PhD Thesis proposal form
Discipline
(Biology)
Doctoral School
ED 145: Plant Sciences / Sciences du Végétal
http://www.ed-sciences-du-vegetal.u-psud.fr/en/ecoledoctorale.htm
Thesis subject title: Studying the Resetting Mechanism of Stress-Induced Epigenetic State of Gene
Expression in Arabidopsis
 Laboratory name and web site:
Plant chromatin and development,
Institut de Biologie des Plantes
http://www.ibp.u-psud.fr/spip.php?rubrique49
 PhD supervisor (contact person):
 Name: Dao-Xiu Zhou
 Position: Professor
 email: dao-xiu.zhou@u-psud.fr
 Phone number: 33 1 6915 3413
 Thesis proposal (max 1500 words):
Studying the Resetting Mechanism of Stress-Induced Epigenetic State of Gene Expression in
Arabidopsis
The sessile life style of plants requires increased developmental plasticity in which flexible
epigenetic regulation is essential for reprogramming of plant gene expression for rapid responses and
adaptation to environmental cues. Closely related species having differences in gene expression in
response to environmental stresses show differences in their epigenetic systems. There exists
epigenetic natural variation between populations or among individuals with a similar genotype. For
instance, DNA methylation in genes is extremely polymorphic among 96 natural accessions of
Arabidopsis thaliana (1). Recent studies by examining spontaneously occurring variation in DNA
methylation in Arabidopsis plants propagated by single seed descent for 30 generations indicate that
transgenerationally stable changes in cytosine methylation occur at a relatively high frequency (2, 3),
suggesting that transgenerational variation in DNA methylation may contribute to phenotypic
variation in plants. A number of recent work have suggested that abiotic and biotic stresses including
DNA damage, drought, high salinity and pathogens are sources of epigenetic variation (reviewed in
(4, 5). Some of the epigenetic adaptive responses to environmental cues can be transmitted to next
generations (e.g. (6, 7). In addition, a number of studies have revealed that epigenetic variability in
plants can be enhanced by different stress treatments. For the long-time transgenerational adaptation
to environmental cues, the perceived information must be memorized in an epigenetic form that is
propagated through mitotic and meiotic divisions, even when the initial signal is removed. However,
multiple epigenetic mechanisms have been suggested to stabilize and buffer the epigenetic states of
gene expression.
Chromatin structure and remodelling are basic components of genetic and epigenetic
regulation of genome expression. They are regulated by an array of proteins or protein complexes,
leading to specific profiles of chromatin modification and remodelling. In addition to DNA
methylation, covalent modifications of the N-terminal tails of the core histones affect nucleosome
positioning and compaction, thus play pivotal roles in chromatin remodelling and in gene regulation.
Histone modifications include acetylation, methylation, phosphorylation and ubiquitinylation, etc.,
among which histone lysine methylation plays essential roles in the epigenetic processes. Histone
lysine residues can be mono- di- or trimethylated. Each methyl state may have different roles for
gene activity or genome function (8, 9). For instance, trimethylation of H3 lysine 4 (H3K4me3) is
associated with active genes and is enriched at the 5’ end of the marked genes. H3K4me3 has been
shown to increase over inducible genes upon application of inductive signals (10). However, recently
results have shown that the increase of H3K4me3 lags behind the gene induction (11), suggesting
that H3K4me3 is more likely to play a role to mark active genes than to have a transcriptional
activation function per se. A number of studies have suggested that H3K4me3 may play a role in
maintaining the induced gene expression state even after removal of the stress signal. However it is
not known whether or not H3K4me3 marking of gene expression state can be maintained through
mitosis or meiosis. Therefore we propose in this PhD thesis project to evaluate the role of H3K4
methylation/demethylation in transgenerational inheritance/resetting of plant stress
adaptation.
1. H3K4me change during stress and after removal of the stress signal. Although it is known
that H3K4me3 is increased during gene induction, it is not clear whether the modification is reset
after the removal of the inductive signals. Arabidopsis seedlings will be treated with salt (100 mM
NaCl) and /or cold (4°C) then transferred to normal growth conditions. Stress-responsive genes (e.g.
RD29A) expression and H3K4me3 will be monitored during and after the stress treatment and in
organs (leaves) grown out post-stress treatment and till a few next generations of the stressed plants
to establish the kinetics of expression and H3K4me3 of the stress-responsive genes. Because histone
modifications and DNA methylation may influence each other, the change of DNA methylation and
other histone modifications (e.g. H3K27me3, H3K9me3) will be tested.
