DNA methyltransferases and DNA methylation
in the pea aphid.
DNA methylation group:
Tom Walsh (CSIRO), Jennifer Brisson (USC), Dayalan Srinivasan
(Princeton), Hugh Robertson (UIUC)
DNA methylation
•
Cytosine is methylated at CpG sites which acts as
a marker for gene suppression (in vertebrates)
•
The DNA methylation status of genes can be
changed by environmental factors
•
Aphids and organophosphate resistance (Green
peach aphid, Myzus persicae)
•
Honey bee, Apis mellifera
– Changes in methylation are associated with
nutrition and the development of queens and
workers
•
Other insects
– Social Hymenoptera
– Lepidoptera
•
Different to vertebrate methylation
– Methylation is in the exons
– Does not suppress gene expression
The companion paper
1.
2.
3.
4.
5.
6.
Annotation and phylogeny of the Dnmts
Global amount of DNA methylation
Which genes/regions are methylated in aphids?
Expression of Dnmts
Changes in methylation associated with morphs, environmental
signals, nutrition etc.
Expression of methylated genes
1. Annotation and phylogeny of the Dnmts
•
•
•
Dnmt1 – maintenance methyltransferase
Dnmt2 – ? methyltransferase
Dnmt3 – de-novo methyltransferase
•
•
DMAP1 – DNA methyltransferase 1 associated protein 1
MBP – methyl binding domain proteins
Diptera
Brachycera
Drosophila melanogaster
Anopheles gambiae
Nematocera
Lepidoptera
Bombyx mori
Coleoptera
Endopterygota
Tribolium castaneum
Apis mellifera
Apoidea
Hymenoptera
Chalcidoidea
Insecta
Heteroptera
Nasonia vitripennis
Rhodnius prolixus
Sternorhyncha
Acyrthosiphon pisum
Hemiptera
Pancrustacea
Exopterygota
Phthiraptera
Pediculus humanus
Daphnia pulex
Ixodes scapularis
Crustacea
Chelicerata
500
400
300
200
100
Approximate Divergence Time
(millions of years ago)
0
1
+
+
+
+
.
+
+
+
+
Dnmt
2 3
+ + + + + +
+ +
. .
+ +
+ + +
+ +
Dnmt1a
Dnmt1b
Dnmt1
(Hugh Robertson)
Dnmt2
Dnmt3
2. Global levels of methylation
Pea aphid
Mauro Mandrioli & Frederica Borsatti, Chromosome research 2007
Honey bee
Digested bee DNA was spiked (a) with exogenous
dCM and analyzed in parallel with the digested DNA
(b), clearly identifying a peak in adult honey bee
coincident with dCM enrichment
Wang et al., Science 2006
3. Which genes/regions are methylated in aphids?
Sequenced 43 clones from the pea
aphid libraries
Broad scale approach -methylation
libraries
16 from the AIMS library
27 from the IP library
Amplification of inter-methylated sites
(AIMS)
Immuno-precipitation (IP)
Aphid
species
13 library clones investigated by
bisulphite sequencing
9 from AIMS
4 from IP
Colonies
AIMS
IP
A. pisum
129
240
M. persicae
112
113
Query
67
Sbjct
13784
TTGTTATTGAAATACGAATTATTTGTTGTACGTTGGGTTGGTGGATTTGAGATTGAAGTA
|||||||||||| |||||||| || ||||||||||||||||||| |||||||||||||
TTGTTATTGAAACACGAATTACCTGCTGTACGTTGGGTTGGTGGACCTGAGATTGAAGTA
T-complex protein 1 subunit epsilon – IP
library
126
13725
Needle in a haystack approach
•
Look for genes that we know are
methylated in other species
– Honey bee genes
• CasK
• Dynactin p62
– Green peach aphid genes
• E4 esterase
•
Look for genes that we would ‘like’ to be
methylated
• Juvenile hormone esterase
• Juvenile hormone binding
protein
4. Expression of Dnmts
• Preliminary results
– One of the Dnmt1s is upregulated in response to crowding (Jennifer Brisson)
– Dnmt3 is expressed at a very low level compared to Dnmt1a and b
5. Changes in methylation associated with morphs, environmental
signals, nutrition etc.
6. Matching expression of methylated genes to methylation changes
The pea aphid, Acyrthosiphon pisum as a model organism for
epigenetics research
•
Advantages
– Developmental plasticity
– Displays both sexual and asexual reproduction
– An important agricultural pest
– Lines can be maintained in the lab
– RNAi has been shown
– There is a genome