Induced and natural epigenetic variation.
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Alleles↗
Biomedical subjects
E J Richards
Publications and source records attributed to E J Richards.
Chromatin remodeling in plants.
In the past two years, a variety of forward genetic screens have revealed predicted plant chromatin remodeling components that are involved in either differential histone acetylation or ATP-dependent SWI2/SNF2-related complexes. Combined with the results of recent reverse genetic studies, these findings have begun to provide the groundwork for determining the function of chromatin-based control in plants.
Adenosine Triphosphatases↗
Mum's the word: MOM and modifiers of transcriptional gene silencing.
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ATPases Associated with Diverse Cellular Activitie↗
Thiamine repression and pyruvate decarboxylase autoregulation independently control the expression of the Saccharomyces cerevisiae PDC5 gene.
The Saccharomyces cerevisiae gene PDC5 encodes the minor isoform of pyruvate decarboxylase (Pdc). In this work we show that expression of PDC5 but not that of PDC1, which encodes the major isoform, is repressed by thiamine. Hence, under thiamine limitation both PDC1 and PDC5 are expressed. PDC5 also becomes strongly expressed in a pdc1delta mutant. Two-dimensional gel electrophoresis of whole protein extracts shows that thiamine limitation stimulates the production of THI gene products and of Pdc5p. Deletion of PDC1 only stimulates production of Pdc5p. We conclude that the stimulation of PDC5 expression in a pdc1delta mutant is not due to a response to thiamine limitation.
Electrophoresis, Gel, Two-Dimensional↗
Maintenance of genomic methylation requires a SWI2/SNF2-like protein.
Altering cytosine methylation by genetic means leads to a variety of developmental defects in mice, plants and fungi. Deregulation of cytosine methylation also has a role in human carcinogenesis. In some cases, these defects have been tied to the inheritance of epigenetic alterations (such as chromatin imprints and DNA methylation patterns) that do not involve changes in DNA sequence. Using a forward genetic screen, we identified a gene (DDM1, decrease in DNA methylation) from the flowering plant Arabidopsis thaliana required to maintain normal cytosine methylation patterns. Additional ddm1 alleles (som4, 5, 6, 7, 8) were isolated in a selection for mutations that relieved transgene silencing (E.J.R., unpublished data). Loss of DDM1 function causes a 70% reduction of genomic cytosine methylation, with most of the immediate hypomethylation occurring in repeated sequences. In contrast, many low-copy sequences initially retain their methylation in ddm1 homozygotes, but lose methylation over time as the mutants are propagated through multiple generations by self-pollination. The progressive effect of ddm1 mutations on low-copy sequence methylation suggests that ddm1 mutations compromise the efficiency of methylation of newly incorporated cytosines after DNA replication. In parallel with the slow decay of methylation during inbreeding, ddm1 mutants accumulate heritable alterations (mutations or stable epialleles) at dispersed sites in the genome that lead to morphological abnormalities. Here we report that DDM1 encodes a SWI2/SNF2-like protein, implicating chromatin remodelling as an important process for maintenance of DNA methylation and genome integrity.
Amino Acid Sequence↗
Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana.
In contrast to mammalian epigenetic phenomena, where resetting of gene expression generally occurs in each generation, epigenetic states of plant genes are often stably transmitted through generations. The Arabidopsis mutation ddm1 causes a 70% reduction in genomic 5-methylcytosine level. We have previously shown that the ddm1 mutation results in an accumulation of a variety of developmental abnormalities by slowly inducing heritable changes in other loci. Each of the examined ddm1-induced developmental abnormalities is stably transmitted even when segregated from the potentiating ddm1 mutation. Here, the inheritance of DNA hypomethylation induced by ddm1 was examined in outcross progeny by HPLC and Southern analyses. The results indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) ddm1 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1 mutation is extremely slow or nonexistent even in wild-type DDM1 backgrounds. The stable transmission of DNA methylation status may be related to the meiotic heritability of the ddm1-induced developmental abnormalities.
Arabidopsis↗
The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis.
To investigate the relationship between cytosine methylation and gene silencing in Arabidopsis, we constructed strains containing the ddm1 hypomethylation mutation and a methylated and silenced PAI2 tryptophan biosynthetic gene (MePAI2) that results in a blue fluorescent plant phenotype. The ddm1 mutation had both an immediate and a progressive effect on PAI gene silencing. In the first generation, homozygous ddm1 MePAI2 plants displayed a weakly fluorescent phenotype, in contrast to the strongly fluorescent phenotype of the DDM1 MePAI2 parent. After two generations of inbreeding by self-pollination, the ddm1/ddm1 lines became nonfluorescent. The progressive loss of fluorescence correlated with a progressive loss of methylation from the PAI2 gene. These results indicate that methylation is necessary for maintenance of PAI gene silencing and that intermediate levels of DNA methylation are associated with intermediate gene silencing. The results also support our earlier hypothesis that ddm1 homozygotes act as "epigenetic mutators" by accumulating heritable changes in DNA methylation that can lead to changes in gene expression.
Arabidopsis↗
Plant centromeres: structure and control.
Recent work has led to a better understanding of the molecular components of plant centromeres. Conservation of at least some centromere protein constituents between plant and non-plant systems has been demonstrated. The identity and organization of plant centromeric DNA sequences are also beginning to yield to analysis. While there is little primary DNA sequence conservation among the characterized plant centromeres and their non-plant counterparts, some parallels in centromere genomic organisation can be seen across species. Finally, the emerging idea that centromere activity is controlled epigenetically finds support in an examination of the plant centromere literature.
Centromere↗
DNA methylation and plant development.
