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