Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing

Proper DNA methylation patterning requires the complementary processes of de novo methylation (the initial methylation of unmethylated DNA sequences) and maintenance methylation (the faithful replication of preexisting methylation). Arabidopsis has two types of methyltransferases with demonstrated maintenance activity: MET1, which maintains CpG methylation and is homologous to mammalian DNMT1, and CHROMOMETHYLASE 3 (CMT3), which maintains CpNpG (N = A, T, C, or G) methylation and is unique to the plant kingdom. Here we describe loss-of-function mutations in the Arabidopsis DOMAINS REARRANGED METHYLASE (DRM) genes and provide evidence that they encode de novo methyltransferases. drm1 drm2 double mutants retained preexisting CpG methylation at the endogenous FWA locus but blocked de novo CpG methylation that is normally associated with FWA transgene silencing. Furthermore, drm1 drm2 double mutants blocked de novo CpNpG and asymmetric methylation and gene silencing of the endogenous SUPERMAN (SUP) gene, which is normally triggered by an inverted SUP repeat. However, drm1 drm2 double mutants did not show reactivation of previously established SUPERMAN epigenetic silenced alleles. Thus, drm mutants prevent the establishment but not the maintenance of gene silencing at FWA and SUP, suggesting that the DRMs encode the major de novo methylation enzymes affecting these genes.     

FON1, an Arabidopsis gene that terminates floral meristem activity and controls flower organ number.

A novel gene that regulates floral meristem activity and controls floral organ number was identified in Arabidopsis and is designated FON1 (for FLORAL ORGAN NUMBER1). The fon1 mutants exhibit normal vegetative development and produce normal inflorescence meristems and immature flowers before stage 6. fon1 flowers become visibly different from wild-type flowers at stage 6, when the third-whorl stamen primordia have formed. The fon1 floral meristem functions longer than does that of the wild type: after the outer three-whorl organ primordia have initiated, the remaining central floral meristem continues to produce additional stamen primordia interior to the third whorl. Prolonged fon1 floral meristem activity also results in an increased number of carpels. The clavata (clv) mutations are known to affect floral meristem activity. We have analyzed the clv1 fon1, clv2 fon1, and clv3 fon1 double mutants. These double mutants all have similar phenotypes, with more stamens and carpels than either fon1 or clv single mutants. This indicates that FON1 and CLV genes function in different pathways to control the number of third- and fourth-whorl floral organs. In addition, to test for possible interactions between FON1 and other floral regulatory genes, we have constructed and analyzed the relevant double mutants. Our results suggest that FON1 does not interact with TERMINAL FLOWER1, APETALA1, APETALA2, or UNUSUAL FLORAL ORGAN. In contrast, normal LEAFY function is required for the expression of fon1 phenotypes. In addition, FON1 and AGAMOUS both seem to affect the domain of APETALA3 function, which also affects the formation of stamen-carpel chimera due to fon1 mutations. Finally, genetic analysis suggests that FON1 interacts with SUPERMAN, which also regulates floral meristem activity.     

拟南芥SUPERMAN基因表观突变的研究进展

本文综述了拟南芥SUPERMAN基因表观突变体clark kent（clk）的表型、基因型、DNA甲基化及表观突变机制等方面的研究进展。主要研究结果有：clk是SUPERMAN(SUP)的等位基因,能遗传但不稳定,其对称位点（CpG和CPXPG）和非对称位点的胞嘧啶高度甲基化,伴随着SUP转录水平的下降;CpG和CPXPG胞嘧啶甲基转移酶分别是METHYLTRANSFERASE1（MET1）和受KRYPTONITE基因调控的CHROMOMETHYLASE3 (CMT3)。 Abstract:The SUPERMAN gene in Arabidopsis has its epigenetic mutants (the clark kent alleles,clk). The phenotype of clk and its genotype and methylated patterns and the epi-mutation mechanisms of SUPERMAN were summarized in the review. Heritable but unstable sup epi-alleles are associated with nearly identical patterns of excess cytosine methylation within the SUP gene and a decreased level of SUP RNA. The methylation of cytosine at CpG and CPXPG is controlled by METHYLTRANSFERASE1（MET1）and CHROMOMETHYLASE3 (CMT3) which is regulated by KRYPTONITE gene,respectively.     

