APOLO

Report
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

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