Translate this page to:
Dutch
In JoVE (1)
Other Publications (6)
Automatic Translation
This translation into Dutch was automatically generated.
English Version | Other Languages
Articles by Wenyan Xiao in JoVE
JoVE General
Bepaling van de DNA-methylatie van genen in Imprinted Arabidopsis Endosperm
Department of Biology, Saint Louis University
Imprinting is een fenomeen in plant en zoogdier reproductie. DNA-methylatie speelt een belangrijke rol in de mechanismen van imprinting. Isoleren endosperm en het bepalen van methylatie status van de ingeprente genen in
Other articles by Wenyan Xiao on PubMed
Imprinting of the MEA Polycomb Gene is Controlled by Antagonism Between MET1 Methyltransferase and DME Glycosylase
The MEA Polycomb gene is imprinted in the Arabidopsis endosperm. DME DNA glycosylase activates maternal MEA allele expression in the central cell of the female gametophyte, the progenitor of the endosperm. Maternal mutant dme or mea alleles result in seed abortion. We identified mutations that suppress dme seed abortion and found that they reside in the MET1 methyltransferase gene, which maintains cytosine methylation. Seeds with maternal dme and met1 alleles survive, indicating that suppression occurs in the female gametophyte. Suppression requires a maternal wild-type MEA allele, suggesting that MET1 functions upstream of, or at, MEA. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell. MET1 is required for DNA methylation of three regions in the MEA promoter in seeds. Our data suggest that imprinting is controlled in the female gametophyte by antagonism between the two DNA-modifying enzymes, MET1 methyltransferase and DME DNA glycosylase.
DNA Methylation is Critical for Arabidopsis Embryogenesis and Seed Viability
DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.
Regulation of Seed Size by Hypomethylation of Maternal and Paternal Genomes
DNA methylation is an epigenetic modification of cytosine that is important for silencing gene transcription and transposons, gene imprinting, development, and seed viability. DNA METHYLTRANSFERASE1 (MET1) is the primary maintenance DNA methyltransferase in Arabidopsis (Arabidopsis thaliana). Reciprocal crosses between antisense MET1 transgenic and wild-type plants show that DNA hypomethylation has a parent-of-origin effect on seed size. However, due to the dominant nature of the antisense MET1 transgene, the parent with a hypomethylated genome, its gametophyte, and both the maternal and paternal genomes of the F(1) seed become hypomethylated. Thus, the distinct role played by hypomethylation at each generation is not known. To address this issue, we examined F(1) seed from reciprocal crosses using a loss-of-function recessive null allele, met1-6. Crosses between wild-type and homozygous met1-6 parents show that hypomethylated maternal and paternal genomes result in significantly larger and smaller F(1) seeds, respectively. Our analysis of crosses between wild-type and heterozygous MET1/met1-6 parents revealed that hypomethylation in the female or male gametophytic generation was sufficient to influence F(1) seed size. A recessive mutation in another gene that dramatically reduces DNA methylation, DECREASE IN DNA METHYLATION1, also causes parent-of-origin effects on F(1) seed size. By contrast, recessive mutations in genes that regulate a smaller subset of DNA methylation (CHROMOMETHYLASE3 and DOMAINS REARRANGED METHYLTRANSFERASES1 and 2) had little effect on seed size. Collectively, these results show that maternal and paternal genomes play distinct roles in the regulation of seed size in Arabidopsis.
Selected Technologies to Control Genes and Their Products for Experimental and Clinical Purposes
"On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990 s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch, coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.
S100A8/9 Induces Cell Death Via a Novel, RAGE-independent Pathway That Involves Selective Release of Smac/DIABLO and Omi/HtrA2
A complex of two S100 EF-hand calcium-binding proteins S100A8/A9 induces apoptosis in various cells, especially tumor cells. Using several cell lines, we have shown that S100A8/A9-induced cell death is not mediated by the receptor for advanced glycation endproducts (RAGE), a receptor previously demonstrated to engage S100 proteins. Investigation of cell lines either deficient in, or over-expressing components of the death signaling machinery provided insight into the S100A8/A9-mediated cell death pathway. Treatment of cells with S100A8/A9 caused a rapid decrease in the mitochondrial membrane potential (DeltaPsi(m)) and activated Bak, but did not cause release of apoptosis-inducing factor (AIF), endonuclease G (Endo G) or cytochrome c. However, both Smac/DIABLO and Omi/HtrA2 were selectively released into the cytoplasm concomitantly with a decrease in Drp1 expression, which inhibits mitochondrial fission machinery. S100A8/A9 treatment also resulted in decreased expression of the anti-apoptotic proteins Bcl2 and Bcl-X(L), whereas expression of the pro-apoptotic proteins Bax, Bad and BNIP3 was not altered. Over-expression of Bcl2 partially reversed the cytotoxicity of S100A8/A9. Together, these data indicate that S100A8/A9-induced cell death involves Bak, selective release of Smac/DIABLO and Omi/HtrA2 from mitochondria, and modulation of the balance between pro- and anti-apoptotic proteins.
Death Receptor 4 is Preferentially Recruited to Lipid Rafts in Chronic Lymphocytic Leukemia Cells Contributing to Tumor Necrosis Related Apoptosis Inducing Ligand-induced Synergistic Apoptotic Responses
Tumor necrosis related apoptosis inducing ligand receptor 1 (TRAIL-R1, death receptor 4 [DR4]) and TRAIL-R2 (DR5) have been proposed as targets for cancer therapy, but which death receptor to target for chemotherapy in chronic lymphocytic leukemia (CLL) is uncertain. Herein, we discovered that Burkitt lymphoma B cell line, BJAB, CLL-like cell line, I-83, and pre-acute lymphocytic leukemia B cell line, NALM-6, underwent apoptosis following TRAIL, whereas a CLL-like cell line, JMV-3, and primary CLL cells failed to undergo apoptosis. In TRAIL resistant CLL cells, only activation of DR4 provided an increase in fludarabine induced apoptosis. This was mediated in part by the localization of DR4 but not DR5 in lipid rafts following TRAIL and fludarabine treatment. This preference for DR4 activation leading to increased fludarabine induced apoptosis was also observed following SAHA, PS-341, and chlorambucil treatment in primary CLL cells. Thus, CLL cells selectively activate DR4 partially mediated through its localization to lipid rafts leading to apoptosis when combined with chemotherapeutic drugs.
