DNA methylation is a crucial, abundant mechanism of gene regulation
in vertebrates. It is less prevalent in many other metazoan organisms and
completely absent in some key model species, such as D. melanogaster and
C. elegans. In this thesis we report on a comprehensive study of the pres-
ence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa
covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada.
We observe that loss of individual DNMTs independently occured multiple
times across ecdysozoan phyla. In several cases, this resulted in a loss of
DNA methylation.
In vertebrates, however, there is no single species known which lost DNA
methylation. Actually, DNA methylation was greatly expanded after the
1R/2R whole genome duplication (WGD) and became a genome-wide phe-
nomena. In our study of vertebrates we are not looking for losses of DNA
methyltransferases and DNA methylation but are rather interested in the
gain of additional DNA methyltransferase genes. In vertebrates there were a
number of WGD. Most vertebrates only underwent two WGD but in the
teleost lineage a third round of WGD occured and in some groups, e.g.
Salmoniformes and some Cypriniformes even a forth WGD occured. The
Carp-specific WGD (4R) is one of the most recent vertebrate WGD and is
estimated to have occured 12.4 mya. We performed the most comprehen-
sive analysis of the evolution of DNA methyltransferases after vertebrate
whole-genome duplications (WGD) so far. We were able to show that the
conservation of duplicated DNMT3 genes in Salmoniformes is more diverse
than previously believed. We were also able to identify DNA methyltrans-
ferases in Cypriniformes which have, due to their recent WGD, quite com-
plex genomes. Our results show that the patterns of retained and lost DNA
methyltransferases after a forth round of WGD differ between Cypriniformes
and Salmoniformes. We also proposed a new nomenclature for teleost DNMT
genes which correctly represents the orthology of DNMT genes for all teleost
species.
Next to these purely computational projects we collaborated with the
Aluru lab to investigate the effects of different disturbances on zebrafish
DNA methylation. One disturbance is the inactivation of DNMT3aa and
DNMT3ab as single knockouts as well as a double knockout. This was the
first double knockout of DNMT genes in zebrafish which was ever generated.
It allows us to study the subfunctionalization of the two DNMT3a genes their
effect on genome-wide DNA methylation. Given our results we hypothesize
that DNMT3aa and DNMT3ab can compensate for each other to a high de-
gree. DNMT3a genes have likely been subfuntionalized but their loss can
be compensated by DNMT3b genes. This compensation by DNMT3b genes
works well enough that no notable phenotype can be observed in double
knockout zebrafish but a difference is notable on the epigenome level. The
second disturbance we studied is the exposure of zebrafish to the toxic chemi-
cal PCB126. We detected a moderate level of DNA methylation changes and
a much larger effect on gene expression. Similar to previous reports we find
little correlation between DNA methylation and gene expression changes.
Therefore, while PCB126 exposure has a negative effect on DNA methyla-
tion it is likely that other gene regulatory mechanisms play a role as well,
possibly even a greater one.
How do genes evolve and how are genes regulated are two of the main
questions of modern molecular biology. In this thesis we have tried to shed
more light on both questions. we have broadly expanded the phylogenetic
range of species with a manually curated set of DNA methyltransferases. We
have done this for ecdysozoan species which have lost all DNA methylating
enzymes as well as for teleost fish which acquired more than ten copies of
the, originally, two genes. We were also able to generate new insight into
the subfunctionalization of the DNA methylation machinery in zebrafish and
how it reacts to environmental effects.:1 Introduction
1.1 Biological introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2 Detecting DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . 7
2 Evolution of DNA methylation across Ecdysozoa
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3 Evolution of DNA methyltransferases after vertebrate whole genome
duplications
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4 The effect of DNMT3aa and DNMT3ab knockout on DNA methyla-
tion in zebrafish
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
5 Role of DNA methylation in altered testis gene expression patterns
in adult zebrafish exposed to Pentachlorobiphenyl
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
6 Conclusions
6.1 Evolution of DNA methylation across Ecdysozoa . . . . . . . . . . . . . 95
6.2 Evolution of DNA methyltransferases after vertebrate whole genome duplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
6.3 Role of DNA methylation in altered testis gene expression patterns in
adult zebrafish (Danio rerio) exposed to Pentachlorobiphenyl (PCB 126). . . 107
6.4 Knockout of DNMT3aa and DNMT3ab in zebrafish (Danio rerio) . . . . . . 108
Bibliography 119
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:74882 |
| Date | 14 May 2021 |
| Creators | Engelhardt, Jan |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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