Chromatin Structure in Water-Deficit Stress in Arabidopsis

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

This work was supported by NSF grant EPS-0701892

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

ChIP-Seq: Chromatin Immunoprecipitation (ChIP) followed by High Throughput DNA Sequencing

Specific histone modification orBound Protein of interest Crosslink protein to DNA and fragment DNA

Immunoprecipitate with antibodies to target modification or bound protein

Specific Antibody

Enriched chromatin after immunoprecipitation

High throughput DNA Sequencing

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

Analyze gene expression levels and chromatin modification for H3K4me1, H3K4me2 and H3K4me3 across Arabidopsis genome

Solexa sequence analysisAffymetrix microarray analysis

Table I. Number of sequencing reads from each chromatin immunoprecipitation experiment

Number of sequencing reads

Treatment H3K4me1 H3K4me2 H3K4me3

Watered 17,451,837 27,354,179 12,285,745

Water deficit 16,972,749 39,299,903 18,012,924

aNumber of sequences that are unique in the Arabidopsis genome and contain 2 or less mismatches

RD29A and RD29B are an adjacent ancient gene duplication

RD29B induced RD29A inducedAT5G52290 No Change

Phosphate responsive protein is repressed by water deficit stress

GAPDH is constitutively expressed

GeneSolexaWatered

Solexa Dry Fold Q-PCR STD

RAB18 155 4229 27.3 10.3 0.57

CBF4 365 1976 5.4 2.7 0.17

LTP 1381 433 0.3 0.4 0.1

GAPC2 208 109 0.5 0.7 0.12

eEF1b2 144 244 1.7 1.6 0.23

XERO2 325 2094 6.4 4.6 0.14

ATHB7 442 5574 12.6 4.5 0.38

ATHB12 3474 8670 2.5 1.6 0.13

SAG29 243 2146 8.8 13.8 2

RD29B 851 3766 4.4 2.4 0.7

RD29A 6295 14096 2.2 1.7 0.6

LR4, LTP4 330 2317 7.0 2.0 0.5

GLP1 5390 1990 0.4 0.5 0.1

Comparison of H3K4me3 levels by Solexa and Q-PCR measurements

Average profiles by expression levels

Table II. Percentage of H3K4 methylation peaks mapping to genes

Treatment and type of H3K4 methylation

Number of H3K4

methylation regions

Number of regions

mapping to genes*

Percentage of regions

mapping to genes

Water: H3K4me1 28271 25501 90.2

Dry: H3K4me1 29780 25612 86.0

Water: H3K4me2 27113 25070 92.5

Dry: H3K4me2 25672 23220 90.4

Water: H3K4me3 20542 19824 96.5

Dry: H3K4me3 22542 21689 96.2

Intersection of regions containing H3K4 me1, me2, and me3 11054 10819 97.9

*Includes 200 bp upstream and downstream of transcribed regions of annotated genes

Table III. Percentage of genes with H3K4 methylation regions

____________________________________________________________________________ Treatment and type of H3K4 methylation

Number of genes with H3K4 methylation

Percentage of genes with H3K4

methylation

Water: H3K4me1 26182 82.4

Dry: H3K4me1 27152 85.5

Water: H3K4me2 26703 84.1

Dry: H3K4me2 26900 84.7

Water: H3K4me3 20593 64.8

Dry: H3K4me3 21852 68.8

Genes with one or more types of H3K4 methylation 29119 91.7

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

Gene ExpressionPercentile

Watered Dry

Number of genes

aPercent of 542 genes

bCalculated percent of 31,762 genes

Number of genes

Percent of 542 genes

Calculated a percent of 31,762 genes

0-19 380 70.1% 5.8% 365 67.3% 5.6%

20-33 101 18.6% 1.6% 108 19.9% 1.7%

34-39 19 3.5% 0.3% 19 3.5% 0.3%

40-59 17 3.1% 0.3% 25 4.6% 0.4%

60-79 12 2.2% 0.2% 9 1.7% 0.1%

80-100 13 2.4% 0.2% 16 3.0% 0.2%

Total genes 542 8.3% 542 8.3%

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?

Median and +/- 1 standard deviation range for changes in H3K4 methylation when gene expression changes

TrendsInducedMe3 upMe2 upMe1 down

RepressedMe3 downMe2 upMe1 up

The broad h3K4me3 profile exists before gene induction and is not dependent on expression level (RD29B has almost undetectable expression before induction)

RD29B induced RD29A inducedNo Change

Inducible genes have broader H3K4me3 profiles along the length of the gene

All expressed genes

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

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

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

Soil drought assay – Yong Ding

Drought treat 9 days Re-water 3 days

WT atx1 WT atx1

45/61 20/66

WT atx1

Water

0

20

40

60

80

100

120

w ater drought

WT

atx1

Plant Survival

ratio (%)

*

Soil drought assay – Yong Ding

The drought response gene expression level

W.t.

atx1

gene expression level

W.t.

atx1

H3K4 Tri-methylation level changes in atx1

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

ATX1: A H3K4 methyltransferase that affects drought sensitivity and chromatin structure

Zoya 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);

Acknowledgements

• NSF EPSCoR

Molecular BiologyYong Ding, Karin van Dijk, Han Chen, M. FrommZhen Wang, Amit Mehra, Heriberto Cerutti, Zoya Avramova

ComputationalSridhar Malkaram, Rong Liu, J.J.M. Riethoven, Jingi Yang, Steve Ladunga, Jamie Davila

Statistics Dong Wang

Microarrays Yuannan Xia