Characterization of the novel POZ-ZF protein Znf131, a Kaiso-specific interaction partner.

摘要

Kaiso is a ubiquitously expressed member of the BTB/POZ zinc finger family of transcription factors with roles in development and tumorigenesis. Like other POZ-ZF proteins, Kaiso possesses an N-terminal POZ domain that mediates protein-protein interactions and three C-terminal DNA-binding zinc fingers used in transcriptional repression or activation of its target genes. During a yeast-two-hybrid screen to identify Kaiso interaction partners, the novel POZ-ZF protein Znf131 was identified as a heterodimerization partner that interacted with Kaiso via its POZ domain. The identification of Znf131 as a Kaiso binding partner led to our hypothesis that Kaiso's interaction with the Znf131 POZ domain regulates Znf131-mediated transcriptional activity.;To elucidate whether Kaiso regulates Znf131 transcriptional activity, we first determined if Znf131 binds DNA. We identified a 12 base pair palindrome (GTCGCR.Xn.YGCGAC) as a sequence-specific Znf131 DNA binding element (ZBE), and demonstrated via electrophoretic mobility shift assay (EMSA) that the entire palindrome was required for Znf131 DNA-binding. Using artificial promoter-reporter assays we demonstrated that Znf131 activates the ZBE in a dose-dependent manner, indicating that Znf131 is a transcriptional activator. We then showed that Kaiso overexpression inhibited Znf131-mediated transcriptional activation of the ZBE reporter, while the Kaiso binding partner p120ctn enhanced Znf131 activation. Furthermore, we found that while Kaiso binds and represses the cyclin D1 promoter, Znf131 activates it.;Collectively, these data demonstrate that Znf131 translocates to the nucleus via the importin-alpha/beta pathway, and while there, binds DNA and activates transcription. Our data also support our hypothesis that interaction of Kaiso with Znf131 POZ domain regulates Znf131 transcriptional activity. Moreover, data generated within this thesis can serve as a stepping stone for identifying Znf131 target genes and for elucidating the physiological relevance of the Kaiso-Znf131 interaction in normal homeostasis and disease states.;To address this hypothesis, we first characterized Znf131 mechanism of nuclear import using a deletion mutagenesis approach. Two nuclear localization signals (NLSs) were identified and characterized. The first NLS is located downstream of the POZ domain while the second NLS lies within the zinc finger region. We found that both NLS's were necessary for Znf131's interaction with the nuclear import receptors importin-alpha-2,-3, -5 and -7.