We have developed a method for mapping unmethylated sites in the human genome based on the resistance of TspRI-digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease HpaII, followed by ExoIII and single-strand DNA nuclease allowed removal of DNA fragments containing unmethylated HpaII sites. We then used array comparative genomic hybridization (CGH) to map the sequences depleted by these procedures in human genomes derived from five human tissues, a primary breast tumor, and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5Ј end of widely expressed genes, including promoter, first exon, and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas the primary tumor exhibits a pattern between those of the normal tissue and the cell lines. A striking characteristic of tumor cell lines is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene-poor regions containing novel tissue-specific genes. Correlation with microarray analysis shows that genes with a hypomethylated sequence 2 kb up-or downstream of the transcription start site are highly expressed, whereas genes with extensive intragenic and 3Ј untranslated region (UTR) hypomethylation are silenced. The method described herein can be used for large-scale screening of changes in the methylation pattern in the genome of interest.
Epigenetic organization represents an important regulation mechanism of gene expression. In this work, we show that the mouse p53 gene is organized into two epigenetic domains. The first domain is fully unmethylated, associated with histone modifications in active genes, and organized in a nucleosome-free conformation that is deficient in H2a/H2b, whereas the second domain is fully methylated, associated with deacetylated histones, and organized in a nucleosomal structure. In mitotic cells, RNA polymerase is depleted in domain II, which is folded into a higher-order structure and is associated with H1 histone, whereas domain I conformation is preserved. Similar results were obtained for cells treated with inhibitors of associated regulatory factors. These results suggest that depletion of RNA polymerase II is the result of a physical barrier due to the folding of chromatin in domain II. The novel chromatin structure in the first domain during mitosis also suggests a mechanism for marking active genes in successive cell cycles.DNA methylation and chromatin organization constitute the major epigenetic control mechanisms of eukaryotic gene expression (14,16,23,28). Methylation of sequences in the promoter region has been shown to repress transcription through the recruitment of methylcytosine-binding protein and chromatin-remodeling enzymes (2, 5). Gene expression is also regulated by histone modifications that alter the conformation of chromatin (13,29,44). In spite of our knowledge of the role of epigenetic modifications in the promoter region, few studies have addressed the epigenetic modifications in the intragenic region and the role of intragenic epigenetic modifications in gene expression. In order to elucidate the biological functions of intragenic epigenetic modifications in gene expression, we analyzed the epigenetic organization of the entire gene region of the mouse p53 gene with respect to DNA methylation, histone modifications, association with regulatory factors, chromatin folding, and transcription. The p53 gene was found to be organized into two epigenetic domains with differential DNA methylation characteristics, histone modifications, and compositions. We showed that in mitotic cells and in cells treated with inhibitors, the loss of RNA polymerase II (pol II) and regulatory factors in the second epigenetic domain was associated with higher-order folding and association with H1 histone in this domain. This result suggests that higher-order folding provides a physical barrier for the elongation of transcription into the second domain. The first domain remained in a loose chromatin conformation, in association with regulatory factors, and deficient in H2a/H2b in mitotic cells, suggesting that this special chromatin organization serves as a memory of active genes to be transcribed in the successive phases of the cell cycle. MATERIALS AND METHODSCell culture and transfection. PT67 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1ϫ gentamicin. ...
We have isolated and characterized an isoform of protein kinase Chk1 gene from rat liver and a rat liver cDNA library by 5-rapid amplification of cDNA ends. The gene (Cil) contains the C-terminal region of the Chk1 gene, but the 5-end is derived from a sequence in the intron of Chk1 preceding the C-terminal domain by differential RNA splicing. The kinase domain of Chk1 gene is absent in this isoform. Tissue RNA and protein blot analyses indicated that Cil was specifically expressed only in rat liver, and its expression increased with liver development. Expression of Cil was found to be reduced in three rat hepatoma cell lines examined. A promoter trap experiment suggested that a promoter was located in the intron preceding the C-terminal domain of Chk1, and transcription from this novel promoter generated the new 5 noncoding exon of Cil. Thus Cil was generated by both alternate promoter usage and differential RNA splicing. UV irradiation induced caffeine-sensitive phosphorylation of both Chk1 and Cil at Ser-345 in Chk1 and its equivalent site in Cil, implying a role for ATR kinase in the phosphorylation of both proteins. We demonstrated the interaction between the kinase domain of Chk1 and Cil using a yeast two-hybrid assay and pull-down technique. In contrast to the effect of Chk1, Cil was found to decrease the transactivating function of p53, and the S63A mutation of Cil abolished this effect. These results suggest that Cil may serve as a dominant negative competitor of Chk1 as suggested previously.Transitions in cell cycle are under the surveillance of regulatory pathways called checkpoints. One of the checkpoints is to ensure the integrity of the genome before entering mitosis (1-6). The mitotic cell cycle checkpoints are conserved from yeast to mammals, and the key target of this surveillance is the Cdc2-cyclin B complex that phosphorylates a number of proteins involved in mitotic processes such as proper chromosome segregation and nuclear disassembly (1, 7-10). When DNA is damaged or DNA replication is unfinished Cdc2-cyclin B is inactivated through inhibitory phosphorylation of Cdc2 by Wee1 and Myt1 kinases (5,11,12). The inhibition is reversed by Cdc25 after completion of DNA replication or repair by removing the inhibitory phosphorylation.
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