Post on 22-Dec-2015
transcript
Non coding transcription by alternative RNA polymerases regulates
chromatin loop dynamics
Federico D. ARIELRAFV 2014
Non coding RNAs in root developmental plasticity
Rinn and Chang, 2012
Long non-coding RNAs: classification
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)
Campalans et al., 2004
Long non-coding RNAs: known functions (plants)
Col-0 35S ASCO
At2G34830
At2G46830
Bardou et al., 2014
RBP1 / ENOD40 RNA NSR / ASCO RNA
Medicago truncatula Arabidopsis thaliana
Franco-Zorrilla et al., 2007
Long non-coding RNAs: known functions (plants)
Heo and Sung, 2011
Long non-coding RNAs: known functions (plants)
Wierzbicki, 2012
24 nt
DCL3
Plant RNA-dependent DNA methylation
Histone deacetylation
Histone acetylation
Histone (de) methylation
Histone (de) methylation
DNA methylation
DNA demethylation
Histone variants
Open configuration Closed configuration
Euchromatin ↔ Heterochromatin
Kasschau et al., 2007
Densely packaged heterochromatin
Less densely packaged euchromatin
CENTROMERE
Chromatin conformation, siRNAs and Epigenetic Marks
Small RNAs
Caudron-Herger and Rippe, 2012
Genome Topology and RNAs
Genome Topology and gene expression regulation
In silico search of A. thaliana ncRNAs
76 lncRNAs
Different from rRNAs, UTR, pseudogene, 3’ cleavage
products
Molecular expertise
J. Hirsch et al., 2006B. BenAmor et al., 2009
Longest ORF < 210 nt
poor coding texture
Arabidopsis thaliana EST
NCBI(172k EST)
Arabidopsis thaliana mRNAs EMBL
(25k mRNAs)
Arabidopsis thaliana Full length cDNAs
URGV (18k mRNAs)
Clustering, Mapping
http://neomorph.salk.edu/epigenome/
The APOLO locus
PID APOLO
siRNAs
AUXIN REGULATED PROMOTER LOOP RNA
Wierzbicki, 2012
PID APOLO
DNA methylation
The APOLO locus
http://neomorph.salk.edu/epigenome/
AUXIN REGULATED PROMOTER LOOP RNA
Wierzbicki, 2012
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’RACEWT polIV/V
CIPNO CIP CIPNO CIP
200 bp
MWM
PolV-ChIP-seqDNA methylation 24nt siRNA production
ATMRP2APOLO
14596839 1460371014598867 14604619 14609779 14619644Chr 2 4960 5160
PID
The APOLO locus
APOLO and PID are induced by auxin
0 3 6 9 12 15 18 21 24
Time (h)
APOLOPID
Auxin kinetics (NPA-NAA)
WT
RNAi APOLO
WT
RNAi APOLO
APOLO PID
APOLO and PID
RNAi
RNAi
WT
WT
WT
RNAi APOLO
Sukumar et al., 2009
pid phenotype?
Angl
e of
cur
vatu
re (°
)
LB RB
P35S KanRRNAi APOLO
35S:RNAi-apolo
http://epigenomics.mcdb.ucla.edu/
The APOLO-PID region
PID APOLO
Time (h)
Auxin kinetics
http://epigenomics.mcdb.ucla.edu/LHP1/
homodimersLHP1 LHP1
Gaudin et al., 2001
LHP1 LHP1LHP1 LHP1LHP1 LHP1 ?
LHP1 binds to APOLO and 5’ PID
Time (h)
PID APOLO
Chromatin Conformation Capture (3C)
LHP1 mediates a chromatin loop between 5’ PID and APOLO, dependent on APOLO RNA levels
Louwers et al., 2009
659 1393M
WM
PCR1
PCR2
C +
C -
PCR1
PCR2
C +
C -
WT lhp1
APOLOPID
PCR1 = 707 bp214 493PCR2 = 2052 bp
C(+) = 600 bpBglIIBglII BglIIBglIIBglII
2000 bp1500 bp
700 bp
Rela
tive
loop
con
form
ation
(%)
WT
RNAi APOLO lhp1
Relative LH
P1-APOLO
interactionRela
tive
Loop
For
mati
on
LHP1-APOLO interaction correlates with loop dynamics
0 3 6 9 12 15 18 21 24
Fold
Cha
nge
0 3 6 9 12 15 18 21 24Time (h)
PID
Chromatin Isolation by RNA Purification (ChIRP)
LHP1-APOLO interaction correlates with loop dynamics
Chu et al., 2011
APOLOPID
A B C D E F G H I
Background
Sign
al /
Back
grou
nd R
atio
J
rdr2 dcl3 ago4nrpd2a
WT
Rela
tive
loop
form
ation
siRNA mediated DNA methylation and loop formation
Wierzbicki, 2012
Fold
Cha
nge
PID
0 3 6 9 12 15 18 21 24Time (h)
0 3 6 9 12 15 18 21 24
Time (h)
DNA methylation and Pol II transcription
WT
RNAi APOLO
.1
% o
f IN
PUT
MeDNA IP
Enzimatically-mediated demethylation?
rdd triple mutant (ros1-3, dml2-1, dml3-1)
APOLO locus
Rela
tive
Loop
For
mati
on
Active DNA methylation and loop opening
0 3 6 9 12 15 18 21 24
0 3 6 9 12 15 18 21 24
Time (h)
Fold
Cha
nge
PID
PID
Pol II and Pol V transcription of the APOLO locus
Ariel
Ariel et al., 2014
Thanks for your attention
Martin CrespiFederico ArielNatali Romero BarriosAurélie Christ
Moussa BenhamedDavid LatrasseTeddy Jegu