Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular 
events associated with cancer. However, KLF8-interacting proteins remain largely 
unidentified. Using co-immunoprecipitation (co-IP), mass spectrometry, and GST 
pulldown assays, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel 
KLF8-interacting protein. Co-IP and Western blotting indicated that KLF8 is also 
a PARP-1 substrate. Mutation of the cysteines in the zinc finger domain of KLF8 
abolished PARP-1 interaction. Surprisingly, immunofluorescent staining revealed 
a cytoplasmic mislocalization of KLF8 in PARP-1(-/-) cells or when the 
interaction was disrupted. This mislocalization was prevented by either PARP-1 
re-expression or inhibition of CRM1-dependent nuclear export. Interestingly, 
co-IP indicated competition between PARP-1 and CRM1 for KLF8 binding. 
Cycloheximide chase assay showed a decrease in the half-life of KLF8 protein 
when PARP-1 expression was suppressed or KLF8-PARP-1 interaction was disrupted. 
Ubiquitination assays implicated KLF8 as a target of ubiquitination that was 
significantly higher in PARP-1(-/-) cells. Promoter reporter assays and 
chromatin immunoprecipitation assays showed that KLF8 activation on the cyclin 
D1 promoter was markedly reduced when PARP-1 was deleted or inhibited or when 
KLF8-PARP-1 interaction was disrupted. Overall, this work has identified PARP-1 
as a novel KLF8-binding and -regulating protein and provided new insights into 
the mechanisms underlying the regulation of KLF8 nuclear localization, 
stability, and functions.