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Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

Wojciechowski M, Rafalski D, Kucharski R, Misztal K, Maleszka J, Bochtler M, Maleszka R - Open Biol (2014)

Bottom Line: AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains.Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species.In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

ABSTRACT
In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

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AmTET gene model and transcript variants. (a) Manually annotated gene model showing all detected exons. zf-CXXC, DNA-binding domain; CD, catalytic domain. (b) Selected transcript models based on RNAseq data. Based on gene assembly OGSv3.2 (www.beebase.org), TET id: GB52555. Genomic location: linkage group LG12 (NCBI reference sequence: NC_007081.3), nucleotides 4 499 630–4 665 644. For more detail on transcript variants detection, see the electronic supplementary material, figure S4.
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RSOB140110F5: AmTET gene model and transcript variants. (a) Manually annotated gene model showing all detected exons. zf-CXXC, DNA-binding domain; CD, catalytic domain. (b) Selected transcript models based on RNAseq data. Based on gene assembly OGSv3.2 (www.beebase.org), TET id: GB52555. Genomic location: linkage group LG12 (NCBI reference sequence: NC_007081.3), nucleotides 4 499 630–4 665 644. For more detail on transcript variants detection, see the electronic supplementary material, figure S4.

Mentions: Domain organization of A. mellifera TET. The core catalytic region of AmTET is located at the C-terminus. It consists of a Cys-rich domain followed by an iron (II)–oxoglutarate-dependent dioxygenase domain (Tet–JBP). This domain harbours an intrinsically disordered 600 amino acid insertion. Known signature motives of Tet–JBP domains from A. mellifera and human TETs are aligned below the domain organization diagram, with critical residues highlighted in red. These motives are: HxD and Hxs (where s is a small residue) responsible for iron coordination and Rx5a (where a is an aromatic residue) responsible for 2-oxo acid coordination. The gene model available via BeeBase (www.beebase.org) does not have an extra exon coding for 25 amino acids and a few mini exons (figure 5) that we found by examining RNAseq datasets. Accession numbers: HsaTET1, NP_085128.2; HsaTET2, NP_001120680.1; HsaTET3, XP_005264244.1; HsaIDAX, NP_079488.2; AmeTET, GB52555 (BeeBase OGSv3.2).


Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

Wojciechowski M, Rafalski D, Kucharski R, Misztal K, Maleszka J, Bochtler M, Maleszka R - Open Biol (2014)

AmTET gene model and transcript variants. (a) Manually annotated gene model showing all detected exons. zf-CXXC, DNA-binding domain; CD, catalytic domain. (b) Selected transcript models based on RNAseq data. Based on gene assembly OGSv3.2 (www.beebase.org), TET id: GB52555. Genomic location: linkage group LG12 (NCBI reference sequence: NC_007081.3), nucleotides 4 499 630–4 665 644. For more detail on transcript variants detection, see the electronic supplementary material, figure S4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150289&req=5

RSOB140110F5: AmTET gene model and transcript variants. (a) Manually annotated gene model showing all detected exons. zf-CXXC, DNA-binding domain; CD, catalytic domain. (b) Selected transcript models based on RNAseq data. Based on gene assembly OGSv3.2 (www.beebase.org), TET id: GB52555. Genomic location: linkage group LG12 (NCBI reference sequence: NC_007081.3), nucleotides 4 499 630–4 665 644. For more detail on transcript variants detection, see the electronic supplementary material, figure S4.
Mentions: Domain organization of A. mellifera TET. The core catalytic region of AmTET is located at the C-terminus. It consists of a Cys-rich domain followed by an iron (II)–oxoglutarate-dependent dioxygenase domain (Tet–JBP). This domain harbours an intrinsically disordered 600 amino acid insertion. Known signature motives of Tet–JBP domains from A. mellifera and human TETs are aligned below the domain organization diagram, with critical residues highlighted in red. These motives are: HxD and Hxs (where s is a small residue) responsible for iron coordination and Rx5a (where a is an aromatic residue) responsible for 2-oxo acid coordination. The gene model available via BeeBase (www.beebase.org) does not have an extra exon coding for 25 amino acids and a few mini exons (figure 5) that we found by examining RNAseq datasets. Accession numbers: HsaTET1, NP_085128.2; HsaTET2, NP_001120680.1; HsaTET3, XP_005264244.1; HsaIDAX, NP_079488.2; AmeTET, GB52555 (BeeBase OGSv3.2).

Bottom Line: AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains.Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species.In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

ABSTRACT
In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

Show MeSH
Related in: MedlinePlus