Transcript APOLO
Non coding RNAs transcription in root developmental by alternative RNA polymerases plasticityregulates chromatin loop dynamics Federico D. ARIEL RAFV 2014 Long non-coding RNAs: classification Rinn and Chang, 2012 Long non-coding RNAs: known functions (animals) PANDA RNA (Hung et al., 2011) XIST, AIR RNAs… (Rinn et al., 2007) HOTAIR RNA (Tsai et al., 2010) HOTTIP RNA (Tsai et al., 2010) Rinn and Chang, 2012 Long non-coding RNAs: known functions (plants) Medicago truncatula Arabidopsis thaliana RBP1 / ENOD40 RNA NSR / ASCO RNA At2G34830 At2G46830 Col-0 Campalans et al., 2004 35S ASCO Bardou et al., 2014 Long non-coding RNAs: known functions (plants) Franco-Zorrilla et al., 2007 Long non-coding RNAs: known functions (plants) Heo and Sung, 2011 Plant RNA-dependent DNA methylation 24 nt DCL3 Wierzbicki, 2012 Euchromatin ↔ Heterochromatin Open configuration Closed configuration DNA demethylation DNA methylation Histone acetylation Histone deacetylation Histone (de) methylation Histone (de) methylation Histone variants Chromatin conformation, siRNAs and Epigenetic Marks Densely packaged heterochromatin CENTROMERE Less densely packaged euchromatin Small RNAs Kasschau et al., 2007 Genome Topology and RNAs Caudron-Herger and Rippe, 2012 Genome Topology and gene expression regulation In silico search of A. thaliana ncRNAs Arabidopsis thaliana EST NCBI (172k EST) Arabidopsis thaliana mRNAs EMBL (25k mRNAs) Arabidopsis thaliana Full length cDNAs URGV (18k mRNAs) Clustering, Mapping Longest ORF < 210 nt poor coding texture Different from rRNAs, UTR, pseudogene, 3’ cleavage products Molecular expertise J. Hirsch et al., 2006 B. BenAmor et al., 2009 The APOLO locus AUXIN REGULATED PROMOTER LOOP RNA PID APOLO siRNAs http://neomorph.salk.edu/epigenome/ Wierzbicki, 2012 The APOLO locus AUXIN REGULATED PROMOTER LOOP RNA PID APOLO DNA methylation http://neomorph.salk.edu/epigenome/ Wierzbicki, 2012 The APOLO locus is transcribed by Pol II and Pol IV/V DNA methylation 24nt siRNA production PolV-ChIP-seq Col0 polV http://neomorph.salk.edu/pol_epigenomes/ APOLO is transcribed also by RNA Pol II 5’RACE WT polIV/V MWM 200 bp The APOLO locus PID Chr 2 5160 4960 14596839 ATMRP2 APOLO 14598867 14603710 14604619 14609779 14619644 APOLO and PID are induced by auxin Auxin kinetics (NPA-NAA) APOLO PID 0 3 6 9 12 15 Time (h) 18 21 24 APOLO and PID P35S RNAi APOLO RNAi KanR RNAi LB RB WT 35S:RNAi-apolo PID Angle of curvature (°) APOLO WT pid phenotype? Sukumar et al., 2009 The APOLO-PID region Auxin kinetics Time (h) PID http://epigenomics.mcdb.ucla.edu/ APOLO LHP1 binds to APOLO and 5’ PID LHP1 LHP1 homodimers Gaudin et al., 2001 Time (h) PID APOLO LHP1 LHP1 LHP1 http://epigenomics.mcdb.ucla.edu/LHP1/ LHP1 LHP1 LHP1 LHP1 mediates a chromatin loop between 5’ PID and APOLO, dependent on APOLO RNA levels Chromatin Conformation Capture (3C) 214 BglII BglIIBglII BglII PCR1 = 707 bp 493 659 PCR2 = 2052 bp 1393 Louwers et al., 2009 lhp1 PCR1 PCR2 C+ C- PCR1 MWM WT 2000 bp 1500 bp 700 bp C(+) = 600 bp PCR2 C+ C- BglII APOLO Relative loop conformation (%) PID 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 PID 0 3 6 9 12 15 Time (h) 18 21 24 LHP1-APOLO interaction correlates with loop dynamics Chromatin Isolation by RNA Purification (ChIRP) PID Signal / Background Ratio A Chu et al., 2011 B C APOLO D E F G H I J Background Wierzbicki, 2012 PID Fold Change Relative loop formation siRNA mediated DNA methylation and loop formation 0 3 6 9 12 15 Time (h) 18 21 24 DNA methylation and Pol II transcription APOLO locus % of INPUT MeDNA IP 0 3 6 9 12 15 18 Time (h) Enzimatically-mediated demethylation? rdd triple mutant (ros1-3, dml2-1, dml3-1) 21 24 Relative Loop Formation Active DNA methylation and loop opening 0 3 6 9 12 15 18 21 24 Fold Change PID PID 0 3 6 9 12 Time (h) 15 18 21 24 Pol II and Pol V transcription of the APOLO locus Ariel et al., 2014 Thanks for your attention Martin Crespi Federico Ariel Natali Romero Barrios Aurélie Christ Moussa Benhamed David Latrasse Teddy Jegu