The phosphotyrosyl phosphatase activator gene is a novel p53 target gene

The minimal promoter of the phosphotyrosyl phosphatase activator (PTPA) gene, encoding a regulator of protein phosphatase 2A contains two yin-yang 1 (YY1)-binding sites, positively regulating promoter activity. We now describe a role for p53 in the regulation of PTPA expression. Luciferase reporter assays in Saos-2 cells revealed that p53 could down-regulate PTPA promoter activity in a dose-dependent manner, whereas four different p53 mutants could not. The p53-responsive region mapped to the minimal promoter. Overexpression of YY1 reverses the repressive effect of p53, suggesting a functional antagonism between p53 and YY1. The latter does not involve competition for YY1 binding, but rather direct control of YY1 function. Inhibition of PTPA expression by endogenous p53 was demonstrated in UVB-irradiated HepG2 cells, both on the mRNA and protein level. Also basal PTPA levels are higher in p53-negative (Saos-2) versus p53-positive (HepG2, U2OS) cells, suggesting "latent" p53 can control PTPA expression as well. The higher PTPA levels in U2OS cells, programmed to overexpress constitutively a dominant-negative p53 mutant, corroborate this finding. Thus, PTPA expression is negatively regulated by p53 in normal conditions and in conditions where p53 is up-regulated, via an as yet unknown mechanism involving the negative control of YY1.     

YY1's role in DNA methylation of Peg3 and Xist

Unusual clusters of YY1 binding sites are located within several differentially methylated regions (DMRs), including Xist, Nespas and Peg3, which all become methylated during oogenesis. In this study, we performed conditional YY1 knockdown (KD) to investigate YY1''s roles in DNA methylation of these DMRs. Reduced levels of YY1 during spermatogenesis did not cause any major change in these DMRs although the same YY1 KD caused hypermethylation in these DMRs among a subset of aged mice. However, YY1 KD during oogenesis resulted in the loss of DNA methylation on Peg3 and Xist, but there were no changes on Nespas and H19. Continued YY1 KD from oogenesis to the blastocyst stage caused further loss in DNA methylation on Peg3. Consequently, high incidents of lethality were observed among embryos that had experienced the reduced levels of YY1 protein. Overall, the current study suggests that YY1 likely plays a role in the de novo DNA methylation of the DMRs of Peg3 and Xist during oogenesis and also in the maintenance of unmethylation status of these DMRs during spermatogenesis.     

EGFR基因启动子区甲基化状态分析

表皮生长因子受体(epidermal growth factor receptor,EGFR)是HER/ERB-B跨膜受体激酶家族成员之一.EGFR的过表达促进细胞的增殖、存活和迁移,与许多实体瘤病人的低存活率相关.EGFR的表达受其启动子DNA甲基化调控.EGFR的转录沉默与CpG岛高甲基化相关.EGFR基因5′调控区包括1个富含GC的启动子,缺保守序列TATA盒和CAAT盒,有多个位点可以起始转录.本实验运用Bisulfite Sequencing PCR(BSP)方法检测了2种肿瘤细胞HeLa(EGFR+)和K562(EGFR)EGFR基因-1300～+600的甲基化状态.所检测目的片段共包含178个CpG位点.发现EGFR阳性与EGFR阴性两种细胞系的甲基化状态不同:宫颈癌细胞系HeLa转录起始点附近包括第一外显子区(-244～+91)处于非甲基化状态,白血病细胞系K562转录起始点附近包括第一外显子区呈嵌合性的高甲基化状态.因此,第一外显子比启动子区的甲基化状态更能反映基因的活化状况.     

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.