Review of previous work: Overview of previous work on DNA methylation and chromatin dynamics

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1 CONTENTS Preface Acknowledgements Lists of Commonly Used Abbreviations Review of previous work: Overview of previous work on DNA methylation and chromatin dynamics Introduction Enzymes involved in DNA methylation in animals Dnmt1 (DNA methyltransferase1) Dnmt3 (DNA methyltransferase3) Enzymes involved in DNA methylation in plants DRM (Domain Rearranged Methyltransferase) MET1 (methyltransferase1) CMT (chromomethyltransferase) Dnmt2 DNA methylation in plants Silencing of transposable elements Gene body methylation Promoter DNA methylation DNA methylation and genomic imprinting Methyl CpG Binding Domain (MBD) proteins in animals MeCP2 MBD1 MBD2 MBD3 MBD4 Kaiso Methyl CpG Binding Domain (MBD) proteins in plants VIM (Variant In Methylation) Role of chromatin remodeling in gene silencing Histone methylation Lysine methylation H3K9 methylation is a silencing mark linked to DNA methylation

2 Role of H3K27 methylation in heterochromatin formation H3K4 methylation H3K36 methylation Arginine methylation Histone demethylation Histone acetylation Histone deacetylation Histone ubiquitination Histone phosphorylation and dephosphorylation ADP-ribosylation RNA-directed DNA Methylation (RdDM) Components of the RNA-directed DNA methylation pathway Targets of RdDM RdDM and Chromatin remodeling DNA demethylation Application of the study MATERIALS AND METHODS Plant material and growth condition Plant growth media preparation Seed sterilization and inoculation Seed inoculation in Soilrite pots Preparation of constructs for Arabidopsis transformation PCR amplification of AtMBD genes Designing of artificial microrna based silencing vector Ligation Preparation of E. coli competent cells Bacterial transformation Screening of desired clone Mini preparation of plasmid DNA Analysis of constructs

3 Arabidopsis transformation and generation of transgenic plants Preparation of Agrobacterium competent cell Transformation of chemically competent Agrobacterium cells Isolation of plasmids from Agrobacterium and selection of clones Floral dip transformation Selection of transgenic plants Analysis of transgenic Arabidopsis plants GUS histochemical assay Verifications of transgenic plants by PCR Transient expression by agroinfiltration Molecular analysis at DNA level Isolation of genomic DNA Methylation analysis Molecular analysis at transcript level Isolation of total RNA Northern analysis Semi-quantitative PCR Real Time PCR (Quantitative RT-PCR) Generation of double transgenic plants Yeast two hybrid screening Cloning of AtMBD genes (bait) in pgbkt7 vector Generation cdna of library Constriction of GAL4AD fusion library Preparation of yeast competent cell Transformations of yeast strain AH109 with ds cdna and SmaI linearized pgadt7- Rec vector Matting of bait and library α and β galactosidase activity Sub-cellular localization and Fluorescence Resonance Energy Transfer (FRET) analysis Cloning in psite Vectors

4 Preparation of gold particles Coating of gold particles with plasmid DNA Particle bombardment of onion epidermal cells CFP/YFP and FRET analysis Microarray cdna synthesis In vitro transcription and purification of arna Hybridation, washing and scanning RESULTS Role of Methyl CpG Binding Domain (MBD) proteins in RNA-mediated gene silencing Cloning and generation of AtMBD11 knockdown Arabidopsis transgenic plants Cloning and generation of AtMBD10 knockdown transgenic plants Construction of a vector for RNAi of gus gene and development of double transgenic plants Generation of transgenic Arabidopsis plants transformed with gus gene (uida) Silencing of gus reporter gene in double transgenic lines Identification of genes involved in RNA directed DNA Methylation Silencing of gus gene using GUSRNAi in mutant background Silencing of gus gene using microrna mediated silencing in mutant background Functional characterization of AtMBD4 Over-expression of AtMBD4 Generation of AtMBD4 over expressing transgenic Arabidopsis plants Characterization of AtMBD4 over-expressing transgenic plants Over-expression of AtMBD4 shows hypomethylation of

5 genomic DNA Characterization of atmbd4 mutant Molecular basis of late flowering phenotype in atmbd4 mutant Transcriptome analysis of atmbd4 mutant AtMBD4 is involved in phosphate starvation Genes involved in oxidative stress showed altered expression Up-regulation of heat stress-related genes in atmbd4 mutant Yeast two hybrid screening for AtMBD4 interacting proteins Florescence Resonance Energy Transfer (FRET) and co- localization of AtMBD4 and AtUBC36 AtMBD4 interacts with AtUBC36 in-vivo Functional characterization of AtMBD6 Over expression of AtMBD6 Generation of AtMBD6 over-expressing Arabidopsis transgenic plants Characterization of AtMBD6 over-expression transgenic plants Characterization of atmbd6 mutant Transcriptome analysis of atmbd6 mutant Decreased transcript level of light reaction-associated genes Up-regulation of phenylpropanoid, lignin and lignans-related genes Ethylene-mediated signaling Up-regulation of cell wall biogenesis genes Yeast two hybrid screening for AtMBD6 interacting proteins Florescence Resonance Energy Transfer (FRET) and co-localization of AtMBD6 with AtNTF2 and AtRPS2C AtMBD6 interacts with AtRPS2C in-vivo AtMBD6 interacts with AtNTF2 in-vivo Functional characterization of AtMBD10 Over-expression of AtMBD10

6 Generation of transgenic plants over-expressing AtMBD10 gene Characterization of AtMBD10 over-expressing transgenic lines Construction of an artificial microrna-based vector to silence AtMBD10 gene Yeast two hybrid screening for AtMBD10-interacting proteins Sub cellular localization and Florescence Resonance Energy Transfer (FRET) analysis of AtMBD10 and AtPAP14 AtMBD10 interacts with AtPAP14 Over-expression of AtMBD11 Characterization of AtMBD11 Generation of transgenic plants over-expressing AtMBD11 Microarray analysis of atmbd11 mutant Nuclear factors Post-translational modification Protein degradation Protein targeting Abiotic stress Chlorophyll biosynthesis and light reaction Lipid metabolism Carbohydrate metabolism Yeast two hybrid screening for AtMBD11-interacting proteins AtMBD11 interacts with At1g03250 DISCUSSIONS AtMBD6 is involved in small RNA mediated gene silencing AtMBD6 interacts with AtRPS2C AtMBD6 interacts with AtNTF2 AtMBD4 interacts with AtUBC36 ATMBD10 interacts with AtPAP14 Characterization of AtMBDs by their silencing Characterization of AtMBDs by their over-expression

7 Microarray analysis of atmbd4 mutant Microarray analysis of atmbd6 mutant Micraoaary analysis of atmbd11 mutant SUMMERY AND CONCLUSIONS TABLES REFERENCES