(A, B) DNA methylation analysis at the AtSN1, IGN5 and solo LTR loci in nrpe1, ago4 and dms3 mutants. Genomic DNA was digested with HaeIII (A) or AluI (B) methylation-sensitive restriction endonucleases followed by PCR. Sequences lacking HaeIII sites (actin 2; panel A) or AluI sites (IGN5, panel B) served as controls to show that equivalent amounts of DNA were tested in all reactions.
(C–D) ChIP analysis of H3K27me1 (C) and H3Ac2 (D) levels in nrpe1, ago4 and dms3 mutants. Histograms show means +/− standard deviations obtained from three independent amplifications.
(E) ChIP analysis of Pol II binding to chromatin in nrpe1, ago4 and dms3 mutants. Histograms show means +/− standard deviations obtained from three independent amplifications.
(F) Control ChIP reactions performed in the absence of antibody reveal background signal levels.

(A) ChIP data showing AGO4 binding to chromatin at solo LTR, IGN5, AtSN1 and IGN6 loci in ago4, nrpe1 and drm2 mutants. DNA purified from input chromatin samples, chromatin subjected to the immunoprecipitation procedure in the absence of antibody (no AB) and chromatin immunoprecipitated using anti-AGO4 antibody (αAGO4) was amplified by PCR using locus-specific primers. Primers amplifying the Actin 2 locus served as an internal control.
(B) ChIP data showing AGO4 binding to chromatin at solo LTR, IGN5, AtSN1 and IGN6 loci in nrpe1 mutant, nrpe1 mutant transformed with a wild type NRPE1 transgene (NRPE1 wt) and nrpe1 mutant transformed with an NRPE1 active site mutant transgene (NRPE1 ASM).
(C) Immunoblot detection of AGO4 in protein extracts of wild-type (Col-0), ago4, nrpe1, or nrpe1 transformed with either a wild type NRPE1 transgene (NRPE1 wt) or an NRPE1 active site mutant transgene (NRPE1 ASM). Ponceau S staining revealed equal loading of lanes. 100% and 50% sample loadings demonstrate that the assay is semi-quantitative.

(A) Strand-specific RT-PCR detection of Pol V transcripts at IGN5 and IGN6 and AtSN1 in wild-type (Col-0) and nrpe1 and dms3 mutants. Derepression of Pol II transcripts at the solo LTR and putative Pol III transcripts at AtSN1 in the nrpe1 and dms3 mutants is shown in the right panel. Actin RT-PCR products and ethidium bromide-stained rRNAs resolved by agarose gel electrophoresis serve as loading controls. To control for background DNA contamination, a reaction using IGN5 bottom strand and AtSN1 (interval B) primers, but no reverse transcriptase (no RT) was performed. No RNA (0 μg) controls are provided for all primer pairs.
(B) ChIP with anti-NRPE1 antibody in Col-0 wild-type, nrpe1 and dms3 mutants followed by real-time PCR. Histograms show means +/− standard deviations obtained from three independent amplifications.

(A) Strand specific RT-PCR of Pol V transcription at IGN5 and IGN6 in ago4 and rdr2 mutants as well as nrpe1 ago4, and rdr2 ago4 double mutants. Wt sibling is a wild type sibling of the ago4 mutant identified in a segregating family. Actin RT-PCR products and ethidium bromide-stained rRNAs resolved by agarose gel electrophoresis serve as loading controls. To control for background DNA contamination, a reaction using IGN5 top strand primers but no reverse transcriptase (no RT) was performed. No RNA (0 μg) controls are provided for all primer pairs.
(B) Densitometric analysis of RT-PCR data for the ago4 mutant presented in panel A. The histogram provides mean band intensities relative to wild type Col-0, +/− standard deviations obtained from three independent experiments.

(A) RNA immunoprecipitation using αAGO4 antibody. Immunoprecipitated RNA isolated from the indicated mutants was digested with DNaseI and amplified by RT-PCR. Total RNA controls show that the Pol V transcripts are present in equivalent amounts in all mutants tested except nrpe1. Ethidium bromide stained rRNAs (bottom left) show that equal amounts of RNA were tested. The no reverse transcriptase (no RT) control was performed with IGN5 bottom strand primers. No RNA controls were performed for all primer pairs tested. RT-PCR amplification of actin RNA serves as a loading control.
(B) Immunoblot detection of AGO4 in protein extracts of wild-type (Col-0) plants or ago4 mutant. Stars denote nonspecific bands.
(C) Immunoblot detection of AGO4 in protein extracts of wild-type (Col-0), rdr2, dcl3, dcl234 or nrpe1 mutants. Asterisks denote nonspecific bands.
(D) Immunoblot detection of AGO4 in protein extracts of wild-type (Col-0), nrpd1 (Pol IV) nrpe1 (Pol V), nrpd2/nrpe2 (shared subunit of Pol IV and Pol V), or rdr2 mutants. Asterisks denote nonspecific bands.

A model for Pol V and siRNA-dependent heterochromatin formation. DMS3 and DRD1 mediate the assembly of Pol V initiation and/or elongation complexes and the production of Pol V transcripts. AGO4-siRNA complexes recognize target loci via base-pairing of siRNAs with nascent Pol V transcripts. AGO4 subsequently recruits chromatin modifying activities including the de novo DNA methyltransferase DRM2 and histone modifying enzymes via unknown mechanisms.