Role of Kruppel-Like Factors in gamma-globin regulation through the CACCC promoter element

The CACCC element is critical for the developmental regulation of the human gamma-globin and beta-globin genes; therefore studies to identify transcription factors that bind this region to alter gene transcription are essential. In this study, we initially screened for candidate Kruppel-Like Factors (KLFs) that might be involved in gamma-globin regulation. Based on the microarray expression profiling study and gamma-globin drug induction, KLF4 emerged as a potential regulator of gamma-globin transcription. To characterize the role of this factor further, siRNA-mediated gene silencing was performed in K562 cells (expressing epsilon- and gamma-globin genes). A 52% reduction in KLF4 expression produced a 56% attenuation in gamma-globin transcription (p<0.05). Furthermore, enforced expression of pMT3-KLF4 (20mug) augmented endogenous gamma-globin expression 2-fold (p<0.01). Collectively, these studies suggest KLF4 acts as a trans-activator of gamma-globin. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. siKLF4 treatment at day 11 and enforced KLF4 expression at day 28 cnfirmed the regulatory role of KLF4 in primary erythroid cells. Since CREB binding protein (CBP) is known to associate with KLF1, 4 and 13, we also tested its role in KLF4 mediated gamma-globin gene regulation. Electrophoretic mobility shift assay established in vitro binding of KLF4 at the gamma-CACCC region; parallel studies at beta-CACCC did not reveal any KLF4 binding, highlighting a gene specific interaction. Interestingly, CBP bound to both these CACCC domains. Co-immunoprecipitation assay demonstrated endogenous interaction between KLF4 and CBP. Furthermore, chromatin immunoprecipitation assay (ChIP) and a subsequent sequential-ChIP confirmed their co-localization at the gamma-CACCC region. Finally we performed luciferase-reporter based transient studies which demonstrated KLF4 trans-activates gamma-globin promoter activity recapitulating our earlier data. In contrast, CBP enforced expression resulted in repression which was rather unexpected. Our data supports a model of antagonistic interactions of KLF4/CBP in gamma-globin gene regulation.