Erythroid Kruppel-like Factor Regulates the G1 Cyclin Dependent Kinase Inhibitor p18INK4c

Erythroid Kruppel-like factor (EKLF, KLF1) is an essential erythroid cell specific C2H2 zinc finger transcription factor that binds CACC box elements in promoters and distant regulatory elements to activate transcription. Forced expression of EKLF arrests cell division. The cyclin dependent kinase (Cdk) inhibitor p18INK4c was identified as a potential novel EKLF target gene from an expression profiling study. The p18INK4c protein functions as an inhibitor of Cdk4 and Cdk6 activity during early G1 phase of the cell cycle, thus acting as a physiological brake on cell division. We confirmed p18INK4c is downregulated in EKLF null mice by real-time PCR and Western blotting, and identified three closely associated and highly conserved EKLF binding sites (CCNCNCCCN) approximately 1 kb upstream of the p18INK4c transcriptional start site. We showed that EKLF binds to one of these elements by gel shift assay and demonstrated this site is capable of driving EKLF dependent transcription. We also determined by chromatin immunoprecipitation (ChIP) that this region of the p18INK4c promoter is bound by EKLF in erythroid cells. Thus, EKLF is a direct regulator of p18INK4c gene expression, and much of EKLF's role in driving erythroid cell differentiation may occur via p18INK4c.