RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway

Abstract

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs (siRNAs) are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and DICER-LIKE 3 (DCL3) are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 (AGO4). Nuclear RNA polymerase V (Pol V) collaborates with DRD1 (DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1) to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drd1 mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

Figure 1.

Major Pericentromere Repeat Organization at Interphase Is Disrupted in nrpe1 and drd1 But Not nrpd1 Mutants. DNA-FISH of 5S genes (red) and 180-bp centromeric repeats (green) (left panel). Organization pattern of pericentromeric regions analyzed by FISH using the 180-bp centromere repeat (green) and the BAC F28D6 (red) DNA probes (middle panel). Immunostaining followed by DNA-FISH performed respectively with an antibody recognizing the A. thaliana centromeric histone H3, HTR12 (red), and 180-bp centromeric repeats (green) (right panel). In all panels, DNA was visualized by DAPI (gray) and size bars indicate 5 μm.

Figure 2.

Pol V, drd1, and met1 Mutations Result in Loss of Heterochromatin at Chromocenters. In the upper panel, representative interphase nuclei of the different mutants are displayed. DNA was counterstained by DAPI (gray). The size bar corresponds to 5 lm. The graph shows the heterochromatin content in wild-type, nrpd1, met1, pol V subunits and drd1 mutants. The mean values and standard deviation are shown in the histogram calculated from Col (n  =  97), met1-1 (n  =  93), nrpd1 (n  =  127), nrpd2/nrpe2 (n  =  90), nrpe1 (n  =  171), drd1-6 (n  =  116).

Figure 3.

H3K9me2 Clustering at the Chromocenters Is Disrupted by nrpe1, drd1, and met1 Loss-of-Function Mutants. The panel shows representative images of immuno-stained interphase nuclei with H3K9me2 (red) combined with DNA-FISH to detect the 180-bp centromeric repeats (green). DNA was visualized by DAPI (gray) and the size bar indicates 5 μm.

Figure 4.

Specific Centromere Sequences Are De-Repressed in pol V and drd1 Mutants. (A) Strand-specific centromere transcription was evaluated by RT–PCR using primers specific for different subsets of 180-bp repeat families: forward conserved (CEN-Fc), reverse conserved (CEN-Rc), forward non-conserved (CEN-F), and reverse non-conserved (CEN-R) 180-bp repeats. (B) Different centromere sequences are up-regulated in nrpe1. The figure displays aligned centromere transcript consensus sequences obtained by cloning RT–PCR products from the following mutants: nrpe1 CEN-Fc 180-bp (n  =  12), nrpe1 CEN-F (n  =  15), and met1 CEN-F 180-bp (n  =  10). (C) Transposable element transcription detected by RT–PCR. (D) Northern blot analysis of centromere-derived siRNAs (siCEN). Ethidium bromide-stained 5S rRNA resolved by agarose gene electrophoresis serves as a loading control.

Figure 5.

Pericentromere-Repeat RNAs Localized by RNA-FISH during Interphase. The probe specifically detects forward transcripts (CEN-F, red). RNase A treatment prior to in-situ hybridization eliminates the FISH signals, indicating that the method predominantly detects RNA (bottom nuclei). DNA was DAPI-counterstained (gray). The size bar corresponds to 5 μm.

Figure 6.

Effects of RNase A Treatment on the Interphase Organization of Centromere Repeats (Green), H3K9me2 (Red), and 5-mC (Red) Detected by DNA-FISH and Immunostaining. Representative images of untreated and nuclease treated (+RNase) nuclei are shown. The right-most nuclei depict immunostaining of HTR12 (red) and 180-bp pericentromeric repeats (green) in non-treated and RNase A-treated nuclei. DNA was visualized by DAPI (gray). Size bars indicate 5 μm.