1. Volume 55, Issue 3, Pages 383-396 (August 2014)
Noncoding Transcription by Alternative RNA Polymerases Dynamically Regulates an Auxin-Driven Chromatin Loop 
Federico Ariel, Teddy Jegu, David Latrasse, Natali Romero-Barrios, Aurélie Christ, Moussa Benhamed, Martin Crespi 
Molecular Cell 
Volume 55, Issue 3, Pages (August 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 55, 383-396DOI: (10.1016/j.molcel.2014.06.011)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
PID and APOLO Are Dynamically Coregulated in Response to Auxin and RNAi-Mediated APOLO Silencing Alters PID Expression
(A) Schematic representation of the PID-APOLO genomic region of chromosome 2 in A. thaliana Col-0 with the PID and APOLO genes indicated in red and the direction of transcription indicated with arrowheads. The PID intron is indicated with a peak. Numbers indicate the position along the chromosome (TAIR version 10). TSS is the transcription starting site.
(B) APOLO and PID transcript levels in response to auxin kinetics.
(C and D) (C) APOLO and (D) PID basal transcript levels in wild-type (WT) and RNAi-APOLO lines. Error bars represent the standard deviation of three biological replicates.
(E–I) Root phenotype of WT and RNAi-APOLO lines. (E and F) APOLO deregulation results in a delayed gravitropic response. Plates with 5-day-old seedlings were turned 90°, and 6 hr later the angle of root curvature was measured. (G) Representative pictures of individual plants in MS/2 agar. (H) Mean and standard deviation of root length measured 0, 7, and 14 days after germination, and (I) lateral root density of each genotype measured 0, 7, and 14 days after germination. The asterisk (∗) indicates a significant difference as determined by Student’s t test <0.05, n = 30. Error bars indicate the standard deviation of 15 plants. See also Figure S1 and Supplemental Experimental Procedures.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 2
The Epigenetic Landscape of the PID-APOLO Region Dynamically Changes in Response to Auxin
(A) Schematic representation of the PID-APOLO genomic region with the PID and APOLO genes indicated in red. A–J indicate the regions amplified by ChIP-qPCR in (B)–(D).
(B–D) (B) The repressive mark H3K27me3, (C) the activation mark H3K9Ac, and (D) the repressive mark H3K9me2 dynamic behavior in response to auxin, measured after 0, 3, 6, and 24 hr of NAA treatment, for the regions indicated in each panel, in wild-type (WT), rdr2, lhp1, and RNAi-APOLO lines. Error bars represent the standard deviation of three biological replicates. Profiles are shown for the regions with the most marked changes, but extended analyses are shown in Figures S3–S5. IgG was used as ChIP-qPCR control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 3
LIKE HETEROCHROMATIN PROTEIN 1, LHP1, Binds to the APOLO and PID Loci, Regulating Their Response to Auxin
(A) Schematic representation of the PID-APOLO genomic region with the PID and APOLO genes indicated in red. A–J indicate the regions amplified by ChIP-qPCR in (B).
(B) LHP1 dynamic deposition in response to auxin, measured after 0, 3, 6, and 24 hr of NAA treatment, for the regions indicated in each panel in wild-type seedlings. Profiles are shown for the regions with the most marked changes, and IgG was used as ChIP-qPCR control.
(C) APOLO and (D) PID transcript levels in wild-type (WT), an RNAi-APOLO line, and lhp1 mutant seedlings in response to a 24 hr auxin time course. Point 0 hr represents seedlings in NPA for 72 hr and then treated with NAA from 3 hr to 24 hr. Error bars represent the standard deviation of three biological replicates. See also Figures S2 and S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 4
A Dynamic Chromatin Loop Encompassing the PID Promoter Region Is Regulated by the APOLO lincRNA and LHP1
(A) Schematic representation of the PID-APOLO genomic region. A–J indicate the amplified regions in ChIRP-qPCR experiments in (E). Blue letters indicate the regions precipitated by ChIRP. BglII and AseI restriction sites and the oligonucleotides (arrows) used for 3C-qPCR (in B–D) also are indicated.
