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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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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).
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RSOB140110F2: 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).

Mentions: By BLAST searching the honeybee genome with mammalian TET proteins, we have identified a large gene, greater than 150 kb, encoding a predicted polypeptide harbouring all the signature domains and motifs characteristic of the TET oxoglutarate-dependent dioxygenase protein family, namely the HxD and Hxs motifs implicated in Fe(II) binding, the oxoglutarate recognition signature Rx5a and the DNA-binding CXXC domain [7]. Like in mammalian TETs, the honeybee protein has a Cys-rich region located upstream of the catalytic domain and a long amino acid insertion. To demonstrate that the putative AmTET protein has dioxygenase activity, we have cloned its C-terminal fragment, spanning the catalytic domain, the Cys-rich domain and a predicted nuclear localization signal (figure 2). We then expressed AmTET in human embryonic kidney (HEK293T) cells and monitored the levels of 5hmC using both dot-blots and immunofluorescence imaging. In vitro expression of AmTET was carried out in HEK293T cells that contain low endogenous 5hmC levels, but have ample 5mC to provide the substrate for oxidation. The AmTET fragment with amino-terminal haemagglutinin (HA) tag was placed under the control of the CMV promoter and transfected into HEK293T cells. In order to ensure that any observed effects were due to the catalytic activity of the AmTET, we used as the negative control cells transfected with a GFP-coding plasmid and a predicted catalytically inactive mutant of AmTET. The inactive AmTET double mutant has the sequence YxA instead of HxD, because this substitution was previously shown to inactivate the mammalian TET1 [19]. As a positive control, we have used a previously described HA-tagged fragment of human TET1, placed in the same vector with the CMV promoter. The expression levels of TET proteins and the abundance of 5hmC were analysed after 48 h or after 16 h upon proteasome inhibition. Protein levels were measured with anti-HA-tag antibody and turned out to be similar for all three constructs. The 5hmC levels were monitored by the dot-blot assay using commercially available anti-5hmC antibody. As expected, we have detected a five to 20-fold increase of 5hmC levels in cells expressing the wild-type honeybee or human TET, relative to cells expressing GFP or mutant TET incapable of iron binding (figure 3). Given the apparent nuclear localization of AmTET in HEK293T cells (figure 3d), the increase in 5hmC levels has to result from the conversion of 5mCs present in nuclear DNA.Figure 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)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140110F2: 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).
Mentions: By BLAST searching the honeybee genome with mammalian TET proteins, we have identified a large gene, greater than 150 kb, encoding a predicted polypeptide harbouring all the signature domains and motifs characteristic of the TET oxoglutarate-dependent dioxygenase protein family, namely the HxD and Hxs motifs implicated in Fe(II) binding, the oxoglutarate recognition signature Rx5a and the DNA-binding CXXC domain [7]. Like in mammalian TETs, the honeybee protein has a Cys-rich region located upstream of the catalytic domain and a long amino acid insertion. To demonstrate that the putative AmTET protein has dioxygenase activity, we have cloned its C-terminal fragment, spanning the catalytic domain, the Cys-rich domain and a predicted nuclear localization signal (figure 2). We then expressed AmTET in human embryonic kidney (HEK293T) cells and monitored the levels of 5hmC using both dot-blots and immunofluorescence imaging. In vitro expression of AmTET was carried out in HEK293T cells that contain low endogenous 5hmC levels, but have ample 5mC to provide the substrate for oxidation. The AmTET fragment with amino-terminal haemagglutinin (HA) tag was placed under the control of the CMV promoter and transfected into HEK293T cells. In order to ensure that any observed effects were due to the catalytic activity of the AmTET, we used as the negative control cells transfected with a GFP-coding plasmid and a predicted catalytically inactive mutant of AmTET. The inactive AmTET double mutant has the sequence YxA instead of HxD, because this substitution was previously shown to inactivate the mammalian TET1 [19]. As a positive control, we have used a previously described HA-tagged fragment of human TET1, placed in the same vector with the CMV promoter. The expression levels of TET proteins and the abundance of 5hmC were analysed after 48 h or after 16 h upon proteasome inhibition. Protein levels were measured with anti-HA-tag antibody and turned out to be similar for all three constructs. The 5hmC levels were monitored by the dot-blot assay using commercially available anti-5hmC antibody. As expected, we have detected a five to 20-fold increase of 5hmC levels in cells expressing the wild-type honeybee or human TET, relative to cells expressing GFP or mutant TET incapable of iron binding (figure 3). Given the apparent nuclear localization of AmTET in HEK293T cells (figure 3d), the increase in 5hmC levels has to result from the conversion of 5mCs present in nuclear DNA.Figure 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