Figure S1. Chromosome-wide density analysis of the AGO4-associated
small RNAs on Chromosome 1. The non-redundant set of small RNAs was
screened against the Arabidopsis genome using WU-BLAST. The density of
small RNAs with perfect matches to the direct strand (upper panel) or the
complementary strand (middle panel) of Chromosome 1, and the density of
repeats (lower panel) on Chromosome 1 were plotted in a 50 Kb sliding window.
The position of the pericentromeric region is marked.
Figure S2. Chromosome-wide density analysis of the AGO4-associated
small RNAs on Chromosome 2. The non-redundant set of small RNAs was
screened against the Arabidopsis genome using WU-BLAST. The density of
small RNAs with perfect matches to the direct strand (upper panel) or the
complementary strand (middle panel) of Chromosome 2, and the density of
repeats (lower panel) on Chromosome 2 were plotted in a 50 Kb sliding window.
The position of the pericentromeric region is marked.
2
Figure S3. Chromosome-wide density analysis of the AGO4-associated
small RNAs on Chromosome 3. The non-redundant set of small RNAs was
screened against Arabidopsis genome using WU-BLAST. The density of small
RNAs with perfect matches to the direct strand (upper panel) or the
complementary strand (middle panel) of Chromosome 3, and the density of
repeats (lower panel) on Chromosome 3 were plotted in a 50 Kb sliding window.
The position of the pericentromeric region is marked.
3
Figure S4. Chromosome-wide density analysis of the AGO4-associated
small RNAs on Chromosome 5. The non-redundant set of small RNAs was
screened against the Arabidopsis genome using WU-BLAST. The density of
small RNAs with perfect matches to the direct strand (upper panel) or the
complementary strand (middle panel) of Chromosome 5, and the density of
repeats (lower panel) on Chromosome 5 were plotted in a 50 Kb sliding window.
The position of the pericentromeric region is marked.
4
Figure S5 Preferential association of a subset of microRNAs with AGO4. a.
Data obtained from 454 sequencing were used to quantify levels of individual
miRNAs, relative to total miRNA content, in AGO1 and AGO4 complexes and in
total RNA. Percent contribution for a subset of microRNAs with preferential
association with either AGO1 or AGO4 is shown graphically. b. Northen
analysis of miR172, miR163, miR390, and miR173 confirm patterns of AGO1 and
AGO4 binding discerned from sequencing. siRNAs from AtSN1 and tasiRNA480 are shown as controls for AGO4 and AGO1-bound species,
respectively.
5
Figure S6 Identification of AGO4-regulated loci through small RNA
sequencing. a, Northern analysis of small RNAs in total RNA and in RNAs
isolated from TAPAGO4 and control purifications were performed using the
indicated probes. Radioactive RNAs of known sizes were included as size
markers. b, Northern analysis of AtREP1/2 and SIMPLEHAT2 siRNAs were
performed on RNA from the indicated plants. miR171 serves as a loading control.
c, Analysis of CpG (left), CpNpG (center), and CpHpH (right) methylation at
ATREP2 and SIMPLEHAT2 loci was carried out by bisulfite sequencing of
genomic DNA prepared from the indicated plants. The methylation level is shown
by the percentage of methylated cytosine in all sequenced clones. Data in Table
S6 were used to generate the histograms.
6
Figure S7. Accumulation of AGO4 wild-type and DDH mutant proteins in
transgenic Arabidopsis plants. Total proteins were extracted from pooled
samples of ~30 T1 transgenic lines for each constructs. Western blot analysis
was performed using anti-myc antibody. The position of myc-AGO4 is indicated
by the arrow. The lower band is a cross-reacting species seen in all samples.
7
Figure S8. AGO4-mediated cleavage of miRNA targets. AGO4 complexes
or control immunoprecipitates were mixed with labeled, synthetic target RNAs for
two AGO-4 associated microRNAs, miR390 and miR172 (TAS3 and AP2 mRNA,
respectively). Cleavage was monitored by electrophoresis of reaction products.
8
Figure S9. Analysis of CpG (left), CpNpG (middle), and CpHpH (right)
methylation at SUP, AtMu1, MEA-ISR and ATREP2 loci in T2 transgenic
lines. Bisulfite sequencing data from two biological replicates were combined.
Methylation levels are shown by the percentage of methylated cytosines in all
sequenced clones. Data from Table S8 were used to generate the histograms.
9
Figure S10. Northern blot analysis of siRNAs derived from MEA-ISR,
AtSN1, and ATREP2 in RNAs prepared from the indicated T2 plants.
miR171 was used as a loading control. The siRNA signals were normalized
relative to miR171 and the relative levels were calculated by comparison to those
in clk-st (see text, arbitrarily set to 1).
10