Anti-MBD1 monoclonal antibody, clone 3C21

Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin. (c) 2006 Elsevier Inc. All rights reserved.

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5' CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in I out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of IS (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P < 0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (-300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.