: PhD
Discipline: Toxicology

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