Chromatin Structure in Water-Deficit Stress in Arabidopsis

1. Yong Ding, Karin van Dijk, Sridhar Malkaram, Rong Liu, J.J.M. Riethoven, Jingi Yang, Han Chen, Yuannan Xia, Dong Wang, S. Ladunga, Zoya Avramova, & M. Fromm NSF EPSCoR Chromatin Biology Grant Chromatin Structure in Water-Deficit Stress in Arabidopsis This work was supported by NSF grant EPS-0701892

2. What we want to learn: how chromatin modifications affect the water-deficit mRNA response • How do mRNA levels correlate with chromatin modifications when comparing many different genes? • How do mRNA levels correlate with chromatin modifications in the same gene when it changes expression during water deficit stress? • How does atx1 mutation in H3K4 methyltransferase affect chromatin and gene expression • How does chromatin affect drought sensitivity of atx1

4. ChIP-Seq: Chromatin Immunoprecipitation (ChIP) followed by High Throughput DNA Sequencing Specific histone modification or Bound Protein of interest Crosslink protein to DNA and fragment DNA Immunoprecipitate with antibodies to target modification or bound protein Specific Antibody High throughput DNA Sequencing Enriched chromatin after immunoprecipitation

5. Experimental Design 4 week old Arabidopsis plants in soil at vegetative stage Watered Watered Deprived to 65% RWC (Wilted leaves) Isolate mRNA for Microarray measurements of gene expression Isolate chromatin for immunoprecipitation with H3K4 methylation specific antibodies Affymetrix microarray analysis Solexa sequence analysis Analyze gene expression levels and chromatin modification for H3K4me1, H3K4me2 and H3K4me3 across Arabidopsis genome

6. Table I. Number of sequencing reads from each chromatin immunoprecipitation experiment aNumber of sequences that are unique in the Arabidopsis genome and contain 2 or less mismatches

7. RD29A and RD29B are an adjacent ancient gene duplication AT5G52290 No Change RD29B induced RD29A induced

8. Phosphate responsive protein is repressed by water deficit stress

9. GAPDH is constitutively expressed

10. Comparison of H3K4me3 levels by Solexa and Q-PCR measurements

11. Average profiles by expression levels

12. Table II. Percentage of H3K4 methylation peaks mapping to genes *Includes 200 bp upstream and downstream of transcribed regions of annotated genes

13. Table III. Percentage of genes with H3K4 methylation regions ____________________________________________________________________________

14. Table V. Expressed Genes without H3K4 methylation comprise only 1% of all expressed genes

15. Focus on the induced or repressed genes • Many induced genes are ABA inducible • What happens to the H3K4 methylation status of individual genes when induced or repressed • Are there unique chromatin profiles of inducible genes?

16. Median and +/- 1 standard deviation range for changes in H3K4 methylation when gene expression changes Trends Induced Me3 up Me2 up Me1 down Repressed Me3 down Me2 up Me1 up

17. The broad h3K4me3 profile exists before gene induction and is not dependent on expression level (RD29B has almost undetectable expression before induction) No Change RD29B induced RD29A induced

18. All expressed genes Inducible genes have broader H3K4me3 profiles along the length of the gene

19. Conclusions • 92% of genes are marked by one or more types of H3K4 methylation • No simple correlation of H3K4 methylation levels with transcription levels for different genes • A change in the transcription of the same gene shows a strong correlation with a change in H3K4me3 levels • Reduced nucleosome density or modification level upstream of TSS

20. What we want to learn: how atx1 mutant affects the water-deficit mRNA response • ATX1 is a H3K4 methyltransferase (Avramova). • Atx1 mutants have pleiotropic phenotypes. • How does atx1 mutation in H3K4 methyltransferase affect chromatin and gene expression • How does chromatin affect drought sensitivity of atx1

21. Arabidopsis ATX1 (Arabidopsis thaliana TRITHORAX ) protein is complex with multiple domains SET peptide [for Su(var)3-9, E(z), Trithorax], encoded by the Drosophila melanogaster Su(var)39-, E(z)-, and Trithorax-related genes, carries histone lysine methyltransferase Conserved Trithorax domains: H3K4 methylases

22. Soil drought assay – Yong Ding WT atx1 WT atx1 WT atx1 Re-water 3 days Drought treat 9 days Water 45/61 20/66

23. Soil drought assay – Yong Ding Plant Survival ratio (%) *

24. W.t. atx1 The drought response gene expression level

25. W.t. atx1 gene expression level

26. H3K4 Tri-methylation level changes in atx1

27. New Genomics Statistics • How to tell the False Discover Rates of differences in peaks in chromatin studies • 1. Variation in replicates to determine frequency of random peaks: two wild type and two atx1 mutant samples • 2. Signal: avg wild type – avg atx1 • 3. FDR = # peaks replicates/signal peaks

29. ATX1: A H3K4 methyltransferase that affects drought sensitivity and chromatin structureZoya Avramova • Small percent of genome shows significant changes in H3K4me3 • New statistical methods for determining False Discover Rate (FDR) • Physiological – drought sensitivity of atx1 • Basis for drought sensitivity – low ABA biosynthesis in Nced3 gene (Nine-cis-epoxycarotenoid Dioxygenase 3);

30. Acknowledgements • NSF EPSCoR Molecular Biology Yong Ding, Karin van Dijk, Han Chen, M. Fromm Zhen Wang, Amit Mehra, Heriberto Cerutti, Zoya Avramova Computational Sridhar Malkaram, Rong Liu, J.J.M. Riethoven, Jingi Yang, Steve Ladunga, Jamie Davila Statistics Dong Wang Microarrays Yuannan Xia