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Judith Bender

Publications and source records attributed to Judith Bender.

18 recordsLinked to original sources
PubMed2006

A histone methylation-dependent DNA methylation pathway is uniquely impaired by deficiency in Arabidopsis S-adenosylhomocysteine hydrolase.

S-adenosylhomocysteine hydrolase (SAH) is a key enzyme in the maintenance of methylation homeostasis in eukaryotes because it is needed to metabolize the by-product of transmethylation reactions, S-adenosylhomocysteine (AdoHcy), which causes by-product inhibition of methyltransferases (MTase's). Complete loss of SAH function is lethal. Partial loss of SAH function causes pleiotropic effects including developmental abnormalities and reduced cytosine methylation. Here we describe a novel partial-function missense allele of the Arabidopsis SAH1 gene that causes loss of cytosine methylation specifically in non-CG contexts controlled by the CMT3 DNA MTase and transcriptional reactivation of a silenced reporter gene, without conferring developmental abnormalities. The CMT3 pathway depends on histone H3 lysine 9 methylation (H3 mK9) to guide DNA methylation. Our results suggest that this pathway is uniquely sensitive to SAH impairment because of its requirement for two transmethylation reactions that can both be inhibited by AdoHcy. Our results further suggest that gene silencing pathways involving an interplay between histone and DNA methylation in other eukaryotes can be selectively impaired by controlled SAH downregulation.

Adenosylhomocysteinase↗
PubMed2006

Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase.

In Arabidopsis thaliana, heterochromatin formation is guided by double-stranded RNA (dsRNA), which triggers methylation of histone H3 at Lys-9 (H3 mK9) and CG plus non-CG methylation on identical DNA sequences. At heterochromatin targets including transposons and centromere repeats, H3 mK9 mediated by the Su(var)3-9 homologue 4 (SUVH4)/KYP histone methyltransferase (MTase) is required for the maintenance of non-CG methylation by the CMT3 DNA MTase. Here, we show that although SUVH4 is the major H3 K9 MTase, the SUVH5 protein also has histone MTase activity in vitro and contributes to the maintenance of H3 mK9 and CMT3-mediated non-CG methylation in vivo. Strikingly, the relative contributions of SUVH4, SUVH5, and a third related histone MTase, SUVH6, to non-CG methylation are locus-specific. For example, SUVH4 and SUVH5 together control transposon sequences with only a minor contribution from SUVH6, whereas SUVH4 and SUVH6 together control a transcribed inverted repeat source of dsRNA with only a minor contribution from SUVH5. This locus-specific variation suggests different mechanisms for recruiting or activating SUVH enzymes at different heterochromatic sequences. The suvh4 suvh5 suvh6 triple mutant loses both monomethyl and dimethyl H3 K9 at target loci. The suvh4 suvh5 suvh6 mutant also displays a loss of non-CG methylation similar to a cmt3 mutant, indicating that SUVH4, SUVH5, and SUVH6 together control CMT3 activity.

Arabidopsis↗
PubMed2005

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases.

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.

Arabidopsis↗
PubMed2004

The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis.

Plants derive a number of important secondary metabolites from the amino acid tryptophan (Trp), including the growth regulator indole-3-acetic acid (IAA) and defense compounds against pathogens and herbivores. In previous work, we found that a dominant overexpression allele of the Arabidopsis (Arabidopsis thaliana) Myb transcription factor ATR1, atr1D, activates expression of a Trp synthesis gene as well as the Trp-metabolizing genes CYP79B2, CYP79B3, and CYP83B1, which encode enzymes implicated in production of IAA and indolic glucosinolate (IG) antiherbivore compounds. Here, we show that ATR1 overexpression confers elevated levels of IAA and IGs. In addition, we show that an atr1 loss-of-function mutation impairs expression of IG synthesis genes and confers reduced IG levels. Furthermore, the atr1-defective mutation suppresses Trp gene dysregulation in a cyp83B1 mutant background. Together, this work implicates ATR1 as a key homeostatic regulator of Trp metabolism and suggests that ATR1 can be manipulated to coordinately control the suite of enzymes that synthesize IGs.

Arabidopsis↗
PubMed2004

RNA-directed DNA methylation.