2. Function of H3K4me3 demethylase genes on stress-responsive gene expression and
H3K4me3. H3K4me3 induced by stresses can be reset by specific histone demethylases, which may
participate in the epigenetic buffering system to stabilized normal gene expression state in plants. In
Arabidopsis there is a group of five genes that potentially encode H3K4me3 demethylases (two of
them have been already confirmed). A few mutant alleles for each gene have been characterized.
These mutant lines will be used for the study. Conversely, this group of genes will be over-expressed
and the over-expression effect on stress-responsive gene expression and H3K4me3 will be studied.
3. Molecular mechanism of H3K4 demethylases in epigenetic resetting. Experiments will be
carried out to study whether the specific H3K4 demethylases respond to the stress signals.
Biochemical and genetic analysis will be performed to study whether the demethylases interact with
other chromatin regulators to reset stress-induced gene expression states and how they are recruited
to specific target genes. For these purposes, antibodies of the demethylases will be prepared and used
in identifying interacting proteins, and genetic crosses between demethylase mutants and other
chromatin mutants (of DNA methylation and/or histone modifications) will be made to study the
epigenetic system of the resetting mechanism.
Our lab has been studying histone modification control of plant development for many years.
We have established a series of chromatin-related techniques and analysis (ChIP, immune-staining,
protein interactions, genetic analysis, DNA microarray etc) readily for the realization of the project.
Mutants of H3K4 demethylase and many other chromatin regulators are available in the lab. This
project will benefit from the financial support of two ongoing ANR projects.
References
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11.
Vaughn MW, et al. (2007) Epigenetic natural variation in Arabidopsis thaliana. PLoS biology
5(7):e174.
Becker C, et al. (2011) Spontaneous epigenetic variation in the Arabidopsis thaliana
methylome. Nature 480(7376):245-249.
Schmitz RJ, et al. (2011) Transgenerational epigenetic instability is a source of novel
methylation variants. Science 334(6054):369-373.
Paszkowski J & Grossniklaus U (2011) Selected aspects of transgenerational epigenetic
inheritance and resetting in plants. Current opinion in plant biology 14(2):195-203.
Richards EJ (2011) Natural epigenetic variation in plant species: a view from the field.
Current opinion in plant biology 14(2):204-209.
Boyko A, et al. (2010) Transgenerational adaptation of Arabidopsis to stress requires DNA
methylation and the function of Dicer-like proteins. PloS one 5(3):e9514.
Verhoeven KJ, Jansen JJ, van Dijk PJ, & Biere A (2010) Stress-induced DNA methylation
changes and their heritability in asexual dandelions. The New phytologist 185(4):1108-1118.
Liu C, Lu F, Cui X, & Cao X (2010) Histone methylation in higher plants. Annual review of
plant biology 61:395-420.
Mosammaparast N & Shi Y (2010) Reversal of histone methylation: biochemical and
molecular mechanisms of histone demethylases. Annu Rev Biochem 79:155-179.
Tsuji H, Saika H, Tsutsumi N, Hirai A, & Nakazono M (2006) Dynamic and reversible
changes in histone H3-Lys4 methylation and H3 acetylation occurring at submergenceinducible genes in rice. Plant & cell physiology 47(7):995-1003.
Hu Y, Shen Y, Conde ESN, & Zhou DX (2011) The Role of Histone Methylation and H2A.Z
Occupancy during Rapid Activation of Ethylene Responsive Genes. PloS one 6(11):e28224.
 Publications of the laboratory in the field (max 5):
Y. Hu, Y. Shen, N. Conde e Silva, DX. Zhou (2011) Role of histone methylation and histone variant H2A.Z
deposition during rapid activation of ethylene-inducible genes. Plos One 2011; 6(11):e28224.
C. Li, L. Huang, C. Xu, Y. Zhao, DX. Zhou (2011) Altered levels of histone deacetylase OsHDT1 affect
differential gene expression patterns in hybrid rice. PloS One 2011; 6(7):e21789.
W. Kim, M. Benhamed, C. Servet, D. Latrasse, W. Zhang, M. Delarue, DX. Zhou (2009) Histone
acetyltransferase GCN5 interferes with the miRNA pathway in Arabidopsis. Cell Res. 19(7): 899-909.
Q. Sun & DX. Zhou (2008) The rice jmjC domain-containing gene JMJ706 encodes a histone H3 lysine 9
(H3K9) demethylase required for floral organ development. Proc. Natl. Acad. Sci. USA 105(36):13679-84.
M. Benhamed, C. Bertrand, C. Servet & DX. Zhou (2006) Arabidopsis GCN5, HD1 and TAF1/HAF2 Interact
to Regulate Histone Acetylation Required for Light-Responsive Gene Expression. Plant Cell 18, 2893-2903.
 Specific requirements to apply, if any: