Modified multiple primer extension method.

A multiple primer extension (MPEX) was originally developed for the hybridization, extension, and amplification of a DNA template on a planar substrate by Kinoshita et al. in 2006. Herein we present a modified MPEX method refined by our group for single nucleotide polymorphism (SNP) detection. In this method, hybridization and extension reactions are performed on a plastic S-BIO PrimeSurface substrate, with a biocompatible polymer. Its surface chemistry offers extraordinarily stable thermal properties, as well as chemical properties advantageous for enzymatic reactions on the surface. To visualize allele-specific PCR products on the surface, biotin-dUTP was incorporated into newly synthesized complementary strands during the extension reaction. The products were ultimately detected by carrying out a colorimetric reaction with a substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. We have further successfully combined this method with multiplex PCR. We demonstrate the advantages of this combined method by analyzing representative SNPs on different linkage disequilibrium blocks of the micro opioid receptor gene (OPRM1), which is a marker gene for pain threshold.

KAP1-independent transcriptional repression of SCAN-KRAB-containing zinc finger proteins.

The Krüppel-associated box-containing zinc finger gene family (KRAB-ZNF) is one of the largest gene families of transcriptional factors in the human genome. Although the functions of most of these genes remain to be determined, it is known that KRAB-mediated transcriptional repression requires a direct interaction with the KAP1 co-repressor. By mammalian one- or two-hybrid experiments in HEK293 cells, we compared transcriptional repression activities of 61 human KRAB-ZNFs. The results showed that six SCAN-KRAB-containing ZNFs are KAP1-independent transcriptional repressors whose SCAN-KRAB domain is unable to associate with KAP1 despite retaining transcriptional repression activity. Transcriptional repression activities of the SCAN-KRAB domain of KAP1-independent KRAB-ZNFs are not influenced by depletion of endogenous KAP1 levels by small interfering RNA. Although the mechanism by which KAP1-independent KRAB-ZNFs repress transcriptional activity remains to be elucidated, it appears that there may be a pathway for transcriptional repression that does not involve KAP1. These results provide new insight into the functions of the members of the KRAB-ZNF family.

PCDH24-induced contact inhibition involves downregulation of beta-catenin signaling.

Elevated expression of the protocadherin LKC (PCDH24) in HCT116 colon carcinoma cells has been shown to induce contact inhibition, thereby completely abolishing tumor formation in vivo (Carcinogenesis, 2002; 23(7):1139-1148). To clarify the molecular mechanism behind this effect, we performed 2-DE/MS and DNA microarray analyses in order to compare protein and gene expression patterns of parental HCT116 and PCDH24-expressing HTC116 derivative cells. The data revealed drastic changes in phenotypic markers between parental and PCDH24-expressing cells. We found that in PCDH24-expressing cells beta-catenin, a major player in TCF/lef signaling, is retained in a submembranous location. beta-catenin retention coincided with a subsequent decrease in downstream targets of beta-catenin such as CD44, PLAUR, Myc, cyclin D1 and Met. From these findings we propose a novel model for the suppression of beta-catenin signaling by PCDH24 that leads to contact inhibition.

The novel protein complex with SMARCAD1/KIAA1122 binds to the vicinity of TSS.

The SMARCAD1/KIAA1122 protein is structurally classified into the SWI2/SNF2 superfamily of DNA-dependent ATPases that are catalytic subunits of chromatin-remodeling complexes. Although the importance of other members of the SWR1-like subfamily in chromatin remodeling (EP400, INOC1, and SRCAP) has already been elucidated, the biological function of SMARCAD1/KIAA1122 in transcriptional regulation remains to be clarified. To gain insight into the role of this protein, we generated a specific antibody against SMARCAD1/KIAA1122 and used it for chromatin and protein immunoprecipitation assays. We employed high-resolution genome tiling microarrays in chromatin immunoprecipitation and found the binding sites of SMARCAD1/KIAA1122 in the vicinity of the transcriptional start site of 69 candidate target genes. In the protein immunoprecipitation assay, we found that endogenous SMARCAD1/KIAA1122 binds with TRIM28, a recently highlighted transcriptional regulator in the cancer field. From these findings, we propose a novel model for gene regulation via the SMARCAD1/KIAA1122 protein complex.