The development of genetic tools to alter DNA methylation has led to a deeper understanding of the importance of DNA modification in the life strategies of different eukaryotic organisms. This review focuses on recent findings that demonstrate a role for cytosine methylation in the development of higher plants. The effects of altering DNA modification are considered in the context of unique aspects of plant development.
Arabidopsis↗
Arabidopsis thaliana centromere regions: genetic map positions and repetitive DNA structure.
The genetic positions of the five Arabidopsis thaliana centromere regions have been identified by mapping size polymorphisms in the centromeric 180-bp repeat arrays. Structural and genetic analysis indicates that 180-bp repeat arrays of up to 1000 kb are found in the centromere region of each chromosome. The genetic behavior of the centromeric arrays suggests that recombination within the arrays is suppressed. These results indicate that the centromere regions of A. thaliana resemble human centromeres in size and genomic organization.
Alleles↗
Developmental abnormalities and epimutations associated with DNA hypomethylation mutations.
A number of aberrant morphological phenotypes were noted during propagation of the Arabidopsis thaliana DNA hypomethylation mutant, ddm1, by repeated self-pollination. Onset of a spectrum of morphological abnormalities, including defects in leaf structure, flowering time, and flower structure, was strictly associated with the ddm1 mutations. The morphological phenotypes arose at a high frequency in selfed ddm1 mutant lines and some phenotypes became progressively more severe in advancing generations. The transmission of two common morphological trait syndromes in genetic crosses demonstrated that the phenotypes are caused by heritable lesions that develop in ddm1 mutant backgrounds. Loss of cytosine methylation in specific genomic sequences during the selfing regime was noted in the ddm1 mutants. Potential mechanisms for formation of the lesions underlying the morphological abnormalities are discussed.
Arabidopsis↗
mCCG methylation in angiosperms.
Two different methods were used to investigate the abundance of cytosine methylation at the outer (5') position in 5'-CCG-3' trinucleotides in angiosperm genomes. Mspl is unable to cut its target site if the outer cytosine is methylated (5'-mCCGG-3'). Using Mspl restriction analysis, it was shown that 5'-mCCG-3' is present in all angiosperm genomes examined, and that the amount of cytosine methylation at this site varies between species. Subsequently, direct measurements were made of the amount of methylation at both cytosines in a subset of 5'-CCG-3' trinucleotides in the Arabidopsis thaliana genome. Based upon these analyses, it was estimated that approximately 20-30% of 5'-CCG-3' trinucleotides in A. thaliana are methylated at the outer cytosine. Approximately 20% of the 5'-CCG-3' trinucleotides contain 5-methyl-cytosine at the inner cytosine position, which corresponds to a previous determination of 5'-mCG-3' methylation in A. thaliana. The implications of 5'-mCCG-3' methylation are discussed.
5-Methylcytosine↗
Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.
We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.
Arabidopsis↗
DNA methylation in eukaryotes.
Recent advances have expanded our understanding of the processes underlying the establishment, maintenance, and elaboration of DNA methylation patterns in eukaryotes. The functional significance of DNA methylation is sought in a comparison of results on a variety of epigenetic phenomena in different eukaryotes. The recent development of DNA methylation mutants in mice, Neurospora, and Arabadopsis will allow traditional genetic dissection to be applied to long-standing problems regarding the function and regulation of eukaryotic DNA methylation. Although methylation appears to be important for maintenance of different epigenetic states, the mechanism that establishes these states is likely to involve additional processes.
Animals↗
Arabidopsis thaliana DNA methylation mutants.
Three DNA hypomethylation mutants of the flowering plant Arabidopsis thaliana were isolated by screening mutagenized populations for plants containing centromeric repetitive DNA arrays susceptible to digestion by a restriction endonuclease that was sensitive to methylated cytosines. The mutations are recessive, and at least two are alleles of a single locus, designated DDM1 (for decrease in DNA methylation). Amounts of 5-methylcytosine were reduced over 70 percent in ddm1 mutants. Despite this reduction in DNA methylation levels, ddm1 mutants developed normally and exhibited no striking morphological phenotypes. However, the ddm1 mutations are associated with a segregation distortion phenotype. The ddm1 mutations were used to demonstrate that de novo DNA methylation in vivo is slow.
5-Methylcytosine↗
Characterization of Arabidopsis thaliana telomeres isolated in yeast.
In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.
Base Sequence↗
The centromere region of Arabidopsis thaliana chromosome 1 contains telomere-similar sequences.
We describe the structure of an Arabidopsis thaliana genomic clone containing two classes of repetitive DNA elements derived from the centromere region of chromosome 1. One class is comprised of tandem arrays of a highly reiterated repeat containing degenerate telomere sequence motifs. Adjacent to these telomere-similar repeats we found a dispersed repetitive element reiterated approximately five times in the A. thaliana genome. The nucleotide sequence of the dispersed repeat is unusual, being extremely AT-rich and composed of numerous, overlapping repeat motifs.
Base Sequence↗
Isolation of a higher eukaryotic telomere from Arabidopsis thaliana.
We have developed a method for constructing genomic libraries enriched for telomeric DNA sequences, enabling the isolation of telomeres from higher eukaryotic organisms with large chromosomes. The method was used to clone telomeric DNA sequences from the flowering plant Arabidopsis thaliana. A. thaliana telomeres are composed primarily of tandemly repeated blocks of the sequence 5'-CCCTAAA-3' and are heterogeneous in size. Genomic sequences that cross-hybridize at high stringency with A. thaliana telomeric repeats are present in other higher plants. In Zea mays (corn), these cross-hybridizing sequences are located at the telomeres. In addition, the A. thaliana telomeric repeats cross-hybridize at low stringency to genomic sequences located at the telomeres of human chromosomes.
Animals↗