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Non coding transcription by alternative RNA polymerases regulates chromatin loop dynamics

Non coding transcription by alternative RNA polymerases regulates chromatin loop dynamics. Non coding RNAs in root developmental plasticity. Federico D. ARIEL RAFV 2014. Long non- coding RNAs : classification. Rinn and Chang, 2012. Long non- coding RNAs : known functions ( animals ).

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Non coding transcription by alternative RNA polymerases regulates chromatin loop dynamics

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

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

  3. Long non-codingRNAs: knownfunctions (animals) PANDARNA (Hung et al., 2011) HOTAIRRNA (Tsai et al., 2010) XIST, AIRRNAs… (Rinn et al., 2007) HOTTIPRNA (Tsai et al., 2010) Rinn and Chang, 2012

  4. At2G34830 At2G46830 35S ASCO Col-0 Long non-codingRNAs: knownfunctions (plants) Medicago truncatula Arabidopsis thaliana RBP1 / ENOD40 RNA NSR / ASCO RNA Campalans et al., 2004 Bardou et al., 2014

  5. Long non-codingRNAs: knownfunctions (plants) Franco-Zorrilla et al., 2007

  6. Long non-codingRNAs: knownfunctions (plants) Heo and Sung, 2011

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

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

  9. Chromatin conformation, siRNAs and Epigenetic Marks Densely packaged heterochromatin CENTROMERE Less densely packaged euchromatin Small RNAs Kasschauet al., 2007

  10. GenomeTopology and RNAs Caudron-Herger and Rippe, 2012

  11. GenomeTopology and gene expression regulation

  12. Arabidopsis thaliana EST NCBI (172k EST) Arabidopsis thaliana mRNAs EMBL (25k mRNAs) Arabidopsis thaliana Full length cDNAs URGV (18k mRNAs) Longest ORF < 210 nt poor coding texture In silico search of A. thaliana ncRNAs Clustering, Mapping Different from rRNAs, UTR, pseudogene, 3’ cleavage products Molecular expertise J. Hirsch et al., 2006 B. BenAmor et al., 2009 76 lncRNAs

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

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

  15. The APOLO locus istranscribed by Pol II and Pol IV/V DNA methylation 24nt siRNA production PolV-ChIP-seq Col0 polV http://neomorph.salk.edu/pol_epigenomes/ APOLOistranscribedalso by RNA Pol II 5’RACE WT polIV/V MWM 200 bp CIP CIP NO CIP NO CIP

  16. The APOLO locus PID ATMRP2 APOLO Chr 2 5160 4960 14596839 14609779 14619644 14604619 14603710 14598867

  17. APOLO and PID are induced by auxin Auxin kinetics (NPA-NAA) APOLO PID 0 3 6 9 12 15 18 21 24 Time (h)

  18. APOLO and PID RNAi WT RNAi WT APOLO PID P35S KanR LB RB 35S:RNAi-apolo RNAi APOLO Angle of curvature (°) pidphenotype? Sukumar et al., 2009 WT WT WT RNAi APOLO RNAi APOLO RNAi APOLO

  19. The APOLO-PIDregion Auxin kinetics Time (h) PID APOLO http://epigenomics.mcdb.ucla.edu/

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

  21. LHP1 mediates a chromatinloopbetween 5’ PID and APOLO, dependent on APOLO RNA levels Chromatin Conformation Capture (3C) Relative loop conformation (%) APOLO PID C(+) = 600 bp BglII BglII BglII BglII BglII PCR1 = 707 bp 214 493 WT lhp1 PCR2 = 2052 bp 659 1393 RNAi APOLO WT lhp1 MWM PCR1 PCR2 PCR1 PCR2 C + C + C - C - Louwers et al., 2009 2000 bp 1500 bp 700 bp

  22. LHP1-APOLO interaction correlateswithloopdynamics Relative LHP1-APOLO interaction Relative Loop Formation 0 3 6 9 12 15 18 21 24 PID Fold Change 0 3 6 9 12 15 18 21 24 Time (h)

  23. LHP1-APOLO interaction correlateswithloopdynamics Chromatin Isolation by RNA Purification (ChIRP) PID APOLO A B C D E F G H I J Signal / Background Ratio Background Chu et al., 2011

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

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

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

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

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

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