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Metilación de ADN dirigida por ARN (ARNmi,
ARNi y horquilla RNA para metilación de ADN)
Dr. Edgar Rodríguez Negrete
CINVESTAV-IRAPUATO
10:00 am, Ciudad de México, México.
31 de octubre de 2014
Ciclo de Seminarios en Nuevas Técnicas de
Mejoramiento Genético
Edgar Antonio Rodríguez Negrete OCTUBRE-2014
Metilación de DNA dirigida por RNA (miRNA, RNAi y
horquilla de RNA para inducir metilación de DNA)
GENE SILENCING.
Developmental and physiological programs, control of transposons
mobility (de novo DNA and histone methylation,
heterochromatin establishment)
Defense mechanism against viruses
Gene silencing pathways
Li and Ding., 2006
(PTGS 21-22 nt)
cytoplasm
(TGS, 24 nt)
nucleus
21-22 nt 24 nt
EPIGENETICS
• The term epigenetics refers to heritable changes in gene expression (active versus inactive genes) that does not involve changes to the underlying DNA sequence; a change in phenotype without a change in genotype.
• Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state.
Arabidopsis ( 5meC) DNA methyltranferases
ESTABLISHMENT
MAINTENANCE
CG Methylation CHG Methylation CHH Methylation
MET1
DDM1 HDA6
CMT3
H3K9me&H3K27me siRNAs
DRM1/2
siRNAs
DRM1/2
siRNAs siRNAs siRNAs
DRM1/2 DRM1/2 DRM1/2 MET1
MET1&CMT3: maintenance
DRM 1/2: de novo
Modified of Schöb and Grossniklaus. 2006. Cell
Experimental design
HpaII
CCGG
CCGG
CH3
No digest
Digest
CCGG
CH3
No digest
Hpa II activity is impaired when
cytosines are methylated
HpaII HpaII HpaII HpaII HpaII HpaII
146 pb 29 pb
183 pb
Methylated cytosine
Non-methylated cytosine
Silencing of key methylation proteins revert silencing of GFP reporter gene
0
20
40
60
80
100
120
MOCK TRV TRV-MET1 TRV-CMT3% o
f m
eth
ylat
ed m
ole
cule
s 35S prom
Mock 4/4 TRV 4/4 TRV-MET1 4/4 TRV-CMT3 4/4
35S GFP 35S GFP
DNA bisulfite treatment
mC C
C U
Bisulfite sequencing primers
3. Bisulfite sequencing
PCR, cloning, and individual clone sequencing
Methylated cytosines
Unmethylated cytosines
TYLCSV_21dpi
TYLCSV-C4m_42dpi
TYLCSV ATG GGC AAC CTC ATC TCC ACA ………
ACG GGC AAC CTC ATC TCC ACA ……… TYLCSV-C4m
TYLCSV C4 mutant
0
100
200
300
1 2 3 4 5 6 7 8 9 10 11 12 13
TYLCSV
7 14 21 28 42dpi
TYLCSV
DN
A v
iral
35 7 14 21 28 42dpi
TYLCSV-C4m 35
qPCR
TYLCSV
C4
*
TYLCSV_21dpi
IR V2
CP
meCHH
meCG
meCHG
CHH
CG
CHG
TYLCSVRep
C2 C3
V2
CP
IR C4
TYLCSV-C4m_35dpi
IR V2
CP
TYLCSV C4 mutation causes IR hypermethylation
TYLCSVRep
C2 C3
V2
CP
IR C4
*
0
10
20
30
40
1 2 3
TYLCSV
TYLCSV-C4m
CG CHG CHH
% 5
me C
TYLCSV C4 mutation causes IR hypermethylation
TYLCSVRep
C2 C3
V2
CP
IR C4
*
• Reverse genetics approaches (loss-of-function-phenotype)
• Ornamental plants modification
• Plant disease resistance (bacteria, fungi, virus)
• Pest resistance
• Crops quality improvement (seedless-ness, enhancing of post-harvest life)
• Abiotic stress resistance (drought, cold/heat, salt tolerance)
RNAi and Biotechnology
Tiwari et al., 2014 Unver and Budak 2009
Virus Induced Gene Silencing (VIGS)
• VIGS is a simple, rapid and transformation free tech- nology.
• VIGS serves as a powerful tool for studying gene functions and metabolic pathways.
