Emiko Kondo

The Thymine DNA Glycosylase MBD4 Represses Transcription and Is Associated with Methylated p16INK4a and hMLH1 Genes

ABSTRACT Epigenetic silencing through methyl-CpG (mCpG) is implicated in many biological patterns such as genome imprinting, X chromosome inactivation, and cancer development. In this process, the mCpG binding domain (MBD) proteins play an essential role in transmitting epigenetic information to downstream regulatory proteins. Among the five MBD proteins identified so far, MBD4 has been the only exception; it has long been thought to be a DNA repair protein. Herein we demonstrate that MBD4 has the ability to repress transcription through mCpG. Transcriptional repression by the MBD4 is histone deacetylase (HDAC) dependent, and MBD4 directly binds to Sin3A and HDAC1 at three central regions that overlap transcriptional repression domains. Furthermore, a chromatin immunoprecipitation assay clearly shows that MBD4 binds to hypermethylated promoters of the p16 INK4a and hMLH1 genes. These results suggest that MBD4 is one of the essential components involved in epigenetic silencing in cancer and its repair activity is necessary for the maintenance of hypermethylated promoters.

The Expression of S100A4 in Human Pancreatic Cancer Is Associated With Invasion

Objectives Pancreatic cancer is one of the most lethal malignancies; its poor prognosis is strongly associated with invasion and metastasis. Expression of S100A4 has been reported to correlate with poor prognosis in various cancers. We have investigated the role of S100A4 in pancreatic cancer tumorigenesis and its clinicopathologic significance. Methods Protein expression of S100A4 was examined by Western blot in pancreatic cancer cell lines and a human pancreatic ductal epithelium cell line, HPDE-6. Then the expressions of S100A4, TP53, and CD133 were examined immunohistochemically in resected specimens from 83 patients with pancreatic cancer to clarify their clinicopathologic significance. Survival analyses were performed using the Kaplan-Meier method and the Mantel-Cox method. Results Forty-eight (58%) of 83 patients with pancreatic cancer positively expressed S100A4, and 50 (60%) and 29 (36%) patients positively expressed TP53 and CD133, respectively. S100A4 expression was significantly correlated with perineural invasion (P = 0.029) and invasion pattern (P = 0.001). Neither TP53 nor CD133 expression showed significant correlations with any other parameters. Conclusions Our present results suggest that S100A4 plays an important role in the invasiveness, particularly with perineural invasion and invasion pattern, of pancreatic cancer. Development of new strategies targeting S100A4 or its downstream effectors is warranted.

Methyl-CpG targeted transcriptional activation allows re-expression of tumor suppressor genes in human cancer cells

DNA methylation and histone modifications are both major features of the epigenetic silencing seen at tumor suppressor genes (TSGs) in cancer. DNA methylation inhibitors, but not, in general, histone deacetylase, can reactivate TSGs. However, DNA methylation inhibitors frequently upregulate genes whose promoters remain unmethylated. Herein we demonstrated that the methyl-CpG targeted transcriptional activation (MeTA), which allows re-expression of TSGs without DNA demethylation, is widely seen in human cancer. We further analyzed MeTA and found that transcriptional coactivators are recruited at hypermethylated promoter regions of TSGs using the methyl-CpG binding domain (MBD). Reactivation of MLH1 by MeTA accompanied acetylation of histone H3 lysine 9/14 at the promoter region. Furthermore, all ten genes analyzed in three cell lines were reactivated by the effect of MeTA. Our present results lead to an efficient way to search for transcriptionally silenced genes with highly methylated CpG islands, particularly TSGs in cancer and developmentally important genes in embryonic stem cells.

Infrequent mutation of APC, AXIN1, and GSK3B in human pituitary adenomas with abnormal accumulation of CTNNB1

We analyzed mutation of the APC, AXIN1, and GSK3genes in 14 pituitary adenomas with abnormal nuclear accumulations of CTNNB1. These tumors did not harbor mutation of the CTNNB1 gene. The genes analyzed encode proteins associated with ubiquitin-mediated degradation of CTNNB1. Although the regions encoding functional domains of these protein products were analyzed, no significant genetic alterations were found. Furthermore, the antibody for the C-terminus of APC detected normal expression of the APC protein in these pituitary adenomas. Our present results imply that an unknown mechanism(s) accelerates the accumulation of CTNNB1 that plays an important role in the pathogenesis of human pituitary adenomas. However, the possibility that mutation of regions outside of our survey or epigenetic mechanism play an important role cannot be excluded.

Methyl-CpG targeted recruitment of p300 reactivates tumor suppressor genes in human cancer cells

Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor suppressor genes (TSGs) in cancer. We have previously shown that the methyl-CpG targeted transcriptional activation (MeTA) that allows re-expression of TSGs in human cancer cells is accomplished by combining a methyl-CpG binding domain (MBD) with a NFkappaB transcriptional activation domain (AD), accompanied by histone H3K9/K14 acetylation. Herein we demonstrate that p300 histone acetyltransferase (HAT), one of the NFkappaB (AD)-associated coactivators, reactivates epigenetically silenced MLH1 in 293T cells. Interestingly, the HAT domain of p300 is not essential for the reactivation of MLH1; instead, the C-terminal transactivation domain (C-TAD) but not the N-terminal one (N-TAD) reactivates MLH1. Furthermore, all ten of the cancer-related genes analyzed in three types of cancer cells were reactivated by the effect of p300 linked to MBD. These results demonstrate that it is possible to reactivate epigenetically silenced TSGs in human cancer cells by direct targeting of a transcriptional coactivator at highly methylated promoters.