Double-stranded RNAs (dsRNAs) and their 'diced' small RNA products can guide key developmental and defense mechanisms in eukaryotes. Some RNA-directed mechanisms act at a post-transcriptional level to degrade target messenger RNAs. However, dsRNA-derived species can also direct changes in the chromatin structure of DNA regions with which they share sequence identity. For example, plants use such RNA species to lay down cytosine methylation imprints on identical DNA sequences, providing a fundamental mark for the formation of transcriptionally silent heterochromatin. Thus, RNA can feed backwards to modulate the accessibility of information stored in the DNA of cognate genes. RNA triggers for DNA methylation can come from different sources, including invasive viral, transgene or transposon sequences, and in some cases are derived from single-stranded RNA precursors by RNA-dependent RNA polymerases. The mechanism by which RNA signals are translated into DNA methylation imprints is currently unknown, but two plant-specific types of cytosine methyltransferase have been implicated in this process. RNA can also direct heterochromatin formation in fission yeast and Drosophila, but in these organisms the process occurs in the absence of DNA methylation.

Animals↗
PubMed2004

The photomorphogenesis-related mutant red1 is defective in CYP83B1, a red light-induced gene encoding a cytochrome P450 required for normal auxin homeostasis.

Previous genetic analysis identified a component, RED1, that is required for normal de-etiolation of Arabidopsis thaliana (L.) Heynh. seedlings in continuous red light (Rc). red1 mutant seedlings exhibit elongated hypocotyls and reduced cotyledon size specifically in Rc and not in continuous far-red light (FRc). Here, we show that red1 is allelic to sur2 and atr4, and is defective in the cytochrome P450 CYP83B1, an enzyme required for normal auxin homeostasis. Two alleles of atr4, like red1, exhibit increased hypocotyl elongation and reduced cotyledon expansion in Rc but not in FRc. We further show that CYP83B1 transcript levels are elevated specifically in Rc-grown seedlings when compared with seedlings grown in darkness or FRc. Hence, the Rc-specific phenotype of the red1 mutant may indicate that Rc-induction of the CYP83B1 transcript is necessary for normal seedling de-etiolation in the wild type.

Amino Acid Sequence↗
PubMed2004

Microbial mats for multiple applications in aquaculture and bioremediation.

Microbial mats occur in nature as stratified communities of cyanobacteria and bacteria, but they can be cultured on large-scale and manipulated for a variety of functions. They are complex systems, but require few external inputs. The functional uses of mats broadly cover the areas of aquaculture and bioremediation. Preliminary research also points to promising uses in agriculture and energy production. Regarding aquaculture, mats were shown to produce protein, via nitrogen fixation, and were capable of supplying nutrition to tilapia (Oreochromis niloticus). Current research is examining the role of mats in the nitrification of nutrient-enriched effluents from aquaculture. Most research has addressed bioremediation, within which two majors categories of contaminants were examined: metals and radionuclides, and organic contaminants. Mats sequester or precipitate metals/radionuclides by surface absorption or by conditioning the surrounding chemical environment, thus bioconcentrating the metal/radionuclide in a small volume. Organic contaminants are degraded and may be completely mineralized. For agriculture mats hold promise as a soil amendment and nitrogen fertilizer. The use of mats in biohydrogen production has been verified, but is in a preliminary phase of development. We propose a comprehensive closed system based on microbial mats for aquaculture and waste management.

Aquaculture↗
PubMed2004

Chromatin-based silencing mechanisms.

Eukaryotic genomes are organized into regions of transcriptionally active euchromatin and transcriptionally inactive heterochromatin. In plant genomes, heterochromatin is marked by methylation of cytosine and methylation of histone H3 at lysine 9. Heterochromatin formation is targeted to transposons as a means of defending the host genome against the deleterious effects of these sequences. Heterochromatin is directed to transposon sequences by transposon-derived aberrant RNA species and functions to prevent unwanted transcription and movement. Formation of heterochromatin at rRNA-encoding genes and centromere-associated repeats might also involve an RNA-based mechanism that is designed to stabilize these potentially labile structures.

Arabidopsis↗
PubMed2004

DNA methylation and epigenetics.

