Download The Epigenetic Memory Different timing of bud burst between epitypes

31st May 2016
IUFRO Genomics and
Forest Tree Genetics
Igor Yakovlev
The Epigenetic Memory
Induced by the Temperature During Embryo Development
Impact on Bud Phenology
Different timing of bud burst between
epitypes
• Related to development
• Involved in adaptation and decease resistance
• Encoded in chromatin and non-coding part of genome
What we know about epigenetic memory so far
The epigenetic memory is defined as a heritable change in
gene expression or behavior that is induced by a previous
stimulus (D’Urso & Brickner 2014)
•
Temperature sum during embryogenesis induce epigenetic
memory
•
The effect lasts for life (at least 25 years……)
•
The effect may be adaptive and may decide life or death
(i.e. frost damage)
•
Epigenetic memory is associated with visible transcriptome
changes both during establishment in embryos and further
maintenance in buds and needles (Yakovlev et al., 2014,
2016)
Multiple molecular mechanisms are involved
•
Epigenetic regulation mechanisms
Non-coding RNAs
Long ncRNAs
DNA Methylation
Cytosine methylation
Adenine methylation
small
small RNAs
RNAs
Gene Expression
Histone Modifications
methylation,
acetylation,
phosphorylation,
ubiquitination,
sumoylation, etc…
Chromatin remodeling
repositioning of nucleosomes
incorporation of histone variants
All these mechanisms realized by the specific pathways and
demands specific enzymes
miRNA pathway
Plant microRNAs (miRNAs), a
class of small non-coding
regulatory RNAs, are
canonically 20–24 nucleotides in
length and bind to
complementary target RNA
sequences, guiding target
attenuation via mRNA
degradation or translation
inhibition.
They should be traceable back
to precursor with a hairpin
structure
Yang & Li, 2012 Biology
Experimental approaches
The main task of current study is to identify and characterize
miRNAs as important regulatory elements involved in the
initiation and maintenance of epigenetic memory
Somatic embryo tissues were induced from one genotype
B10W of the full-sib family of Picea abies greenhouse and
tested in the field conditions. Obtained epitypes show clear
phenotypic differences related to memory of temperature
during embryogenesis, confirming as obsereved differences in
timing of bud burst
We constructed 9 small RNA and 9 mRNA libraries
Temperature
conditions
cold
(18oC),
normal
(23oC)
warm
(28oC)
+
+
+
Intermediate – E2
+
+
+
Mature embryos – E3
+
+
+
Stages of
embryo
development
Early – E1
miRNA profiling in somatic embryos of Norway spruce
under different growth temperatures
We defined about 2300 miRNA candidates with prevailing length
21 nt (41%) and 22 nt (34%).
Among them we selected 1115 highly expressed miRNAs (>100
reads).
Allowing up to 2 mismatches, we defined 522 conserved miRNAs
belonged to 58 miRNA families and 593 novel miRNAs.
Differential expression analysis revealed 676 differential
expressed miRNAs
676 DE miRNAs could be grouped to 12 main clusters
202 DE miRNAs
23 DE miRNAs
423 DE miRNAs
Differences in abundant transcript families between
18°C compared to the epitype inducing
temperatures of 23°C and 28°C were largely
qualitative, while the differences between 23°C
and 28°C conditions were mostly quantitative
Prediction of targets for highly expressed miRNAs
We defined 470 miRNAs targeting to 1139 gene models, incl.
930 annotated gene models from around 212 gene families with diverse
biological functions and
209 gene models without match at the Databases
We found high redundancy in miRNA – mRNA target pairs
We found that 317 DEGs could be regulated by several miRNAs and 290
miRNAs could regulate more than one gene
a.
