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Regulation of environmental responses in the Pacific oyster, Crassostrea gigas Mackenzie Gavery University of Washington School of Aquatic and Fishery.

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Presentation on theme: "Regulation of environmental responses in the Pacific oyster, Crassostrea gigas Mackenzie Gavery University of Washington School of Aquatic and Fishery."— Presentation transcript:

1 Regulation of environmental responses in the Pacific oyster, Crassostrea gigas Mackenzie Gavery University of Washington School of Aquatic and Fishery Sciences Committee Meeting: April 15 th, 2014

2 Agenda: Research update Proposed timeline

3 Environmental Response in Oysters 20092010201120122013

4 Environmental Response in Oysters 20092010201120122013 Cyclooxygenase characterization

5 Environmental Response in Oysters 20092010201120122013 Gene expression: disease resistance Cyclooxygenase characterization

6 Environmental Response in Oysters 20092010201120122013 Whole transcriptome Chapter I. CBP: Part D Gene expression: disease resistance Cyclooxygenase characterization

7 Chapter I. RNA-Seq HIGH IMPACTLOW IMPACT

8 HIGH IMPACTLOW IMPACT 742 novel contigs 427 differentially expressed Chapter I. RNA-Seq

9 Ultra-short read technology is a powerful tool for gene discovery and expression analysis in organisms with limited genomic resources HIGH IMPACTLOW IMPACT 742 novel contigs 427 differentially expressed Chapter I. RNA-Seq

10 Environmental Response in Oysters 20092010201120122013 Whole transcriptome Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D

11 Environmental Response in Oysters 20092010201120122013 Chapter II. BMC Genomics Frontiers Whole transcriptome DNA methylation: single gene/in silico Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D

12 Chapter II. in silico analysis ‘housekeeping’ ‘inducible’ Predicted degree of DNA methylation

13 ‘housekeeping’ ‘inducible’ DNA methylation detected by targeted bisulfite sequencing and methylation specific PCR DNA methylation patterns are different between functionally distinct classes of genes Chapter II. in silico analysis Predicted degree of DNA methylation

14 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics

15 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics

16 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics

17 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring bringing it all together! meth & expression transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics

18 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring bringing it all together! meth & expression Ch III. PeerJ transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics

19 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring bringing it all together! meth & expression Chapter IV. Brief Func Gen transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics Ch III. PeerJ

20 Chapter III: whole methylome genomic DNA Approach High-throughput bisulfite sequencing

21 genomic DNA Approach High-throughput bisulfite sequencing Chapter III: whole methylome

22 genomic DNA Approach High-throughput bisulfite sequencing RNA-Seq data Chapter III: whole methylome

23 Gill tissue: > 2.5 million CG dinucleotides Chapter III: whole methylome

24 scaffold 86 (Galaxy Trackster) ex CG genes exons %methylation 0bp200,000bp 100% 0% Chapter III: whole methylome

25 ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster) Chapter III: whole methylome

26 ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster) Chapter III: whole methylome

27 ex CG genes exons %methylation 0bp200,000bp 100% 0% scaffold 86 (Galaxy Trackster) Chapter III: whole methylome

28 Distribution in genomic elements Chapter III: whole methylome

29 Distribution in genomic elements Chapter III: whole methylome

30 Relationship with expression Chapter III: whole methylome

31 RNA-Seq data (Zhang et al., 2012) Relationship with expression Gene expression (Deciles) DNA methylation/gene Chapter III: whole methylome

32 Relationships with other gene specific attributes? Chapter III: whole methylome

33 Relationships with other gene specific attributes? Spatial e.g. number of exons, length of mRNA Gene expression (Zhang et al 2012) : Mean expression level across 9 tissues Variation in expression between tissues (%CV) Chapter III: whole methylome