In many eukaryotes, including plants, DNA methylation provides a heritable mark that guides formation of transcriptionally silent heterochromatin. In plants, aberrant RNA signals direct DNA methylation to target sequences, sometimes appropriately and sometimes inappropriately. This chapter discusses the generation of RNA signals for epigenetic changes, the factors that mediate those changes, and some of the consequences of those changes for plant gene expression and genome integrity.

DNA Methylation↗
PubMed2004

An internal rearrangement in an Arabidopsis inverted repeat locus impairs DNA methylation triggered by the locus.

In plants, transcribed inverted repeats trigger RNA interference (RNAi) and DNA methylation of identical sequences. RNAi is caused by processing of the double-stranded RNA (dsRNA) transcript into small RNAs that promote degradation of complementary RNA sequences. However, the signals for DNA methylation remain to be fully elucidated. The Arabidopsis tryptophan biosynthetic PAI genes provide an endogenous inverted repeat that triggers DNA methylation of PAI-identical sequences. In the Wassilewskija strain, two PAI genes are arranged as a tail-to-tail inverted repeat and transcribed from an unmethylated upstream promoter. This locus directs its own methylation, as well as methylation of two unlinked singlet PAI genes. Previously, we showed that the locus is likely to make an RNA signal for methylation because suppressed transcription of the inverted repeat leads to reduced PAI methylation. Here we characterize a central rearrangement in the inverted repeat that also confers reduced PAI methylation. The rearrangement creates a premature polyadenylation signal and suppresses readthrough transcription into palindromic PAI sequences. Thus, a likely explanation for the methylation defect of the mutant locus is a failure to produce readthrough dsRNA methylation triggers.

Arabidopsis↗
PubMed2003

Transcription from an upstream promoter controls methylation signaling from an inverted repeat of endogenous genes in Arabidopsis.

In plants, replication of RNA viruses and RNA from highly transcribed transgenes can trigger DNA methylation. These systems accumulate diced small RNA(smRNA) products of double-stranded RNA(dsRNA) precursors, but it is not known which RNA species directs methylation. The methylated PAI tryptophan biosynthetic genes in Arabidopsis allow the study of methylation signals for endogenous genes with lower expression levels. The PAI genes are arranged as a tandem inverted repeat plus two singlet genes at unlinked loci. Here we show that the predominant PAI transcript initiates at a novel unmethylated promoter that lies upstream of one of the inverted repeat PAI genes. Suppressed transcription from the upstream promoter using transgene-directed silencing reduces methylation on the singlet PAI genes, but not on the inverted repeat, consistent with an RNA methylation signal. RNA gel blots detect normal PAI transcripts and dsRNA read-through species, but not diced smRNAs, suggesting that either precursor dsRNAs or subdetectable levels of smRNAs, below the threshold to effectively degrade PAI transcripts, serve as the PAI methylation signal. Thus, the lower expression endogenous gene system allows dissection of a RNA-directed methylation pathway distinct from RNA degradation pathways.

Arabidopsis↗
PubMed2003

Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis.

Methylation of cytosine residues in eukaryotic genomes is often associated with repeated sequences including transposons and their derivatives. Methylation has been implicated in control of two potential deleterious effects of these repeats: (1) uncontrolled transcription, which often disturbs proper expression of nearby host genes, and (2) changes in genome structure by transposition and ectopic recombination. Arabidopsis thaliana provides a genetically tractable system to examine these possibilities, since viable mutants in DNA methyltransferases are available. Arabidopsis MET1 (METHYLTRANSFERASE1, ortholog of mammalian DNA methyltransferase Dnmt1) is necessary for maintaining genomic cytosine methylation at 5'-CG-3' sites. Arabidopsis additionally methylates non-CG sites using CHROMOMETHYLASE3 (CMT3). We examined the mobility of endogenous CACTA transposons in met1, cmt3, and cmt3-met1 mutants. High-frequency transposition of CACTA elements was detected in cmt3-met1 double mutants. Single mutants in either met1 or cmt3 were much less effective in mobilization, despite significant induction of CACTA transcript accumulation. These results lead us to conclude that CG and non-CG methylation systems redundantly function for immobilization of transposons. Non-CG methylation in plants may have evolved as an additional epigenetic tag dedicated to transposon control. This view is consistent with the recent finding that CMT3 preferentially methylates transposon-related sequences.

Arabidopsis↗
PubMed2002

An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation.

