Studying the biological functions of the TET and TDG proteins during zebrafish embryonic development
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
5'-methylcytosine (5mC) is a transcriptionally repressive epigenetic mark, enriched at CpG dinucleotides
within vertebrates. The TET proteins (TETs 1, 2 and 3) oxidize 5mC to 5'-hydroxymethylcytosine (5hmC) then
subsequently to 5'-formylcytosine (5fC) and 5'-carboxylcytosine (5caC)1. 5fC and 5caC are excised by
thymine-DNA glycosylase (TDG)2. This process is known as active DNA demethylation.
Active demethylation is essential during embryonic development. It prevents aberrant repression of
pluripotency factor expression and allows for the de-repression of lineage specification factors' expression -
essential for regulating stem cell differentiation and organogenesis. Worth considering is that passive
demethylation (linked to cell division) may contribute significantly to embryonic development too; however the
importance relative to active demethylation is unknown.
The relationship between TET expression, 5hmC presence and pluripotency varies across vertebrate species
- big differences exist between mice and zebrafish (see Figs.1&2)3. This suggests a phylogenetically varied
developmental role for the TET proteins. Why these variations exist and the differences in their
developmental functions are unknown.
In mouse embryos, the knockout of individual TET genes leads to phenotypes of varying severity, with TET1
causing a very mild impact upon development and viability4 and TET3 being lethal post-implantation5. TDG
knockout is also lethal6. Combinatorial knockout of TETs 1&2 results in a severe phenotype4. Little reliable
data has been obtained regarding the consequences of TET1 and TET3 knockout upon zebrafish embryonic
development and none for TDG. However available evidence suggests both phenotypic similarities and
differences exist between mouse4 and zebrafish7 TET-depleted embryos.
Generating CRISPR/Cas9-mediated knockout lines for TETs 1,2,3 and TDG in zebrafish is a crucial step in
elucidating the functions of both the enzymes and the epigenetic marks which they deposit/process.
within vertebrates. The TET proteins (TETs 1, 2 and 3) oxidize 5mC to 5'-hydroxymethylcytosine (5hmC) then
subsequently to 5'-formylcytosine (5fC) and 5'-carboxylcytosine (5caC)1. 5fC and 5caC are excised by
thymine-DNA glycosylase (TDG)2. This process is known as active DNA demethylation.
Active demethylation is essential during embryonic development. It prevents aberrant repression of
pluripotency factor expression and allows for the de-repression of lineage specification factors' expression -
essential for regulating stem cell differentiation and organogenesis. Worth considering is that passive
demethylation (linked to cell division) may contribute significantly to embryonic development too; however the
importance relative to active demethylation is unknown.
The relationship between TET expression, 5hmC presence and pluripotency varies across vertebrate species
- big differences exist between mice and zebrafish (see Figs.1&2)3. This suggests a phylogenetically varied
developmental role for the TET proteins. Why these variations exist and the differences in their
developmental functions are unknown.
In mouse embryos, the knockout of individual TET genes leads to phenotypes of varying severity, with TET1
causing a very mild impact upon development and viability4 and TET3 being lethal post-implantation5. TDG
knockout is also lethal6. Combinatorial knockout of TETs 1&2 results in a severe phenotype4. Little reliable
data has been obtained regarding the consequences of TET1 and TET3 knockout upon zebrafish embryonic
development and none for TDG. However available evidence suggests both phenotypic similarities and
differences exist between mouse4 and zebrafish7 TET-depleted embryos.
Generating CRISPR/Cas9-mediated knockout lines for TETs 1,2,3 and TDG in zebrafish is a crucial step in
elucidating the functions of both the enzymes and the epigenetic marks which they deposit/process.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M008770/1 | 01/10/2015 | 31/10/2024 | |||
| 1943438 | Studentship | BB/M008770/1 | 01/10/2015 | 30/09/2019 |
| Description | I have discovered the consequences of TET gene knockout upon the genomic levels of 5-methylcytosine and its derivatives in zebrafish. I have observed the developmental phenotypes caused by these knockouts. |
| Exploitation Route | This project requires more work to further develop the zebrafish lines necessary to characterise the impact of TET gene knockout in more detail |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Science in the Park |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | I used a block building game to simulate the effects of DNA damage for children |
| Year(s) Of Engagement Activity | 2016 |