Ciclo de Seminarios en Nuevas Técnicas de Mejoramiento Genético
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1 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
2 Metilación de DNA dirigida por RNA (mirna, RNAi y horquilla de RNA para inducir metilación de DNA) Edgar Antonio Rodríguez Negrete OCTUBRE-2014
3 GENE SILENCING. Developmental and physiological programs, control of transposons mobility (de novo DNA and histone methylation, heterochromatin establishment) Defense mechanism against viruses
4 Gene silencing pathways nt 24 nt (TGS, 24 nt) nucleus (PTGS nt) cytoplasm Li and Ding., 2006
5 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.
6 Nucleosome is the basic unit of Chromatin
7 Heterochromatin vs Euchromatin
8 DNA methylation in Arabidopsis ESTABLISMENT MAINTENANCE Law and Jacobsen Nature Reviews
9 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 Cell
10 Methods for DNA methylation detection
11 1. DNA methylation sensitive enzymes monomer Cytosine methylation HRS60 repeats
12 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
13 SOUTHERN BLOT USING DNA METHYLATION SENSITIVE RESTRICTION ENZYMES Undigested MspI Hpa II Sau3A I
14 2. DNA methylation dependent enzymes McrBC recognition site
15 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/ S prom MOCK TRV TRV-MET1 TRV-CMT3
16 3. Bisulfite sequencing DNA bisulfite treatment mc C C U Bisulfite sequencing primers Methylated cytosines PCR, cloning, and individual clone sequencing Unmethylated cytosines
17 TYLCSV C4 mutant DNA viral TYLCSV TYLCSV-C4m ATG GGC AAC CTC ATC TCC ACA ACG GGC AAC CTC ATC TCC ACA C4 * TYLCSV TYLCSV_21dpi 100 TYLCSV qpcr dpi dpi TYLCSV TYLCSV-C4m TYLCSV-C4m_42dpi
18 TYLCSV DNA methylation levels C4 IR V2 Rep TYLCSV CP C2 C3 IR V2 CP CG CHG CHH mecg mechg mechh
19 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
20 TYLCSV C4 mutation causes IR hypermethylation % 5me C Rep C4 * IR TYLCSV V2 CP 20 TYLCSV TYLCSV-C4m C2 C CG 1 CHG 2 CHH 3
21 ChIP: Chromatin immunoprecipitation
22 TGS PTGS
23 Cosuppression: Gene silencing discovery
24 trans DNA methylation
25 TGS PTGS
26 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)
27 Gene silencing and Biotechnology Ossowiski et al., 2008
28 RNAi Ossowiski et al., 2008
29 Gene silencing and Biotechnology Ossowiski et al., 2008
30 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
31 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.
32 Gene silencing and Biotechnology Ossowiski et al., 2008
33 mirnas Biogenesis Tiwari et al., 2014
34 Delivering of amirnas Highly specific Minimizing off-target effects
35 Gene silencing in plants using artificial mirnas (amirnas) Tiwari et al., 2014
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39 2013 VIGS and amirnas
40 Micro RNAs (mirnas) mediate DNA methylation Chellapan et al., 2010
41 24nt mirnas Biogenesis Wu et al., 2010
42 How generate amirnas to induce DNA methylation? Mutation of 21nt mirna Replace of 24nt mirna OK X
43 VIGS induced DNA methylation Otagaki et al., 2011
44 RNAi Ossowiski et al., 2008
45 hprnas Tiwari et al., 2014
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49 Kanasawa et al., 2010
50 Jones et al., 2001 VIGS-mediated TGS silencing TRV vector 35S 35S GFP 35S GFP
51 % OF METHYLATED CYTOSINES CHOP-qPCR. McRBC. 35S methylation level analysis. % OF METHYLATED CYTOSINES % OF METHYLATED CYTOSINES S S C S C 16C-TGS G 16C-TGS R 0 16C 16C-TGS G 16C-TGS R 0 16C 16C-TGS G 16C-TGS R
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53 El silenciamiento génico como mecanismo de defensa Transcriptional gene silencing(tgs) Postranscriptional gene silencing (PTGS) Ruiz-Ferrer and Voinnet., 2010
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55 GRACIAS POR SU ATENCIÓN
56 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
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59 ACRE (Advisory Committee on Releases to the Environment) advice: New technique used in plant breeding
60 % 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 * PepGMV Mock
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62 Kanasawa et al., 2010
63 Saze et al., 2012
64 Las plantas son atacadas por diversos patógenos.
65 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
66 Tool for TGS suppression assay TRV TRV:sulfur 35S Epigenetic effect? Jones et al., 2001
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68 Micro RNAs (mirnas) mediate DNA methylation Wu et al., 2010
69 Tiwari et al., 2014
70 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 Hinf I GANTC Digest No digest CH3 GANTC Dig. Hinf I 35 cycles Undigested control 30 cycles
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72 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 Cell
73 Virus Induced Gene Silencing (VIGS) of NB MET1 and NB CMT3 transcripts Relative mrna levels TRV Vector TRV TRV:sulfur NB MET1, NB CMT MET CMT ** ** 0 TRV TRV-MET1 0 TRV TRV-CMT3
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76 RNAi Ossowiski et al., 2008
77 Histone code
The world of non-coding RNA. Espen Enerly
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