(B) 3C Interaction frequency considering 100% the amplification product of the BglII-3C using primers 1F and 1R. J was used a qPCR housekeeping product.
(C) Relative loop conformation measured by BglII-3C-qPCR, considering the WT level as 100%. Two independent lines RNAi-APOLO lines and lhp1: plant PRC1 component mutant. Error bars represent the standard deviation of three biological replicates.
(D) Auxin-responsive loop dynamics in WT (black, taken as 100% at time 0 hr) and lhp1 (red) seedlings compared with variations on LHP1-APOLO RNA interactions measured by RNA-IP (RIP; blue triangles). RIP values are expressed relative to WT 0 hr.
(E) Chromatin Isolation by APOLO RNA purification (APOLO ChIRP). APOLO-associated chromatin was detected by purifying APOLO RNA with biotinylated DNA probes. Background levels were determined using a set of probes matching the LacZ RNA. For APOLO, two sets of independent intercalated probes (odd and even numbered) were used for purification. Three biological replicates gave similar results, and one of the replicates is shown, for both sets of probes. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 5
RNA-Dependent DNA Methylation Is Crucial for Loop Formation and Impacts APOLO and PID Pol II Oscillating Transcription
(A) Relative loop conformation measured by BglII-3C-qPCR in wild-type seedlings (WT), as well as in nrpd2a (common subunit of PolIV and PolV), rdr2, dcl3, and ago4: si-RNA mediated DNA methylation pathway mutants, see Figure S1A.
(B and C) (B) PID and (C) APOLO transcript levels in the same lines as in (A), plus the triple mutant dcl2/3/4.
(D and E) (D) PID and (E) APOLO transcript levels in the 24 hr auxin treatment time course in WT and rdr2 seedlings.
(F) DNA methylation profile (5mC) of the whole PID-APOLO region in WT and RNAi-APOLO seedlings.
(G) Chromatin condensation profile measured by formaldehyde-assisted isolation of regulatory elements (FAIRE) in WT and RNAi-APOLO seedlings. Higher values express less condensed chromatin. Error bars represent the standard deviation of three biological replicates. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 6
Loop Dynamic Conformation Is Altered in the Demethylation Defective Mutant rdd and RNAi-APOLO Plants, Impairing the Response of APOLO and PID to Auxin
(A) DNA methylation (5mC) dynamics of the APOLO locus (region G of Figure 4A) in wild-type (WT), RNAi-APOLO, and the triple mutant rdd seedlings, in response to auxin treatment. Similar results were obtained for region H.
(B) Relative loop formation dynamics measured by BglII-3C-qPCR for WT, RNAi-APOLO, and rdd mutant seedlings. WT 0 hr was set at 100%.
(C and D) (C) PID and (D) APOLO transcript levels in response to auxin for WT, RNAi-APOLO, and rdd seedlings.
(E and F) (E) PID and (F) APOLO 5′ deposition of Pol II and Pol V throughout auxin treatment. Error bars represent the standard deviation of three biological replicates. See also Figures S6 and S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

9. Figure 7
APOLO Transcription by Alternative Polymerases Controls Chromatin Loop Dynamics to Fine-Tune PID Promoter Activity
(A) Auxin activates RDD-mediated APOLO DNA demethylation and opens the loop encompassing the PID promoter region.
(B) The H3K27me3 mark (pale blue symbols) decreases, whereas H3K9Ac levels (pink symbols) increase. Pol II divergent transcription leads to increased accumulation of PID and APOLO transcripts.
(C) Pol II APOLO transcripts gradually recruit LHP1 to mediate loop formation, whereas Pol IV/V transcription triggers DNA methylation (red symbols), and PRC2 likely redeposits repressive marks in the region.
(D) Pol II APOLO-LHP1 mediated loop is finally conformed and maintained by Pol IV/V-dependent DNA methylation to downregulate levels of PID transcripts.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions