Kaiso Represses the Cell Cycle Gene cyclin D1 via Sequence-Specific and Methyl-CpG-Dependent Mechanisms

Kaiso is the first member of the POZ family of zinc finger transcription factors reported to bind DNA with dual-specificity in both a sequence- and methyl-CpG-specific manner. Here, we report that Kaiso associates with and regulates the cyclin D1 promoter via the consensus Kaiso binding site (KBS), and also via methylated CpG-dinucleotides. The methyl-CpG sites appear critical for Kaiso binding to the cyclin D1 promoter, while a core KBS in close proximity to the methyl-CpGs appears to stabilize Kaiso DNA binding. Kaiso's binding to both sites was demonstrated in vitro using electrophoretic mobility shift assays (EMSA) and in vivo using Chromatin immunoprecipitation (ChIP). To elucidate the functional relevance of Kaiso's binding to the cyclin D1 promoter, we assessed Kaiso overexpression effects on a minimal cyclin D1 promoter-reporter that contains both KBS and CpG sites. Kaiso repressed this minimal cyclin D1 promoter-reporter in a dose-dependent manner and transcriptional repression occurred in a KBS-specific and methyl-CpG-dependent manner. Collectively our data validates cyclin D1 as a Kaiso target gene and demonstrates a mechanism for Kaiso binding and regulation of the cyclin D1 promoter. Our data also provides a mechanistic basis for how Kaiso may regulate other target genes whose promoters possess both KBS and methyl-CpG sites.