Transcript NexGen Sequencing Applicaitons to Whole Genome Chromatin

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 or
Bound Protein of interest
Specific Antibody
High throughput DNA
Sequencing
Crosslink protein to DNA and fragment DNA
Immunoprecipitate with antibodies to
target modification or bound protein
Enriched chromatin after
immunoprecipitation
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
Table I. Number of sequencing reads from each
chromatin immunoprecipitation experiment
Number of sequencing reads
Treatment H3K4me1
Watered
17,451,837
Water
deficit
16,972,749
aNumber
H3K4me2
27,354,179
H3K4me3
12,285,745
39,299,903
18,012,924
of sequences that are unique in the Arabidopsis genome
and contain 2 or less mismatches
RD29A and RD29B are an adjacent ancient
gene duplication
AT5G52290 No Change
RD29B induced
RD29A induced
Phosphate responsive protein is repressed by water
deficit stress
GAPDH is constitutively expressed
Comparison of H3K4me3 levels by Solexa and Q-PCR measurements
Solexa
Solexa
Gene
Watered
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
Average profiles by expression levels
Table II. Percentage of H3K4 methylation peaks
mapping to genes
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
Treatment and type of H3K4
methylation
*Includes 200 bp upstream and downstream of transcribed regions of annotated genes
Table III. Percentage of genes with H3K4 methylation regions
Treatment
and
Percentage of genes
____________________________________________________________________________
type of H3K4
Number of genes with
with H3K4
methylation
H3K4 methylation
methylation
Water: H3K4me1
26182
82.4
Dry: H3K4me1
27152
85.5
Water: H3
K4me2
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
Watered
Dry
bCalculated
Gene
Expression
Percentile
Number
of
genes
aPercent
of 542
genes
percent of
31,762
genes
Percent
Number of 542
of genes 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
Trends
Induced
Me3 up
Me2 up
Me1 down
Repressed
Me3 down
Me2 up
Me1 up
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
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
WT
atx1
Water
WT
atx1
Drought treat 9 days
WT
atx1
Re-water 3 days
45/61
20/66
Soil drought assay – Yong Ding
120
100
Plant Survival
80
ratio (%)
60
WT
atx1
40
*
20
0
w ater
drought
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-cisepoxycarotenoid Dioxygenase 3);
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