siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Allele-specific DNA methylation analyses associated with siRNAs in Arabidopsis hybrids

Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.     

Mesodermal determination genes: evidence from DNA methylation studies

Mouse embryo cells, primed to differentiate with the hypomethylating agent 5-azacytidine (5-aza-CR), provide an excellent model system in which cellular differentiation can be studied at the molecular level. An inherent advantage of this system is the availability of clonal populations of cells representative of the non-differentiated precursor, those whose determinative state is that of a specific lineage, and the end stage, phenotypically mature cell. Analysis of these cultures at the cellular and molecular level will advance our understanding of requirements for and the mechanism of action of growth modulators as well as the necessity for controlled expression of certain proto-oncogenes. At a more fundamental level, the system can be used to identify, isolate and characterize genes whose expression alters the phenotypic fate of cells.     

Plant phosphoglucose isomerase genes lack introns and are expressed in Escherichia coli

Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Human cytomegalovirus infection is sensitive to the host cell DNA methylation state and alters global DNA methylation capacity

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects and establishes latency in the majority of the human population and may cause fatal infections in immunocompromised patients. Recent data implies a close interaction between HCMV encoded proteins and cellular epigenetic mechanisms such as histone acetylation and deacetylation. In this study, we investigated the interactions between HCMV infection and the DNA methylation machinery in different host cells using several approaches. We found that colon cancer cell line HCT-116 lacking the DNMT1 and DNMT3b methyltransferases was susceptible to HCMV-AD169 infection, while wild-type cells were non-susceptible. Treatment of wild-type HCT-116 cells with 5-azacytidine rendered them susceptible to infection. Further investigation of HCMV infected MRC-5 fibroblasts demonstrated significant global hypomethylation, a phenomenon that was virus strain-specific and associated with the re-localization of DNMT1 and DNMT3b from the nucleus to the cytoplasm. The cytoplasmic accumulation of DNMT1 was also evident in in vitro infected macrophages and in epithelial cells in tissue samples from patients with inflammatory bowel disease and concomitant HCMV infection. Foscavir treatment of virus infected fibroblasts did not affect the majority of the virus induced nuclear exclusion of DNMT1, which suggest that it is dependent on viral IE gene products. In conclusion, HCMV infection results in profound effects on the host cell DNA methylation machinery and is associated with inflammation in vivo. Our results improve the understanding of cytomegalovirus pathogenesis and open the search for new antiviral therapy targets. These findings may also contribute to the further understanding of mechanisms involved in DNA methylation abnormalities in physiological and pathological conditions.     

Viral small interfering RNAs target host genes to mediate disease symptoms in plants

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.     

LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range

Legume-Rhizobium symbiosis is an example of selective cell recognition controlled by host/non-host determinants. Individual bacterial strains have a distinct host range enabling nodulation of a limited set of legume species and vice versa. We show here that expression of Lotus japonicus Nfr1 and Nfr5 Nod-factor receptor genes in Medicago truncatula and L. filicaulis, extends their host range to include bacterial strains, Mesorhizobium loti or DZL, normally infecting L. japonicus. As a result, the symbiotic program is induced, nodules develop and infection threads are formed. Using L. japonicus mutants and domain swaps between L. japonicus and L. filicaulis NFR1 and NFR5, we further demonstrate that LysM domains of the NFR1 and NFR5 receptors mediate perception of the bacterial Nod-factor signal and that recognition depends on the structure of the lipochitin-oligosaccharide Nod-factor. We show that a single amino-acid variation in the LysM2 domain of NFR5 changes recognition of the Nod-factor synthesized by the DZL strain and suggests a possible binding site for bacterial lipochitin-oligosaccharide signal molecules.     

Wolbachia mediate variation of host immunocompetence

Background

After decades during which endosymbionts were considered as silent in their hosts, in particular concerning the immune system, recent studies have revealed the contrary. In the present paper, we addressed the effect of Wolbachia, the most prevalent endosymbiont in arthropods, on host immunocompetence. To this end, we chose the A. vulgare-Wolbachia symbiosis as a model system because it leads to compare consequences of two Wolbachia strains (wVulC and wVulM) on hosts from the same population. Moreover, A. vulgare is the only host-species in which Wolbachia have been directly observed within haemocytes which are responsible for both humoral and cellular immune responses.

Methodology/Principal Findings

We sampled gravid females from the same population that were either asymbiotic, infected with wVulC, or infected with wVulM. The offspring from these females were tested and it was revealed that individuals harbouring wVulC exhibited: (i) lower haemocyte densities, (ii) more intense septicaemia in their haemolymph and (iii) a reduced lifespan as compared to individuals habouring wVulM or asymbiotic ones. Therefore, individuals in this population of A. vulgare appeared to suffer more from wVulC than from wVulM. Symbiotic titer and location in the haemocytes did not differ for the two Wolbachia strains showing that these two parameters were not responsible for differences observed in their extended phenotypes in A. vulgare.

Conclusion/Significance

The two Wolbachia strains infecting A. vulgare in the same population induced variation in immunocompetence and survival of their hosts. Such variation should highly influence the dynamics of this host-symbiont system. We propose in accordance with previous population genetic works, that wVulM is a local strain that has attenuated its virulence through a long term adaptation process towards local A. vulgare genotypes whereas wVulC, which is a widespread and invasive strain, is not locally adapted.