DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of ...regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic ...regulation is very limited. Using the Arabidopsis thaliana repressor of silencingi mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation.
In this study, a rapid diagnosis platform was developed for the detection of Escherichia coli O157:H7. An electrical double layer (EDL)-gated field-effect transistor-based biosensor (BioFET) as a ...point-of-care testing device is demonstrated with its high sensitivity, portability, high selectivity, quick response, and ease of use. The specially designed ssDNA probe was immobilized on the extended gate electrode to bind the target complementary DNA segment of E. coli, resulting in a sharp drain current change within minutes. The limit of detection for target DNA is validated to a concentration of 1 fM in buffer solution and serum. Meanwhile, the results of a Kelvin probe force microscope were shown to have reduced surface potential of the DNA immobilized sensors before and after the cDNA detection, which is consistent with the decreased drain current of the BioFET. A 1.2 kb E. coli duplex DNA synthesized in plasmid was sonicated and detected in serum samples with the sensor array. Gel electrophoresis was used to confirm the efficiency of sonication by elucidating the length of DNA. Those results show that the EDL-gated BioFET system is a promising platform for rapid identification of pathogens for future clinical needs.
Summary
Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA‐induced silencing complex have been ...well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the ARGONAUTE1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold‐stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene FLOWERING LOCUS C (FLC). In a genetic screen for enhancers of sic–1, we isolated a loss‐of‐function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1–7 mutant flowered early and was smaller in stature than the wild‐type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri‐MIR168b and pre‐MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.
Significance Statement
Proper accumulation and function of miRNAs is essential for plant growth and development. Here we show a new role of previously identified HOS1 in regulating miR168a/b. We discovered HOS1 regulate MIR168b gene during transcription and it is required for proper expression of MIR168b. This role of HOS1 helps explain its broad function in plant growth, development and stress tolerance.
Biliary Atresia (BA) is a severe liver disease that affects newborns. This disease may cause cholestasis and progressive hepatic failure or even death if not treated immediately. ELISA blood tests ...for metabolic screening are the current method of diagnosing BA. However, Newborns need rapid BA detection for treatment in the future. Level increase of intrahepatic matrix metalloproteinase-7 (MMP7) is a factor known to cause BA-related liver fibrosis. We brought out a platform to detect BA using an electric-double-layer (EDL)-gated field-effect transistor (FET). MMP7 concentration differences affect gate voltages then consequent in the sensitivity of this platform. EDL-gated FETs are planned to display in hospitals for medical personnel to accurately detect BA in infants.
Summary
Proper accumulation and function of mi
RNA
s is essential for plant growth and development. While core components of the mi
RNA
biogenesis pathway and mi
RNA
‐induced silencing complex have ...been well characterized, cellular regulators of mi
RNA
s remain to be fully explored. Here we report that
HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES
1 (
HOS
1) is a regulator of an important mi
RNA
, mi168a/b, that targets the
ARGONAUTE
1
(
AGO
1
) gene in Arabidopsis.
HOS
1 functions as an ubiquitin E3 ligase to regulate plant cold‐stress responses, associates with the nuclear pores to regulate
mRNA
export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene
FLOWERING LOCUS
C
(
FLC
). In a genetic screen for enhancers of
sic–
1, we isolated a loss‐of‐function Arabidopsis mutant of
HOS
1
that is defective in mi
RNA
biogenesis. Like other
hos1
mutant alleles, the
hos1–7
mutant flowered early and was smaller in stature than the wild‐type. Dysfunction in
HOS
1 reduced the abundance of miR168a/b but not of other mi
RNA
s. In
hos1
mutants,
pri‐
MIR
168b
and
pre‐
MIR
168b
levels were decreased, and
RNA
polymerase
II
occupancy was reduced at the promoter of
MIR
168b
but not that of
MIR
168a
. Chromatin immunoprecipitation assays revealed that
HOS
1 protein is enriched at the chromatin of the
MIR
168b
promoter. The reduced miR168a/b level in
hos1
mutants results in an increase in the
mRNA
and protein levels of its target gene,
AGO
1
. Our results reveal that
HOS
1 regulates miR168a/b and
AGO
1 levels in Arabidopsis by maintaining proper transcription of
MIR
168b
.
Significance Statement
Proper accumulation and function of miRNAs is essential for plant growth and development. Here we show a new role of previously identified HOS1 in regulating miR168a/b. We discovered HOS1 regulate
MIR168b
gene during transcription and it is required for proper expression of
MIR168b
. This role of HOS1 helps explain its broad function in plant growth, development and stress tolerance.
DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of ...regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Argonaute (AGO) family proteins are conserved key components of small RNA-induced silencing pathways. In the RNA-directed DNA methylation (RdDM) pathway in Arabidopsis,AGO6 is generally considered to ...be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4- and AGO6-dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co-localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co-localized. In the nucleoplasm, AGO4 displays a strong co-localization with Pol II, whereas AGO6 co-localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V-dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA-directed DNA methylation. Synopsis AGO4 and AGO6 have distinct, but cooperative functions in RNA-directed DNA methylation in Arabidopsis and show spatially segregated interactions with RNA polymerases II and V, respectively. Genome-wide DNA methylation analysis reveals that AGO4 and AGO6 are mutually required in the majority of AGO4- and AGO6-dependent methylated loci; AGO4 and AGO6 display different co-localization patterns with DNA-dependent RNA polymerase; RNA Pol II physically interacts with AGO4 but not AGO6; RNA Pol V chromatin occupancy and the accumulation of Pol V-dependent scaffold RNAs requires AGO6 but not AGO4
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