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.

Nucleosome is the basic unit of Chromatin

Heterochromatin vs Euchromatin

MAINTENANCE

DNA methylation in Arabidopsis

Law and Jacobsen. 2010. Nature Reviews

ESTABLISMENT

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

Methods for DNA methylation detection

1. DNA methylation sensitive enzymes

HRS60 repeats

monomer

Cytosine methylation

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

SOUTHERN BLOT USING DNA METHYLATION SENSITIVE RESTRICTION ENZYMES

Undigested MspI Hpa II Sau3A I

2. DNA methylation dependent enzymes

McrBC recognition site

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

*

IR V2

CP

meCHH

meCG

meCHG

CHH

CG

CHG

TYLCSVRep

C2 C3

V2

CP

IR C4

TYLCSV DNA methylation levels

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

*

ChIP: Chromatin immunoprecipitation

PTGS TGS

Cosuppression: Gene silencing discovery

trans DNA methylation

PTGS TGS

• 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

Ossowiski et al., 2008

Gene silencing and Biotechnology

Ossowiski et al., 2008

RNAi

Ossowiski et al., 2008

Gene silencing 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.

Ossowiski et al., 2008

Gene silencing and Biotechnology

miRNAs Biogenesis

Tiwari et al., 2014

• Highly specific • Minimizing off-target

effects

Delivering of amiRNAs

Gene silencing in plants using artificial miRNAs (amiRNAs)

Tiwari et al., 2014

VIGS and amiRNAs

2013

Chellapan et al., 2010

Micro RNAs (miRNAs) mediate DNA methylation

Wu et al., 2010

24nt miRNAs Biogenesis

How generate amiRNAs to induce DNA methylation?

Mutation of 21nt miRNA

Replace of 24nt miRNA

X

OK

VIGS induced DNA methylation

Otagaki et al., 2011

Ossowiski et al., 2008

RNAi

Tiwari et al., 2014

hpRNAs

Kanasawa et al., 2010

Jones et al., 2001

35S GFP 35S GFP

TRV vector

VIGS-mediated TGS silencing

35S

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)

GRACIAS POR SU ATENCIÓN

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

Kanasawa et al., 2010

Saze et al., 2012

Las plantas son atacadas por diversos patógenos.

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

Jones et al., 2001

Tool for TGS suppression assay

TRV TRV:sulfur

35S

Epigenetic effect?

Wu et al., 2010

Micro RNAs (miRNAs) mediate DNA methylation

Tiwari et al., 2014

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

**

Ossowiski et al., 2008

RNAi

Histone code