Clin D1 was never validated as a bona fide Kaiso target gene and it was unknown whether the changes in cyclin D1 mRNA and protein levels were a direct or indirect effect of transcriptional regulation by Kaiso. Our lab has identified numerous CpG dinucleotide-rich regions and three KBSs (at positions 22336, 21067 and +69 in the cyclin D1 promoter, ID#:6842 from the Transcriptional Regulatory Element Database) relative to the transcriptional start site (Figure 1A). As a first step towards 3687-18-1 web validating cyclin D1 as a Kaiso target gene and determining the mechanism by which it is regulated, we examined get Gracillin Kaiso’s ability to bind the human cyclin D1 promoter in vitro. We performed EMSA studies using various bacterially-expressed and purified GST-Kaiso fusion proteins and nine oligonucleotides that individually corresponded to the KBS found at position -1067 and various CpG rich regions of the cyclin D1 promoter (Figures 1, 2 S1). The 21067 KBS oligonucleotide used in Figure 1 possessed the full KBS (TCCTGCNA) and two CpG sites while one of the CpG oligonucleotides (CpG7) used in Figure 2 contained a core KBS (CTGCNA) and three CpG sites (see Figure 2). We employed GST-Kaiso fusion proteins lacking the Kaiso POZ domain for our EMSA studies, since we and others have found that the presence of the POZ domain in most full-length POZ-ZF proteins resulted in weak or no association with DNA in 26001275 vitro, [23] and our unpublished data. In repeated experiments, we found that GST-Kaiso-DPOZ (lacking the N-terminal POZ domain) and GST-Kaiso-ZF domain could bind the 21067 KBS region of the cyclin D1 promoter (Figure 1C, lanes 3 4 ). As expected, no binding was observed with the GST alone or GSTKaiso-DPOZDZF negative controls (Figure 1C, lanes 1 2). To confirm that Kaiso was binding the 21067 region in a KBSspecific manner, three point mutations were introduced into the core KBS (CTGCNA to ATTACA) sequence. When this mutated oligonucleotide (21067 mut.) was tested in EMSA, Kaiso DNA binding was completely abolished (Figure 1C, lanes 7 8). This confirmed that Kaiso was binding directly to the 21067 cyclin D1 promoter region in a KBS-specific manner. To confirm that Kaiso bound the 21067 KBS region of the cyclin D1 promoter endogenously, we next performed ChIP experiments using chromatin isolated from MCF7 and HCT 116 cells, which express moderate to high levels of Kaiso respectively, and immunoprecipitated the DNA-protein complexes with the Kaiso-specific monoclonal antibody 6F [22]. PCR wasKaiso Binds Specifically to the +69 core KBS Region in a Methyl-CpG Dependent MannerSince Kaiso bound to the methylated CpG7 but not to the nonmethylated CpG7 which possessed a core KBS (CTGCNA) and three single CpG dinucleotides (Figure 2B, compare lanes 13 14), we sought to determine the relevance of this core KBS in the cyclin D1 promoter and whether it contributed to Kaiso’s binding to this probe. EMSA experiments were performed with an oligonucleotide named “+69 core KBS” that encompassed most of the CpG7 probe and seven additional nucleotides at the 39 end. We included the full-length GST-Kaiso fusion protein in these EMSA experiments after determining that full-length Kaiso canKaiso Represses cyclin D1 via KBS and Me-CpG SitesFigure 1. Kaiso binds specifically to the 21067 KBS site of the cyclin D1 promoter in vitro and in vivo. (A) Schematic representation of the ,1748 cyclin D1 promoter fragment showing the 21067 KBS, the +69 core KBS (in blue and underline.Clin D1 was never validated as a bona fide Kaiso target gene and it was unknown whether the changes in cyclin D1 mRNA and protein levels were a direct or indirect effect of transcriptional regulation by Kaiso. Our lab has identified numerous CpG dinucleotide-rich regions and three KBSs (at positions 22336, 21067 and +69 in the cyclin D1 promoter, ID#:6842 from the Transcriptional Regulatory Element Database) relative to the transcriptional start site (Figure 1A). As a first step towards validating cyclin D1 as a Kaiso target gene and determining the mechanism by which it is regulated, we examined Kaiso’s ability to bind the human cyclin D1 promoter in vitro. We performed EMSA studies using various bacterially-expressed and purified GST-Kaiso fusion proteins and nine oligonucleotides that individually corresponded to the KBS found at position -1067 and various CpG rich regions of the cyclin D1 promoter (Figures 1, 2 S1). The 21067 KBS oligonucleotide used in Figure 1 possessed the full KBS (TCCTGCNA) and two CpG sites while one of the CpG oligonucleotides (CpG7) used in Figure 2 contained a core KBS (CTGCNA) and three CpG sites (see Figure 2). We employed GST-Kaiso fusion proteins lacking the Kaiso POZ domain for our EMSA studies, since we and others have found that the presence of the POZ domain in most full-length POZ-ZF proteins resulted in weak or no association with DNA in 26001275 vitro, [23] and our unpublished data. In repeated experiments, we found that GST-Kaiso-DPOZ (lacking the N-terminal POZ domain) and GST-Kaiso-ZF domain could bind the 21067 KBS region of the cyclin D1 promoter (Figure 1C, lanes 3 4 ). As expected, no binding was observed with the GST alone or GSTKaiso-DPOZDZF negative controls (Figure 1C, lanes 1 2). To confirm that Kaiso was binding the 21067 region in a KBSspecific manner, three point mutations were introduced into the core KBS (CTGCNA to ATTACA) sequence. When this mutated oligonucleotide (21067 mut.) was tested in EMSA, Kaiso DNA binding was completely abolished (Figure 1C, lanes 7 8). This confirmed that Kaiso was binding directly to the 21067 cyclin D1 promoter region in a KBS-specific manner. To confirm that Kaiso bound the 21067 KBS region of the cyclin D1 promoter endogenously, we next performed ChIP experiments using chromatin isolated from MCF7 and HCT 116 cells, which express moderate to high levels of Kaiso respectively, and immunoprecipitated the DNA-protein complexes with the Kaiso-specific monoclonal antibody 6F [22]. PCR wasKaiso Binds Specifically to the +69 core KBS Region in a Methyl-CpG Dependent MannerSince Kaiso bound to the methylated CpG7 but not to the nonmethylated CpG7 which possessed a core KBS (CTGCNA) and three single CpG dinucleotides (Figure 2B, compare lanes 13 14), we sought to determine the relevance of this core KBS in the cyclin D1 promoter and whether it contributed to Kaiso’s binding to this probe. EMSA experiments were performed with an oligonucleotide named “+69 core KBS” that encompassed most of the CpG7 probe and seven additional nucleotides at the 39 end. We included the full-length GST-Kaiso fusion protein in these EMSA experiments after determining that full-length Kaiso canKaiso Represses cyclin D1 via KBS and Me-CpG SitesFigure 1. Kaiso binds specifically to the 21067 KBS site of the cyclin D1 promoter in vitro and in vivo. (A) Schematic representation of the ,1748 cyclin D1 promoter fragment showing the 21067 KBS, the +69 core KBS (in blue and underline.