Sisi Qin1, Baozhen Zhang1, Wei Tian1, Liankun Gu1, Zheming Lu1, Dajun Deng1. 1. Beijing Key Laboratory of Carcinogenesis and Translational Research, Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
Abstract
OBJECTIVE: Kaiso is upregulated in many cancers and proposed to bind with both methylated- and unmethylated-DNA in the nucleus as a transcriptional repressor. The objective is to define its subcellular localization in vivo and exact binding DNA sequences in cells. METHODS: Compartmentalization of exogenous Kaiso in cells was tracked with enhanced green fluorescence protein (EGFP) tag. The endogenous Kaiso expression in gastric carcinoma tissue was examined with immunohistochemical staining. Kaiso-DNA binding was tested using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). RESULTS: Kaiso mainly localized in the nucleus of cancer and stromal cells in vivo, but remained in the cytoplasm of cultured cells. Most importantly, nuclear Kaiso can bind with the methylated-CGCG-containing sequence in the CDKN2A promoter, but not with the hydroxymethylated-CGCG sequence in HCT116 cells. CONCLUSIONS: Kaiso locates mainly in the nucleus in vivo where it binds with the methylated-CGCG sequences, but does not bind with the hydroxymethylated-CGCG sequences.
OBJECTIVE: Kaiso is upregulated in many cancers and proposed to bind with both methylated- and unmethylated-DNA in the nucleus as a transcriptional repressor. The objective is to define its subcellular localization in vivo and exact binding DNA sequences in cells. METHODS: Compartmentalization of exogenous Kaiso in cells was tracked with enhanced green fluorescence protein (EGFP) tag. The endogenous Kaiso expression in gastric carcinoma tissue was examined with immunohistochemical staining. Kaiso-DNA binding was tested using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). RESULTS: Kaiso mainly localized in the nucleus of cancer and stromal cells in vivo, but remained in the cytoplasm of cultured cells. Most importantly, nuclear Kaiso can bind with the methylated-CGCG-containing sequence in the CDKN2A promoter, but not with the hydroxymethylated-CGCG sequence in HCT116 cells. CONCLUSIONS: Kaiso locates mainly in the nucleus in vivo where it binds with the methylated-CGCG sequences, but does not bind with the hydroxymethylated-CGCG sequences.
Entities:
Keywords:
CDKN2A; DNA binding; Kaiso; compartmentalization; hydroxymethylation; methylation
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