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis: This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.

Aldose-Ketose Isomerases↗
PubMed2002

Glucose conjugation of anthranilate by the Arabidopsis UGT74F2 glucosyltransferase is required for tryptophan mutant blue fluorescence.

Plant mutants with defects in intermediate enzymes of the tryptophan biosynthetic pathway often display a blue fluorescent phenotype. This phenotype results from the accumulation of the fluorescent tryptophan precursor anthranilate, the bulk of which is found in a glucose-conjugated form. To elucidate factors that control fluorescent tryptophan metabolites, we conducted a genetic screen for suppressors of blue fluorescence in the Arabidopsis trp1-100 mutant, which has a defect in the second enzymatic step of the tryptophan pathway. This screen yielded loss-of-function mutations in the UDP-glucosyltransferase gene UGT74F2. The bacterially expressed UGT74F2 enzyme catalyzed a conjugation reaction, with free anthranilate and UDP-glucose as substrates, that yielded the same fluorescent glucose ester compound as extracted from the trp1-100 mutant. These results indicate that sugar conjugation of anthranilate by UGT74F2 allows its stable accumulation in plant tissues. A highly related Arabidopsis enzyme UGT74F1 could also catalyze this reaction in vitro and could complement the ugt74F2 mutation when overexpressed in vivo. However, the UGT74F1 gene is expressed at a lower level than the UGT74F2 gene. Therefore, even though UGT74F1 and UGT74F2 have redundant conjugating activities toward anthranilate, UGT74F2 is the major source of this activity in the plant.

Amino Acid Sequence↗
PubMed2002

Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes.

Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.

Acetyltransferases↗
PubMed2002

Arabidopsis cytochrome P450 cyp83B1 mutations activate the tryptophan biosynthetic pathway.

In plants, the tryptophan biosynthetic pathway provides a number of important secondary metabolites including the growth regulator indole-3-acetic acid (IAA) and indole glucosinolate defense compounds. Genes encoding tryptophan pathway enzymes are transcriptionally induced by a variety of stress signals, presumably to increase the production of both tryptophan and secondary metabolites during defense responses. To understand the mechanism of transcriptional induction, we isolated altered tryptophan regulation (atr) mutants in Arabidopsis thaliana with activated transcription of tryptophan genes. One atr complementation group consisted of mutations in the cytochrome P450 gene CYP83B1. Mutant plants had constitutively activated expression of the ATR1 Myb factor gene, which was identified as a positive regulator of tryptophan genes via the atr mutant screen. cyp83B1 mutants were previously characterized as having defects in IAA homeostasis due to perturbation of secondary tryptophan metabolism. Our findings indicate that the upregulation of tryptophan pathway genes might also contribute to the overaccumulation of IAA in mutant plants. Moreover, we show that cyp83B1 mutants have lesion-mimic phenotypes, suggesting that multiple stress pathways are activated by loss of CYP83B1 function.

Arabidopsis↗
PubMed2002

Dominant alleles of the basic helix-loop-helix transcription factor ATR2 activate stress-responsive genes in Arabidopsis.

Members of the R/B basic helix-loop-helix (bHLH) family of plant transcription factors are involved in a variety of growth and differentiation processes. We isolated a dominant mutation in an R/B-related bHLH transcription factor in the course of studying Arabidopsis tryptophan pathway regulation. This mutant, atr2D, displayed increased expression of several tryptophan genes as well as a subset of other stress-responsive genes. The atr2D mutation creates an aspartate to asparagine change at a position that is highly conserved in R/B factors. Substitutions of other residues with uncharged side chains at this position also conferred dominant phenotypes. Moreover, overexpression of mutant atr2D, but not wild-type ATR2, conferred pleiotropic effects, including reduced size, dark pigmentation, and sterility. Therefore, atr2D is likely to be an altered-function allele that identifies a key regulatory site in the R/B factor coding sequence. Double-mutant analysis with atr1D, an overexpression allele of the ATR1 Myb factor previously isolated in tryptophan regulation screens, showed that atr2D and atr1D have additive effects on tryptophan regulation and are likely to act through distinct mechanisms to activate tryptophan genes. The dominant atr mutations thus provide tools for altering tryptophan metabolism in plants.

Alleles↗