34 PC1 (50.2%) PC2 (26.1%) Chapter III: whole methylome

35 PC1 (50.2%) PC2 (26.1%) Chapter III: whole methylome

36 Chapter III: summary

37 unmethylatedmethylated Chapter III: summary

38 Gene function: unmethylatedmethylated Chapter III: summary

39 Gene function: unmethylated inducible housekeeping methylated Chapter III: summary

40 Gene function: Expression: unmethylated inducible housekeeping methylated Chapter III: summary

41 Gene function: Expression: unmethylated inducible low housekeeping high methylated Chapter III: summary

42 Gene function: Expression: Tissue specific expression: unmethylated inducible low housekeeping high methylated Chapter III: summary

43 Gene function: Expression: Tissue specific expression: unmethylated inducible low housekeeping high methylated variable conserved Chapter III: summary

44 Gene function: Expression: Tissue specific expression: unmethylated inducible low housekeeping high methylated variable conserved A context dependent role of DNA methylation in bivalves (Gavery & Roberts 2014) Chapter IV. Chapter III: summary

45 Summary of Chapters: I. Transcriptomic approaches to understanding environmental response II. First characterization of DNA methylation in oysters (in silico analysis) III. DNA methylation & gene expression: whole genome analysis IV. Perspective/Review: DNA methylation in bivalves

46 Environmental Response in Oysters 20092010201120122013 Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring bringing it all together! meth & expression transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Chapter I. CBP: Part D Chapter II. BMC Genomics Ch III. PeerJ Chapter IV. Brief Func Gen

47 Environmental Response in Oysters 20092010201120122013 BMC Genomics Frontiers Whole transcriptome DNA methylation: single gene/in silico Nanostring bringing it all together! meth & expression transgenerational (5-aza pesticide) EE2 Gene expression: disease resistance Cyclooxygenase characterization Authorea link Chapter I. CBP: Part D Ch III. PeerJ Chapter IV. Brief Func Gen

48 Agenda: Research update Proposed timeline

49 Proposed Timeline: Draft proposal to reading committee: April 21 st

50 Proposed Timeline: Mem Day Spring End

51 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves?

52 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx)

53 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f

54 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank) control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control

55 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank) control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control 6m / 7f / 1? – EE2

56 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank) control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control 6m / 7f / 1? – EE2

57 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank) control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control 6m / 7f / 1? – EE2

58 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control 6m / 7f / 1? – EE2

59 Estrogen Experiment Does DNA methylation mediate the phenotypic effects of estrogen exposure in bivalves? EE2 (n=50/tank control (n=50/tank) Day 0 (n=10) Day 7 (n=15/tx) Day 35 (n=15/tx) Day 60 (n=120/tx) 7m / 3f10m / 5f - control 6m / 7f / 1? – EE2

60 Estrogen Experiment Identifying differential methylation in day 7 females Approach: MBD-Chip Results: 45 differentially methylated regions 42 in genes, 3 upstream of genes 38 different genes identified E.g.: CRHR, serotonin receptor, acetylcholine receptor, various DNA binding proteins Next steps: validate DMRs by pyrosequencing

61 Appendices: Gene expression: 1. Characterization of cycloxygenase in C. gigas 2. Gene expression in disease resistant oysters DNA methylation: 4. Nanostring report 5. Transgenerational Experimental Design 6. EE2 experiment: phenotypic data & DNA methylation results

62 Prostaglandins in oyster immunity Sequenced full length gene Phylogenetic analysis Tissue Distribution Immune Response

63 Gene Expression in Disease Resistant oysters Crassostrea virginica Resistant and Naïve population Immune Related Genes Major Findings: Resistant population appeared to be ‘primed’ with higher level of baseline expression of genes prior to exposure

64 Nanostring Project Targeted methylation analysis nCounter system Methylation at 48 target genes About 30% of the targets performed well Major findings Identified first differential methylation between tissue types

65 Transgenerational Experiment Do methylation patterns persist across generations? Expsure to either: 5-azacytidine Vinclozolin Spawned and raised larvae Parents sampled: nanostring assay Growout Samples 18 months later (gametes) Findings: size difference, no meth difference by Nanostring

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