Abstract
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates proliferation of various cell types, including fibroblasts, smooth muscle cells, and intestinal epithelial cells. To identify proteins that interact with KLF5, we performed a yeast two-hybrid screen of a 17-day mouse embryo cDNA library with KLF5 as bait. The screen revealed 21 preys clustered in four groups as follows: proteins mediating gene expression, metabolism, trafficking, and signaling. Among them was protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) ligase that regulates transcription factors through SUMOylation or physical interaction. Association between PIAS1 and KLF5 was verified by co-immunoprecipitation. Structural determination showed that the acidic domain of PIAS1 bound to both the amino- and carboxyl-terminal regions of KLF5 and that this interaction was inhibited by the amino terminus of PIAS1. Indirect immunofluorescence demonstrated that PIAS1 and KLF5 co-localized to the nucleus. Furthermore, the PIAS1-KLF5 complex was co-localized with the TATA-binding protein and was enriched in RNA polymerase II foci. Transient transfection of COS-7 cells by PIAS1 and KLF5 significantly increased the steady-state protein levels of each other. Luciferase reporter and chromatin immunoprecipitation assays showed that PIAS1 significantly activated the promoters of KLF5 and PIAS1 and synergistically increased the transcriptional activity of KLF5 in activating the cyclin D1 and Cdc2 promoters. Importantly, PIAS1 increased the ability of KLF5 to enhance cell proliferation in transfected cells. These results indicate that PIAS1 is a functional partner of KLF5 and enhances the ability of KLF5 to promote proliferation.
Highlights
Them is Kruppel-like factor (KLF)2 5, which belongs to the Sp/KLF family of transcription factors [1,2,3,4]
TFIID binding initiates the recruitment of other factors required for polymerase II (pol II) to begin transcription [32]. pol II is mainly localized to nuclear speckles termed pol II foci [33, 34], which overlie three spatially connected nuclear compartments, perichromatin fibrils, interchromatin granule clusters (ICGCs), and Cajal bodies [35]
Cajal bodies are initial assembly sites for pol II transcription machineries [35], which are delivered to ICGCs for storage and subsequently transported to active transcription sites located at the periphery of perichromatin fibrils, which represent nascent transcripts [35, 36]
Summary
Plasmid Constructs—The bait plasmid, pGBKT7-KLF5, was constructed by fusing the coding region of mouse KLF5 [5] to the GAL4 DNA-binding domain present in the pGBKT7 plasmid (Clontech). The pGL3-mPIAS1 luciferase reporter plasmid was generated by cloning 2 kb of the mouse PIAS1 promoter from genomic DNA using PCR. To generate the KLF5 luciferase reporter plasmid, pGL3-mKLF5, a 1.6-kb mouse KLF5 promoter was amplified from a bacterial artificial chromosome clone, RPCI-23 302C3 (ResGen, Invitrogen), and inserted into pGL3-Basic (Promega). The bait and prey were co-transformed into AH109, and the specificity of the interactions was confirmed by growth on SD/Ϫade/Ϫhis/Ϫleu/Ϫtrp plates. The various KLF5 bait and PIAS1 prey plasmids were cotransformed into AH109, and interactions were confirmed by growth on SD/Ϫade/Ϫhis/Ϫleu/Ϫtrp in the absence or presence of X-gal. The immune complexes were washed four times, and the precipitated proteins were detected by Western blotting with a monoclonal anti-HA (Covance), anti-FLAG (Sigma), or rabbit polyclonal anti-KLF5 antibody (raised against residues 95–111 of mouse KLF5). The symbols used are as follows: ϩϩ, the prey is primarily localized to the nucleus; ϩ, the prey has been reported to be localized to the nucleus the nucleus is not the primary location; (Ϫ), the prey is localized in the cytoplasm but in a perinuclear location
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
