Ciclo de Seminarios en Nuevas Técnicas de Mejoramiento Genético 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
Metilación de DNA dirigida por RNA (mirna, RNAi y horquilla de RNA para inducir metilación de DNA) Edgar Antonio Rodríguez Negrete OCTUBRE-2014
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 21-22 nt 24 nt (TGS, 24 nt) nucleus (PTGS 21-22 nt) cytoplasm Li and Ding., 2006
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
DNA methylation in Arabidopsis ESTABLISMENT MAINTENANCE Law and Jacobsen. 2010. Nature Reviews
Arabidopsis ( 5me C) DNA methyltranferases ESTABLISHMENT DRM 1/2: de novo sirnas sirnas sirnas MET1 DRM1/2 DRM1/2 DRM1/2 CG Methylation CHG Methylation CHH Methylation MET1 CMT3 DRM1/2 DRM1/2 DDM1 HDA6 H3K9me&H3K27me sirnas sirnas MAINTENANCE MET1&CMT3: maintenance Modified of Schöb and Grossniklaus. 2006. Cell
Methods for DNA methylation detection
1. DNA methylation sensitive enzymes monomer Cytosine methylation HRS60 repeats
Experimental design HpaII HpaII HpaII HpaII HpaII HpaII 183 pb 146 pb 29 pb Methylated cytosine Non-methylated cytosine Hpa II activity is impaired when cytosines are methylated HpaII No digest CH3 CCGG CCGG Digest No digest CH3 CCGG
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 % of methylated molecules 35S GFP 35S GFP Mock 4/4 TRV 4/4 TRV-MET1 4/4 TRV-CMT3 4/4 120 35S prom 100 80 60 40 20 0 MOCK TRV TRV-MET1 TRV-CMT3
3. Bisulfite sequencing DNA bisulfite treatment mc C C U Bisulfite sequencing primers Methylated cytosines PCR, cloning, and individual clone sequencing Unmethylated cytosines
TYLCSV C4 mutant DNA viral TYLCSV TYLCSV-C4m ATG GGC AAC CTC ATC TCC ACA ACG GGC AAC CTC ATC TCC ACA C4 * TYLCSV 300 200 TYLCSV_21dpi 100 TYLCSV qpcr 0 7 14 21 28 35 42dpi 7 14 21 28 35 42dpi 1 2 3 4 5 6 7 8 9 10 11 12 13 TYLCSV TYLCSV-C4m TYLCSV-C4m_42dpi
TYLCSV DNA methylation levels C4 IR V2 Rep TYLCSV CP C2 C3 IR V2 CP CG CHG CHH mecg mechg mechh
TYLCSV C4 mutation causes IR hypermethylation TYLCSV_21dpi C4 IR V2 Rep TYLCSV CP C2 C3 IR TYLCSV-C4m_35dpi V2 CP CG CHG CHH mecg mechg mechh Rep C4 * IR TYLCSV V2 CP IR V2 CP C2 C3
TYLCSV C4 mutation causes IR hypermethylation % 5me C 40 30 Rep C4 * IR TYLCSV V2 CP 20 TYLCSV TYLCSV-C4m C2 C3 10 0 CG 1 CHG 2 CHH 3
ChIP: Chromatin immunoprecipitation
TGS PTGS
Cosuppression: Gene silencing discovery
trans DNA methylation
TGS PTGS
RNAi and Biotechnology Reverse genetics approaches (loss-of-functionphenotype) 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)
Gene silencing and Biotechnology Ossowiski et al., 2008
RNAi Ossowiski et al., 2008
Gene silencing and Biotechnology Ossowiski et al., 2008
Virus Induced Gene Silencing (VIGS) 1 amirna 2 sirna 3 hprna VIGS is a simple, rapid and transformation free technology. 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) Tiwari et al., 2014 Unver and Budak 2009
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.
