Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

A non-methylated CpG-rich island associated with the human muscle-specific carbonic anhydrase III gene

A cluster of CpG dinucleotides immediately upstream from exon 1 in the muscle-specific carbonic anhydrase III gene (CAIII) resembles the 'HpaII tiny fragment' (HTF) islands characteristic of mammalian 'housekeeping' genes. Since this CAIII gene shows tissue-specific expression we have carried out a detailed examination of methylation status within the CpG cluster using a polyacrylamide gel/electroblot procedure to extend the range of conventional Southern blotting. None of the clustered CpGs are methylated in DNA from muscle or other somatic tissues or in DNA from spermatozoa although flanking CpGs are methylated. Comparison with a candidate HTF island from the more ubiquitously expressed carbonic anhydrase II gene (CAII) shows that the CAII CpG cluster is markedly more CpG-rich than that from the strictly tissue-specific CAIII gene.     

Comprehensive Biostatistical Analysis of CpG Island Methylator Phenotype in Colorectal Cancer Using a Large Population-Based Sample

Background

The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel.

Metholodology/Principal Findings

DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and β-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.

Conclusions

Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.     

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

DNA methylation as an epigenetic regulator of neural 5-lipoxygenase expression: evidence in human NT2 and NT2-N cells

Increased expression of 5-lipoxygenase is associated with various neuropathologies and may be related to epigenetic gene regulation. DNA methylation in promoter regions is typically associated with gene silencing. We found that human NT2 cells, which differentiate into neuron-like NT2-N cells, express 5-lipoxygenase and we investigated the relationship between 5-lipoxygenase expression and the methylation state of the 5-lipoxygenase core promoter. We used the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor valproate to alter DNA methylation and to induce histone modifications. 5-Lipoxygenase expression and DNA methylation were assayed with RT-PCR and bisulfite genomic sequencing, respectively. Neuronal differentiation of proliferating NT2 precursors decreased 5-lipoxygenase expression. 5-Aza-2'-deoxycytidine increased 5-lipoxygenase mRNA levels only in proliferating cells, whereas valproate increased 5-lipoxygenase mRNA levels in a cell cycle-independent manner. In both precursors and differentiated cells, CpG dinucleotides of the promoter were poorly methylated. In precursors, both 5-aza-2'-deoxycytidine and valproate further reduced the number of methylated CpGs. Moreover, we found evidence for cytosine methylation in CpWpG (W=adenine or thymine) and other asymmetrical sequences; CpWpG methylation was reduced by valproate in NT2-N but not in NT2 cells. This is the first report demonstrating that the dynamics of DNA methylation relates to neural 5-lipoxygenase gene expression.     

The oocyte-specific methylated region of the U2afbp-rs/U2af1-rs1 gene is dispensable for its imprinted methylation

Imprinted genes harbor discrete regions which are differentially methylated in gametes; usually the final differential methylation patterns in adults are established during embryogenesis through modifications of the initial methylation patterns in gametes. Previous reports have shown that a 200-bp region termed region II within the CpG island of the mouse imprinted U2afbp-rs gene is methylated in oocytes but not in sperm, suggesting that this region is a center for the propagation of methylated CpGs on the maternal allele and is also a candidate for an imprinting control element. To determine whether region II is required for the imprinted methylation of this gene at the endogenous locus, we generated mice carrying a deletion of this region. We herein show that parental methylation differences still exist in the CpG island on the region II-deleted allele. These findings suggest that region II is dispensable for the imprinted methylation of the U2afbp-rs gene.     

CpG甲基化与基因调控

CpG双核苷酸中的胞嘧啶甲基化和去甲基化在哺乳动物的基因表达中有重要的调控作用.哺乳动物基因组中有两类启动子:CpG岛启动子和CpG缺乏启动子.两种蛋白质因子通过与甲基化CpG的相互作用影响基因表达,CpG岛在基因组分析中也有广泛的用途.