p53, the most commonly mutated gene in cancer cells, directs cell cycle arrest or induces programmed cell death (apoptosis) in response to stress. It has been demonstrated that p53 activity is up-regulated in part by posttranslational acetylation. In agreement with these observations, here we show that mammalian histone deacetylase (HDAC)-1, -2, and -3 are all capable of downregulating p53 function. Down-regulation of p53 activity by HDACs is HDAC dosage-dependent, requires the deacetylase activity of HDACs, and depends on the region of p53 that is acetylated by p300/CREB-binding protein (CBP). These results suggest that interactions of p53 and HDACs likely result in p53 deacetylation, thereby reducing its transcriptional activity. In support of this idea, GST pull-down and immunoprecipitation assays show that p53 interacts with HDAC1 both in vitro and in vivo. Furthermore, a pre-acetylated p53 peptide was significantly deacetylated by immunoprecipitated wild type HDAC1 but not deacetylase mutant. Also, coexpression of HDAC1 greatly reduced the in vivo acetylation level of p53. Finally, we report that the activation potential of p53 on the BAX promoter, a natural p53-responsive system, is reduced in the presence of HDACs. Taken together, our findings indicate that deacetylation of p53 by histone deacetylases is likely to be part of the mechanisms that control the physiological activity of p53.

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

The IE2 gene of human cytomegalovirus has been implicated in the development of coronary restenosis, and the gene product appears to inhibit p53-dependent transactivation. Here we describe an analysis of the IE2-p53 interaction. Repression of p53 function by IE2 requires two separable domains of IE2. The N terminus of IE2 interacts with p53. IE2 has little effect on the ability of p53 to bind specific DNA sequences. Reduction of the transactivation activity of p53 is caused by a transcriptional repression function contributed by the C-terminal domain of IE2. These findings suggest that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53.

T cell factor 4 (TCF4) interacts with ␤-catenin in the WNT signaling pathway and transactivates downstream target genes involved in cancer progression. To identify proteins that regulate TCF4-mediated biological responses, we performed a yeast two-hybrid screen to search for a TCF4-binding protein(s) and found that MAD2B interacts with TCF4. We confirmed that MAD2B is a TCF4-binding protein by co-immunoprecipitation. Using the TOPFLASH reporter assay, we found that MAD2B blocks TCF4-mediated transactivation. The MAD2B binding regions of TCF4 were identified by TCF4 deletion mapping and electrophoretic mobility shift assay analysis. TCF4 and MAD2B interactions abolished the DNA binding ability of TCF4. Knockdown of MAD2B in SW480 colorectal cancer cells led to the conversion of epithelial cells to a mesenchymal fibroblastoid phenotype (epithelial-mesenchymal transdifferentiation). An E-cadherin promoter reporter analysis showed that MAD2B modulates TCF4-mediated E-cadherin expression. MAD2B knockdown blocked E-cadherin expression and significantly induced mesenchymal markers, such as N-cadherin and vimentin. Mesenchymal induction was accompanied by F-actin redistribution and the appearance of a fibroblastoid phenotype. MAD2B knockdown also increased both mRNA and protein levels of Slug, a known TCF4-induced E-cadherin transcriptional repressor. A chromatin immunoprecipitation assay showed that MAD2B silencing enhances the ability of TCF4 to bind the Slug promoter. Thus, MAD2B is a novel TCF4-interacting protein. This study provides the first evidence for the involvement of MAD2B in TCF4-mediated epithelial-mesenchymal transdifferentiation.The WNT signaling cascade is a crucial cell development and growth regulatory pathway (1-3). The key event of WNT signaling is the activation of the T cell factor (TCF) 2 -␤-catenin complex. WNT binding to transmembrane Frizzled receptors is followed by phosphorylation and functional deactivation of glycogen synthase kinase-3␤. Then membrane-associated ␤-catenin is released and stabilized in the cytoplasm. Accumulated ␤-catenin associates with TCFs and translocates into the nucleus, leading to transcriptional activation of downstream target genes (3, 4). Inappropriate activation of the WNT signaling pathway, such as by mutation of adenomatous polyposis coli and glycogen synthase kinase-3␤, is often involved in early premalignant lesions of the intestine and tumorigenesis of several cell types (5). Human germ line mutations in the adenomatous polyposis coli result in familial adenomatous polyposis, which is characterized by multiple intestinal adenomas (6). Up to 80% of colorectal cancers (CRCs) show mutations in both adenomatous polyposis coli alleles (7). Accumulation of ␤-catenin protein and transactivation of a certain set of TCF4 target genes by accumulated ␤-catenin are crucial for colorectal carcinogenesis (8).Within the TCF family proteins, TCF4, which is encoded by the Tcf7L2 gene, is predominantly expressed in CRC cells. TCF4 binds directly to DNA through its high mobil...

We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.