Functional And Molecular Mechanism Study Of A KRAB-containing Zinc Finger Protein ZNF268in The Development Of Human Cervical Cancer

Zinc finger proteins are the most common transcription factors in Eukaryotes. About one thid of Zinc finger proteins contain a transcription repressor domain KRAB at the N-terminus (KRAB-ZNF). KRAB is found only in tetrapod vertebrate genomes and the number increases as organisms evolve, suggesting this type of protein evolved to play crucial role for higher organism development. However, little was known of the functions of KRAB-ZNF proteins. ZNF268is a typical KRAB containing zinc finger protein identified by our laboratory. Previous studies suggest that ZNF268is implicated in human fetal liver development, blood cell development and hematological diseases. However, the function of ZNF268remains largely unknown. To investigate the function of ZNF268in human development, we first profiled the expression pattern of total ZNF268protein in various human normal and cancer tissues. The expression patterns suggest that ZNF268may be a multifunctional molecule that plays different roles in different tissues. Take cervical tissue for example, we observe higher expression of total ZNF268in squamous cervical cancer (SCC) than in normal cervical tissue and we further demonstrate that ZNF268b2shows higher expression than ZNF268a in SCC, suggesting that ZNF268b2levels contribute to the molecular mechanism of ZNF268action in cervical cancer development.To investigate the effort of the high expression of ZNF268in cervical cancer tissues on cervical carcinogenesis, we establish ZNF268knockdown HeLa cells. Knockdown of ZNF268suppresses HeLa cell growth, reduces colony formation, causes cell cycle arrest at the G0/G1phase, and increases sensitivity to TNFa-induced apoptosis. In addition, HeLa cell growth in xenograft nude mice is suppressed by ZNF268knockdown. These results suggest that ZNF268overexpression may contribute to human cervical cancer development.NF-κB signalling been implicated in the development and progression of various cancers. In our study, we observe a high frequency of NF-κB activation in ZNF268overexpressing cervical cancer tissues, suggesting that ZNF268may act as a regulator of NF-κB signaling in cervical cancer development. Furthermore, ZNF268b2promotes TNFa induced NF-κB pathway by interaction with the key kinase IKK complex and ZNF268knockdown HeLa cells also display impaired NF-κB activity in vivo xenografts. Finally, reconstitution of NF-κB activity restores proliferation in ZNF268knockdown HeLa cells. These results suggest that ZNF268functions by NF-κB signalling.ZNF268encodes ZNF268a and ZNF268b2isoforms. ZNF268a contains the KRAB and zinc finger domain while ZNF268b2contains only the zinc finger domain. Our results demonstrate that ZNF268a localizes in the nucleus and ZNF268b2distributes both in the cytoplasm and nucleus, suggest that the KRAB maybe a potential nuclear localization signal (NLS). We further characterize KRAB as a functional and universal NLS for zinc finger proteins. The A box of KRAB plays the main role for the NLS activity and contains most of the conserved functional amino residues (Va19, Valll, Phe13, Glu16, Glul7, Trp18, Leu40and Lys49) essential for the NLS activity. Finally, we demonstrate interaction with KAP1mediates the NLS activity of KRAB.Taken together, ZNF268is overexpressed in human cervical cancer and its knockdown suppresses HeLa cell growth, suggesting that ZNF268may contribute to human cervical cancer development and may serve as a novel therapeutic target. In addition, we identify and characterize KRAB, the repressor domain as a novel and universal NLS, which provides novel mechanistic information on nuclear localization of KRAB-ZNF proteins and helps us understand the functions of KRAB-ZNF proteins.