High-throughput bisulfite sequencing reveals relationships between gene expression and DNA methylation in the bivalve, Crassostrea gigas Mackenzie Gavery.

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Presentation transcript:

High-throughput bisulfite sequencing reveals relationships between gene expression and DNA methylation in the bivalve, Crassostrea gigas Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences Seattle, WA USA

Open Science Slides, links and related materials can be found at: oystergen.es/norway

Background DNA methylation in invertebrates Results Characterization of DNA methylation in Pacific oysters Discussion & Future Directions Outline

GENES (DNA) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens temperature Background

GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens Background temperature

GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT nutrition pathogens Background temperature

Me C G C G DNA Methylation

Me C G C G Gene A TF X DNA Methylation

Me C G C G Gene A TF X DNA Methylation VERTEBRATE

Gene A TF X VERTEBRATE DNA Methylation

Gene A TF X VERTEBRATE Gene A INVERTEBRATE DNA Methylation

Gene A TF X VERTEBRATE Gene A INVERTEBRATE DNA Methylation

Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation

Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation

Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation Ancestral pattern

Gene A TF X VERTEBRATE Gene A ? INVERTEBRATE DNA Methylation Ancestral pattern Important regulator of phenotype

DNA Methylation Objectives: Characterize DNA methylation in C. gigas Gain an understanding of the functional role

Part 1

Approach In silico analysis Experimental analysis: MBD-Seq

CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

CpG O/E Predicted degree of DNA methylation Measured degree of DNA methylation Enrichment level in MBD library (Gavery & Roberts, 2010) (Roberts & Gavery, 2011) Part 1: Results

Part 2

genomic DNA Approach High-throughput bisulfite sequencing: Gill tissue Additional resources: RNA-seq data: gill tissue (Zhang et al, 2012)

genomic DNA Part 2 Approach High-throughput bisulfite sequencing: Gill tissue Additional resources: RNA-seq data: gill tissue (Zhang et al, 2012)

> 2.5 million CG dinucleotides Part 2: Results

scaffold 86 (Galaxy Trackster) Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0%

Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

Part 2: Results ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster)

Distribution in genomic elements Part 2: Results

Distribution in genomic elements Part 2: Results

Relationship with expression

RNA-Seq data (Zhang et al., 2012) Part 2: Results Relationship with expression Gene expression (Deciles) DNA methylation/gene

Part 3

Approach: High-throughput bisulfite sequencing: Gill tissue Part 3

Approach: High-throughput bisulfite sequencing: Gill tissue Male gamete (sperm) tissue Part 3

Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

Part 3: Results genes %methylation: gill CG %methylation: sperm 0bp 6,000bp

Part 3: Results Identify differential methylation (DM)

Part 3: Results Identify differential methylation (DM)

Part 3: Results Identify differential methylation 1/3 of all genes had DM

Summary

unmethylatedmethylated

Gene function: Summary unmethylatedmethylated

Gene function: Summary unmethylated inducible housekeeping methylated

Gene function: Expression: Summary unmethylated inducible housekeeping methylated

Gene function: Expression: Summary unmethylated inducible low housekeeping high methylated

Gene function: Expression: Summary unmethylated inducible low housekeeping high methylated Tissue specific methylation:

Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated patterns change at fine-scale

Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated patterns change at fine-scale Role of methylation in introns:

Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated unknown Role of methylation in introns: patterns change at fine-scale

Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated Role of methylation in inter-genic regions: unknown Role of methylation in introns: patterns change at fine-scale

Gene function: Expression: Tissue specific methylation: Summary unmethylated inducible low housekeeping high methylated Role of methylation in inter-genic regions: unknown Role of methylation in introns: unknown patterns change at fine-scale

Next Steps GENES (DNA) EPIGENOME (DNA methylation)

Explore relationships between DNA methylation and alternative splicing Determine if DNA methylation mediates response to environmental stress in shellfish Tool development: DNA tiling array (MeDIP-Chip) Next Steps GENES (DNA) EPIGENOME (DNA methylation)

Acknowledgements Roberts Lab: Steven Roberts Samuel White Brent Vadopalas Emma Timmins-Schiffman Claire Ellis Lisa Crosson Taylor Shellfish: Jonathan Davis Molly Jackson website: oystergen.es/norway