The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size

Most plant organs develop from meristems. Rice FON1, which is an ortholog of Clv1, regulates stem cell proliferation and organ initiation. The point muta-tions, fon1-1 and fon1-2, disrupt meristem balance, resulting in alteration of floral organ numbers and the architecture of primary rachis branches. In this study, we identified two knockout alleles, fon1-3 and fon1-4, generated by T-DNA and Tos17 insertion, respectively. Unlike the previously isolated point mutants, the null mutants have alterations not only of the reproductive organs but also of vegetative tissues, producing fewer tillers and secondary rachis branches. The mutant plants are semi-dwarfs due to delayed leaf emergence, and leaf senescence is delayed. SEM analysis showed that the shoot apical meristems of fon1-3 mutants are enlarged. These results indicate that FON1 controls vegetative as well as reproductive development by regulating meristem size.     

Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.     

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.     

Characterization of the Rice Floral Organ Number Mutant fon3

A spontaneous rice mutant named floral organ number 3 (fon3) had major mutations in floral organ numbers. Genetic analysis indicated that fort3 acted as a single recessive gene. Microscopic observation showed that the number of floral organs infon3 increased centripetally. For example, the number of pistils was the more frequently increased than organs in the outer whorls. Homeotic conversion of lodicules and glumes into palea/lemma-like organs was observed in some flowers. Scanning electron microscopy observation showed that the size of flower meristems was maintained the same or similar until the lemma primordium started to differentiate, at which time the floral meristem became enlarged, suggesting abnormal development of the inner whorls of rice florets. The relationship of fort3 with other similar rice mutants is discussed.     

Differential promoter methylation and histone modification contribute to the brain specific expression of the mouse Mbu-1 gene

Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expression to the central nervous system. In this study, to elucidate the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052–0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39-93%. Treatment of 5-aza-2′-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression.     

Genetic and molecular analyses of the SUP201 gene: a tRNA(3Arg) nonsense suppressor of yeast cyrl-2.

The temperature-sensitive cyr1-2 mutant in Saccharomyces cerevisiae produces low levels of adenylate cyclase and cyclic AMP at 25 degrees C and is unable to synthesize repressible acid phosphatase at 25 degrees C. Suppressor mutants of cyr1-2 were isolated by detecting acid phosphatase activity. One of the dominant suppressor mutations isolated was designated SUP201 and characterized. The SUP201 mutant gene was isolated from a gene library made from cyr1-2 SUP201 mutant DNA. Nucleotide sequence analysis of the cloned SUP201 gene revealed that the SUP201 gene was a mutated tRNA gene flanking GCN4, which worked as a UGA suppressor.     

Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading

The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides. Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity. Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1. Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501. Peptides from the N- and C-termini bound methylated DNA independently. Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation. Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A). Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.     

Two CpG islands in the <Emphasis Type="Italic">SEMA3B</Emphasis> gene: Methylation in clear cell renal cell carcinoma

Earlier, methylation of a CpG island in the SEMA3B gene (3p21.31) was observed in cell lines of small-cell and non-small-cell lung carcinoma. According to NCBI (Build 36), that island belonged to intron 1 of the gene. Our study concerns the methylation of two CpG islands, promoter and intronic, in the SEMA3B gene in patients with clear cell renal cell carcinoma (RCC). Methylation-specific PCR and bisulfite sequencing revealed a high frequency of methylation in the promoter CpG island (34/61, 56%) and somewhat lower, in the intronic (17/48, 35%). A significant inverse correlation was found between the SEMA3B mRNA level and methylation of the promoter CpG island in RCC (P < 0.05 according to Fisher’s exact test). The intronic island showed no such correlation. Thus, we suggest that the methylation of the promoter CpG island contributes to the inactivation of the SEMA3B suppressor gene in RCC tissue.     

Tissue-specific hypomethylation and expression of rat phosphoenolpyruvate carboxykinase gene induced by in vivo treatment of fetuses and neonates with 5-azacytidine

Rat fetuses of 17-19-day gestation were injected in utero with 5-azacytidine (two to three daily injections of 40 micrograms/fetus). Neonates were injected with seven daily injections (1 mg/kg). DNA samples were isolated from the fetal and neonatal livers and neonatal spleen and subjected to analysis of their methylation status. Overall methylation was analyzed by the nearest-neighbor analysis (at CpG sites) and the pattern of methylation at CCGG sites by Southern blot analysis using phosphoenolpyruvate carboxykinase (PEPCK) sequences as probes. While DNAs from the liver and spleen undergo hypomethylation to the same extent in response to the 5-azacytidine treatment, the changes in the methylation patterns of the PEPCK gene in the two tissues are strikingly different. The changes observed indicate that a decrease in the methylase activity (inhibition by 5-azacytidine) results in site- and tissue-specific hypomethylation. The tissue-specific changes in the methylation pattern are associated with a tissue-specific expression of the PEPCK gene. Although the gene is hypomethylated by azacytidine in both liver and spleen, it is expressed only in the liver. The expression of already active genes (PEPCK in the kidney and albumin in the liver) is not further enhanced by the drug.