Gardening the genome: DNA methylation in Arabidopsis thaliana.

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading.

Plants use siRNAs to target cytosine DNA methylation to both symmetrical CG and nonsymmetrical (CHG and CHH) sequence contexts. DNA methylation and siRNA clusters most frequently overlap with transposons in the Arabidopsis thaliana genome. However, a significant number of protein-coding genes also show promoter DNA methylation, and this can be used to silence their expression. Loss of the majority of non-CG DNA methylation in drm1 drm2 cmt3 triple mutants leads to developmental phenotypes. We identified the gene responsible for these phenotypes as SUPPRESSOR OF drm1 drm2 cmt3 (SDC), which encodes an F-box protein and possesses seven promoter tandem repeats. The SDC repeats show a unique silencing requirement for non-CG DNA methylation directed redundantly by histone methylation and siRNAs, and display spreading of siRNAs and methylation beyond the repeated region. In addition to revealing the complexity of DNA methylation control in A. thaliana, SDC has important implications for how plant genomes utilize gene silencing to repress endogenous genes.

RNAi, DRD1, and histone methylation actively target developmentally important Non-CG DNA methylation in Arabidopsis

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes. © 2006 Chan et al.

Comparative analysis of dna methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

Previous reports have shown that DNA methylation profiles within primary human malignant tissues are altered when these cells are transformed into cancer cell lines. However, it is unclear if similar differences in DNA methylation profiles exist between DNA derived from peripheral blood leukocytes (PBLs) and corresponding Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs). To assess the utility of LCLs as a resource for methylation studies we have compared DNA methylation profiles in promoter and 5' regions of 318 genes in PBL and LCL sample pairs from patients with type 1 diabetes with or without nephropathy. We identified a total of 27 (similar to 8%) genes that revealed different DNA methylation profiles in PBL compared with LCL-derived DNA samples. In conclusion, although the profiles for most promoter regions were similar between PBL-LCL pairs, our results indicate that LCL-derived DNA may not be suitable for DNA methylation studies at least in diabetic nephropathy.

DNA methylation profiling in cell models of diabetic nephropathy

We have previously identified differentially expressed genes in cell models of diabetic nephropathy and renal biopsies. Here we have performed quantitative DNA methylation profiling in cell models of diabetic nephropathy. Over 3,000 CpG units in the promoter regions of 192 candidate genes were assessed in unstimulated human mesangial cells (HMCs) and proximal tubular epithelial cells (PTCs) compared to HMCs or PTCs exposed to appropriate stimuli. A total of 301 CpG units across 38 genes (similar to 20%) were identified as differentially methylated in unstimulated HMCs versus PTCs. Analysis of amplicon methylation values in unstimulated versus stimulated cell models failed to demonstrate a >20% difference between amplicons. In conclusion, our results demonstrate that specific DNA methylation signatures are present in HMCs and PTCs, and standard protocols for exposure of renal cells to stimuli that alter gene expression may be insufficient to replicate possible alterations in DNA methylation profiles in diabetic nephropathy.

The tumor suppressor gene DLEC1 is frequently silenced by DNA methylation in hepatocellular carcinoma and induces G1 arrest in cell cycle

Background/Aims: The chromosome locus 3p21.3 is a

The topography of DNA methylation in the non-neoplastic colonic mucosa surrounding colorectal cancers

The degree of gene hypermethylation in non-neoplastic colonic mucosa (NNCM) is a potentially important event in the development of colorectal cancer (CRC), particularly for the subgroup with a CpG island methylator phenotype (CIMP). In this study, we aimed to use an unbiased and high-throughput approach to evaluate the topography of DNA methylation in the non-neoplastic colonic mucosa (NNCM) surrounding colorectal cancer (CRC). A total of 61 tissue samples comprising 53 NNCM and 8 tumor samples were obtained from hemicolectomy specimens of two CRC patients (Cases 1 and 2). NNCM was stripped from the underlying colonic wall and samples taken at varying distances from the tumor. The level of DNA methylation in NNCM and tumor tissues was assessed at 1,505 CpG sites in 807 cancer-related genes using Illumina GoldenGate® methylation arrays. Case 1 tumor showed significantly higher levels of methylation compared to surrounding NNCM samples (P?