MA_10433003g0010
Disease resistance
protein (TIR-NBSLRR class) family;
PF00931 - NB-ARC domain; PF01582; PF13676 TIR domain; PF01637 - Archaeal ATPase;
PF01656 - CobQ/CobB/MinD/ParA nucleotide
binding domain; PF03205-Molybdopterin guanine
dinucleotide synthesis protein B; PF03308-ArgK
protein; PF04665-Poxvirus A32 protein; PF05729NACHT domain; PF06745-KaiC; PF08477-Mirolike protein; PF13191-AAA ATPase domain;
PF13173; PF13207; PF13238; PF13401-AAA
domain
Comparison of transcription profiles of selected differentially
expressed miRNA–gene functional pairs, involved in
temperature-dependent regulatory processes
MA_97097g0010
MA_10433968g0020
MA_8090186g0010
MA_2193g0020
PF00097 - Zinc finger, C3HC4 type (RING
finger); PF03031 - NLI interacting factor-like
phosphatase; PF11793 - FANCL C-terminal
domain; PF12678 - RING-H2 zinc finger;
PF12861 - Anaphase-promoting complex subunit
11 RING-H2 finger
PF03031 - NLI interacting factor-like
phosphatase; PF07527 - Hairy Orange domain
PF00847 - AP2 domain
PF00847 - AP2 domain
Comparison of transcription profiles of selected differentially
expressed miRNA–gene functional pairs, involved in
temperature-dependent regulatory processes
MA_10436505g0010
MA_17313g0010
MA_88843g0010
PF08744 - Plant
transcription factor NOZZLE
PF02045 - CCAAT-binding
transcription factor (CBFB/NF-YA) subunit B
PF00145 - C-5 cytosine-specific DNA methylase;
PF01426 - BAH domain; PF12047 - Cytosine
specific DNA methyltransferase replication foci
domain
Characterization of numbers of differentially expressed miRNAs and
their putative differentially expressed targets* preliminary annotated
based on Pfam domains
##
Pfam ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
PF00560
PF00069
PF00931
PF01535
PF01582
PF00004
PF00515
PF00637
PF00394
PF02536
PF00646
PF00249
PF00847
PF01397
PF00418
PF06345
PF00566
PF01715
PF00046
PF00201
PF08263
PF11721
PF04937
PF08744
PF03110
PF00106
…
Pfam description
Leucine Rich Repeat (LRR)
Protein kinase domain
NB-ARC (nucleotide-binding adaptor R-gene shared) domain
Pentatricopeptide (PPR) repeat
Toll-Interleukin receptor (TIR) domain
ATPase family associated with various cellular activities (AAA)
Tetratricopeptide (TPR) repeat
Clathrin heavy chain/ VPS (vacuolar protein sorting-associated)
Multicopper oxidase
mTERF (Mitochondrial transcription termination factor)
F-box domain
Myb-like DNA-binding domain
AP2 domain
Terpene synthase, N-terminal domain
Microtubule-associated protein (MAP) Tau, tubulin-binding repeat
DRF (Diaphanous-related formins) autoregulatory domain
Rab-GTPase-TBC domain
tRNA Delta(2)-isopentenylpyrophosphate transferase (IPP transferase)
Homeobox domain
UDP-glucoronosyl and UDP-glucosyl transferase
Leucine rich repeat N-terminal domain
Di-glucose binding within endoplasmic reticulum
Protein of unknown function (DUF 659)
Plant transcription factor NOZZLE
SBP (SQUAMOSA promoter binding protein-like) domain
short chain dehydrogenase
Number of Number of
miRNAs gene models
274
139
169
105
138
85
57
40
44
15
11
12
8
8
7
11
5
15
11
5
4
4
3
3
40
7
163
97
90
84
52
52
44
30
15
11
11
10
8
8
5
4
4
3
3
3
3
3
3
3
2
2
* - evaluation
based on variants
with negative
correlation (-0.6
… -1.0) between
DE miRNAs target gene
expression
patterns. Targets
were predicted by
psRNATarget
Differentially expressed miRNAs targeting epigenetic
regulators
Number of
Number of
DEGs
miRNAs
DNA methylation
4
4
Histone methylation
41
38
Histone acetylation
7
7
136
70
Histone ubiquitination
9
9
Chromatin remodeling
7
7
sRNA pathways
4
4
Thermosensing
1
1
Histone phosphorylation
Total 212
differentially
expressed miRNA –
target pairs,
composed by 125
miRNAs and 204
gene models
Conclusions
• We showed that Norway spruce poses a variety of miRNAs
and their isoforms with distinct temperature dependent
expression patterns
• We found around 500 differentially expressed miRNAs
targeting to 1139 gene models
• Target genes are mostly represented by transcripts of
multiple repeats proteins like TIR, NBS-LRR protein genes,
PPR and TPR repeat, Clathrin heavy chains/VPS proteins,
etc. , including also genes involved in epigenetic regulation
• We consider that TIR, NBS and LRR domain containing
proteins could fulfill more general functions for signals
transduction from external environment and conversion
them into molecular response of any type
Conclusions
• More than 200 DEGs of epigenetic regulators (from about 700) could
be post-transcriptionally regulated by 125 miRNAs
• Except the protein kinases, miRNAs mostly involved in regulation of
genes related to methylation modifications, both in histones and DNA
• Unknown mechanisms provide fine-tuning of miRNAs pool content
participating in developmental regulation and epigenetic memory
formation.
• Taking together, our data confirm significant involvement and
importance of miRNAs into post-transcriptional gene regulation and
epigenetic memory formation
People involved:
NIBIO:
Igor Yakovlev, Vivian-Smith A. , Carneros E., and Carl G. Fossdal
NMBU (Norwegian University
of life Sciences) :
Jorunn E. Olsen, Lee Y.K., Marcos Viejo
Funding: FRIPRO (NFR), EU FP7, TOPPFORSK (NFR)