1. Non coding transcription by alternative RNA polymerases regulates chromatin loop dynamics Federico D. ARIEL RAFV 2014 Non coding RNAs in root developmental plasticity

2. Rinn and Chang, 2012 Long non-coding RNAs: classification

3. Rinn and Chang, 2012 PANDA RNA (Hung et al., 2011)HOTAIR RNA (Tsai et al., 2010) XIST, AIR RNAs… (Rinn et al., 2007)HOTTIP RNA (Tsai et al., 2010) Long non-coding RNAs: known functions (animals)

4. Campalans et al., 2004 Long non-coding RNAs: known functions (plants) Col-0 35S ASCO At2G34830 At2G46830 Bardou et al., 2014 RBP1 / ENOD40 RNANSR / ASCO RNA Medicago truncatulaArabidopsis thaliana

6. Franco-Zorrilla et al., 2007 Long non-coding RNAs: known functions (plants)

7. Heo and Sung, 2011 Long non-coding RNAs: known functions (plants)

8. Wierzbicki, 2012 24 nt DCL3 Plant RNA-dependent DNA methylation

9. Histone deacetylation Histone acetylation Histone (de) methylation DNA methylation DNA demethylation Histone variants Open configuration Closed configuration Euchromatin ↔ Heterochromatin

10. Kasschau et al., 2007 Densely packaged heterochromatin Less densely packaged euchromatin CENTROMERE Chromatin conformation, siRNAs and Epigenetic Marks Small RNAs

11. Caudron-Herger and Rippe, 2012 Genome Topology and RNAs

12. Genome Topology and gene expression regulation

13. In silico search of A. thaliana ncRNAs Different from rRNAs, UTR, pseudogene, 3’ cleavage products Molecular expertise J. Hirsch et al., 2006 B. BenAmor et al., 2009 Longest ORF < 210 nt poor coding texture Longest ORF < 210 nt poor coding texture Arabidopsis thaliana EST NCBI (172k EST) Arabidopsis thaliana EST NCBI (172k EST) Arabidopsis thaliana mRNAs EMBL (25k mRNAs) Arabidopsis thaliana mRNAs EMBL (25k mRNAs) Arabidopsis thaliana Full length cDNAs URGV (18k mRNAs) Arabidopsis thaliana Full length cDNAs URGV (18k mRNAs) Clustering, Mapping

14. http://neomorph.salk.edu/epigenome/ The APOLO locus PIDAPOLO siRNAs AUXIN REGULATED PROMOTER LOOP RNA Wierzbicki, 2012

15. PIDAPOLO DNA methylation The APOLO locus http://neomorph.salk.edu/epigenome/ AUXIN REGULATED PROMOTER LOOP RNA Wierzbicki, 2012

16. The APOLO locus is transcribed by Pol II and Pol IV/V http://neomorph.salk.edu/pol_epigenomes/ Col0 polV APOLO is transcribed also by RNA Pol II 5’RACE WTpolIV/V CIP NO CIP CIP NO CIP 200 bp MWM PolV-ChIP-seqDNA methylation24nt siRNA production

17. ATMRP2 APOLO 14596839 14603710 14598867 14604619 1460977914619644 Chr 2 4960 5160 PID The APOLO locus

18. APOLO and PID are induced by auxin 03691215182124 Time (h) APOLO PID Auxin kinetics (NPA-NAA)

19. WT RNAi APOLO WT RNAi APOLO APOLOPID APOLO and PID RNAi WT RNAi APOLO Sukumar et al., 2009 pid phenotype? Angle of curvature (°) LB RB P35SKan R RNAi APOLO 35S:RNAi-apolo

20. http://epigenomics.mcdb.ucla.edu/ The APOLO-PID region PIDAPOLO Time (h) Auxin kinetics

21. http://epigenomics.mcdb.ucla.edu/LHP1/  homodimers LHP1 Gaudin et al., 2001 LHP1 LHP1 binds to APOLO and 5’ PID Time (h) PIDAPOLO

22. Chromatin Conformation Capture (3C) LHP1 mediates a chromatin loop between 5’ PID and APOLO, dependent on APOLO RNA levels Louwers et al., 2009 6591393 MWM PCR1PCR2 C + C - PCR1PCR2 C + C - WTlhp1 APOLOPID PCR1 = 707 bp 214493 PCR2 = 2052 bp C(+) = 600 bp BglII 2000 bp 1500 bp 700 bp Relative loop conformation (%) WT RNAi APOLO lhp1

23. Relative LHP1-APOLO interaction Relative Loop Formation LHP1-APOLO interaction correlates with loop dynamics 0 3 6 9 12 15 18 21 24 Fold Change 0 3 6 9 12 15 18 21 24 Time (h) PID

24. Chromatin Isolation by RNA Purification (ChIRP) LHP1-APOLO interaction correlates with loop dynamics Chu et al., 2011 APOLOPID ABCDEFGHI Background Signal / Background Ratio J

25. rdr2 dcl3 ago4 nrpd2a WT Relative loop formation siRNA mediated DNA methylation and loop formation Wierzbicki, 2012 Fold Change PID 0 3 6 9 12 15 18 21 24 Time (h)

26. 0 3 6 9 12 15 18 21 24 Time (h) DNA methylation and Pol II transcription WT RNAi APOLO.1 % of INPUT MeDNA IP Enzimatically-mediated demethylation? rdd triple mutant (ros1-3, dml2-1, dml3-1) APOLO locus

27. Relative Loop Formation Active DNA methylation and loop opening 0 3 6 9 12 15 18 21 24 Time (h) Fold Change PID

28. Pol II and Pol V transcription of the APOLO locus Ariel et al., 2014

29. Thanks for your attention Martin Crespi Federico Ariel Natali Romero Barrios Aurélie Christ Moussa Benhamed David Latrasse Teddy Jegu