• VIGS vectors are mainly derived from viruses such as tobacco mosaic virus (TMV), potato virus X (PVX), tobacco rattle virus (TRV), cabbage leaf curl virus (CaLCuV), tomato yellow leaf curl china virus (TYLCV)
amiRNA 1
siRNA 2
hpRNA 3
RNA-directed DNA methylation and biotechnology
• Primary DNA sequence is not altered.
• DNA methylation is highly specific.
• DNA methylation is heritable.
• DNA methylation is reversible
• Non coding RNA from promoters avoid the production of allergenic peptides.
0
20
40
60
80
100
16C 16C-TGS G 16C-TGS R
% O
F M
ETH
YLA
TED
CY
TOSI
NES
35S 5´
0
20
40
60
80
100
16C 16C-TGS G 16C-TGS R
% O
F M
ETH
YLA
TED
CY
TOSI
NES
35S C
0
20
40
60
80
100
16C 16C-TGS G 16C-TGS R
% O
F M
ETH
YLA
TED
CY
TO
SIN
ES 35S 3´
CHOP-qPCR. McRBC. 35S methylation level analysis.
Ruiz-Ferrer and Voinnet., 2010
El silenciamiento génico como mecanismo de defensa
Postranscriptional gene silencing (PTGS)
Transcriptional gene silencing(TGS)
Tiwari et al., 2014 Unver and Budak 2009
Virus Induced Gene Silencing (VIGS)
• VIGS is a simple, rapid and transformation free tech- nology.
• VIGS serves as a powerful tool for studying gene functions and metabolic pathways.
• VIGS vectors are mainly derived from viruses such as tobacco mosaic virus (TMV), potato virus X (PVX), tobacco rattle virus (TRV), cabbage leaf curl virus (CaLCuV), tomato yellow leaf curl china virus (TYLCV)
amiRNA 1
siRNA 2
hpRNA 3
ACRE (Advisory Committee on Releases to the Environment) advice: New technique used in plant breeding
PepGMV systemic infection induces DNA demethylation in 16C-TGS plants
0
20
40
60
80
100
120
PepGMV Mock
% O
F M
ETH
YLA
TED
CY
TOSI
NES
*
0
10
20
30
40
50
60
70
80
90
100
% O
F M
ETH
YLA
TED
CY
TOC
INES
35S GFP
DNA hypermethylation
No GFP expression
35S GFP
DNA hypomethylation
GFP expression
Componentes del sistema pasivo
tricoma
cutícula
Metabolitos preformados: -fenoles -alcaloides -glicósidos -saponinas -taninos -resinas
Proteínas de defensa: -hidrolasas -inhibidores enzimáticos -unión a quitina -péptidos antifúngicos -tioninas -inactivadoras de ribosomas
Componentes del sistema
activo
Sistemas de defensa en plantas.
-calosa -suberina -calcio -lignina
Respuesta Hipersensible (HR)
Silenciamiento génico mediado por RNA
PepGMV systemic infection induces DNA demethylation in 16C-TGS plants
35S GFP
DNA hypermethylation
No GFP expression
35S GFP
DNA hipomethylation
GFP expression
Hinf I
GANTC
GANTC
CH3
No digest
Digest
Hinf I activity is impaired when
cytosines are methylated
Undigested control 30 cycles
Dig. Hinf I 35 cycles
1 2 3 4 5 6 7 8 1 2
PepGMV Mock
Arabidopsis ( 5meC) DNA methyltranferases
ESTABLISHMENT
MAINTENANCE
CG Methylation CHG Methylation CHH Methylation
MET1
DDM1 HDA6
CMT3
H3K9me&H3K27me siRNAs
DRM1/2
siRNAs
DRM1/2
siRNAs siRNAs siRNAs
DRM1/2 DRM1/2 DRM1/2 MET1
MET1&CMT3: maintenance
DRM 1/2: de novo
Modified of Schöb and Grossniklaus. 2006. Cell
Virus Induced Gene Silencing (VIGS) of NB MET1 and NB CMT3 transcripts
TRV Vector TRV TRV:sulfur
NB MET1, NB CMT3
Rel
ativ
e m
RN
A le
vels
TRV TRV-MET1
0
20
40
60
80
100
120
140MET1
**
0
20
40
60
80
100
120CMT3
TRV-CMT3 TRV
**