Gene silencing and Biotechnology Ossowiski et al., 2008
mirnas Biogenesis Tiwari et al., 2014
Delivering of amirnas Highly specific Minimizing off-target effects
Gene silencing in plants using artificial mirnas (amirnas) Tiwari et al., 2014
2013 VIGS and amirnas
Micro RNAs (mirnas) mediate DNA methylation Chellapan et al., 2010
24nt mirnas Biogenesis Wu et al., 2010
How generate amirnas to induce DNA methylation? Mutation of 21nt mirna Replace of 24nt mirna OK X
VIGS induced DNA methylation Otagaki et al., 2011
RNAi Ossowiski et al., 2008
hprnas Tiwari et al., 2014
Kanasawa et al., 2010
Jones et al., 2001 VIGS-mediated TGS silencing TRV vector 35S 35S GFP 35S GFP
% OF METHYLATED CYTOSINES CHOP-qPCR. McRBC. 35S methylation level analysis. % OF METHYLATED CYTOSINES % OF METHYLATED CYTOSINES 100 80 35S 5 100 80 35S C 100 80 35S 3 60 60 60 40 40 40 20 20 20 0 16C 16C-TGS G 16C-TGS R 0 16C 16C-TGS G 16C-TGS R 0 16C 16C-TGS G 16C-TGS R
El silenciamiento génico como mecanismo de defensa Transcriptional gene silencing(tgs) Postranscriptional gene silencing (PTGS) Ruiz-Ferrer and Voinnet., 2010
GRACIAS POR SU ATENCIÓN
Virus Induced Gene Silencing (VIGS) 1 amirna 2 sirna 3 hprna VIGS is a simple, rapid and transformation free technology. 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) Tiwari et al., 2014 Unver and Budak 2009
ACRE (Advisory Committee on Releases to the Environment) advice: New technique used in plant breeding
% OF METHYLATED CYTOCINES % OF METHYLATED CYTOSINES PepGMV systemic infection induces DNA demethylation in 16C-TGS plants 35S GFP 35S GFP DNA hypermethylation No GFP expression DNA hypomethylation GFP expression 100 90 80 70 60 50 40 30 20 10 0 120 100 80 60 40 20 0 * PepGMV Mock
Kanasawa et al., 2010
Saze et al., 2012
Las plantas son atacadas por diversos patógenos.
Sistemas de defensa en plantas. Componentes del sistema activo -calosa -suberina -calcio -lignina Componentes del sistema pasivo tricoma cutícula Respuesta Hipersensible (HR) Silenciamiento génico mediado por RNA Metabolitos preformados: -fenoles -alcaloides -glicósidos -saponinas -taninos -resinas Proteínas de defensa: -hidrolasas -inhibidores enzimáticos -unión a quitina -péptidos antifúngicos -tioninas -inactivadoras de ribosomas
Tool for TGS suppression assay TRV TRV:sulfur 35S Epigenetic effect? Jones et al., 2001
Micro RNAs (mirnas) mediate DNA methylation Wu et al., 2010
Tiwari et al., 2014
PepGMV systemic infection induces DNA demethylation in 16C-TGS plants 35S GFP 35S GFP DNA hypermethylation No GFP expression DNA hipomethylation GFP expression Hinf I activity is impaired when cytosines are methylated PepGMV Mock 1 2 3 4 5 6 7 8 1 2 Hinf I GANTC Digest No digest CH3 GANTC Dig. Hinf I 35 cycles Undigested control 30 cycles
Arabidopsis ( 5me C) DNA methyltranferases ESTABLISHMENT DRM 1/2: de novo sirnas sirnas sirnas MET1 DRM1/2 DRM1/2 DRM1/2 CG Methylation CHG Methylation CHH Methylation MET1 CMT3 DRM1/2 DRM1/2 DDM1 HDA6 H3K9me&H3K27me sirnas sirnas MAINTENANCE MET1&CMT3: maintenance Modified of Schöb and Grossniklaus. 2006. Cell
Virus Induced Gene Silencing (VIGS) of NB MET1 and NB CMT3 transcripts Relative mrna levels TRV Vector TRV TRV:sulfur NB MET1, NB CMT3 140 120 MET1 120 100 CMT3 100 80 60 40 20 ** 80 60 40 20 ** 0 TRV TRV-MET1 0 TRV TRV-CMT3
RNAi Ossowiski et al., 2008
Histone code