Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of Receptor-regulated Smads (R-Smads) to Ensure Transforming Growth Factor ß (TGFß)-mediated Germ Layer Induction in Xenopus Embryos.

Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos.

Nucleosomes correlate with in vivo progression pattern of de novo methylation of p16 CpG islands in human gastric carcinogenesis.

BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP) assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC) samples (36/40) was significantly higher than that observed in gastritis (19/45) or normal samples (7/13) (P<0.01). Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01). In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.

[Downregulation of chromobox protein homolog 7 expression in multiple human cancer tissues].

OBJECTIVE: To investigate the relationship between chromobox protein homolog 7 (cbx7) expression and the occurrence and development of colorectal carcinoma (CRC), gastric carcinoma (GC) and hepatocarcinoma (HCC) tissues. METHODS: The samples of neoplastic tissues and the corresponding cutting-edge normal tissues from 22 cases of CRC, 20 cases of GC, 30 cases of HCC were surgically collected. Level of cbx7 mRNA was detected with a fluorescent quantitative RT-PCR assay, and the correlationship among expression of cbx7 mRNA, the patients' clinicopathologic features and the surviving time after surgery was analyzed. RESULTS: The relative copy number of cbx7 mRNA in carcinomas and the normal tissues was 0.010 ± 0.015 vs 0.053 ± 0.042 for CRCs, 0.197 ± 0.195 vs 1.891 ± 1.254 for GCs, and 0.008 ± 0.008 vs 0.030 ± 0.021 for HCCs, respectively. Compared with the corresponding normal tissues, cbx7 expression was significantly downregulated in CRCs, GCs, and HCCs (t = -7.351, -5.417 and -6.680, respectively, P < 0.01). The expression of cbx7 mRNA in CRCs had significant differences not only between two age groups (the relative copy number of cbx7 mRNA in age > 55 group was 0.007 ± 0.015, but 0.017 ± 0.012 in age ≤ 55 group, t = -2.586, P = 0.022); but also between vascular embolus-positive and negative groups (the level of cbx7 mRNA in positive and negative group was 0.022 ± 0.021 vs 0.006 ± 0.011, t = -3.175, P = 0.010). The area under the receiver operating characteristics (ROC) curve is 0.769 (P = 0.033). when the Cut-off value of the relative copy number of cbx7 mRNA was 0.002 in CRCs. The values less-than 0.002 were defined as low expression. The CRC patients with low expression of cbx7 had a shorter overall survival time; whose 5 years survival rate was only 30.8% (4/13); while the rate was 77.8% (7/9) in high expression of cbx7 group. The difference had statistical significance (χ(2) = 4.329, P = 0.037). The similar differences could not be found among GC and HCC patients. CONCLUSION: Downregulation of cbx7 expression was very common among multiple carcinomas cases, and the downregulation influenced the prognosis of CRC patients.

Polycomb CBX7 directly controls trimethylation of histone H3 at lysine 9 at the p16 locus.

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter.