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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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Related in: MedlinePlus

In vitro expression of AmTET in human embryonic kidney (HEK293T) cells. (a) Western blot analysis of the expression levels of TET-HA proteins were analysed 48 h after transfection. GAPDH level is a protein loading control. (b) Genomic DNA from these cells was isolated and dot-blotted with specific anti-5hmC antibody. About 24–190 ng of each genomic DNA and the same amounts of PCR product with dCTP swapped for d5mCTP were used for this experiment. About 0.25–2ng of PCR product with dCTP swapped for d5hmCTP was used as a positive control. Signal obtained from cells expressing wild-type TETs is stronger than from cells expressing GFP or a catalytically inactivated honeybee TET. (c) Data obtained from three independent dot-blots were quantified by densitometry. Results were normalized with DNA obtained from cells expressing GFP (set as 1). (d) TET localization and 5hmC presence in transfected HEK293T cells was analysed via immunofluorescence. HA-tagged TET proteins (orange) localize mainly in the nuclei (blue). Cells that express catalytically competent TETs also have more 5hmC (red). The increase of hydroxymethylation in transfected cells is statistically significant (the p-value of the  hypothesis is 0.054). Scale bar, 20 µm. The arrows point to cells with TETs. The image represents a single slice of a confocal stack. The ring for 5hmC staining is expected because of the penetration depth of denaturation affecting the 5hmC detection. In contrast, both TET detection and DAPI staining do not require denaturation of the DNA, and hence do not show the same ring feature. Although HEK293 cells have very low endogenous TET and 5hmC, their residual amounts result in a small background.
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RSOB140110F3: In vitro expression of AmTET in human embryonic kidney (HEK293T) cells. (a) Western blot analysis of the expression levels of TET-HA proteins were analysed 48 h after transfection. GAPDH level is a protein loading control. (b) Genomic DNA from these cells was isolated and dot-blotted with specific anti-5hmC antibody. About 24–190 ng of each genomic DNA and the same amounts of PCR product with dCTP swapped for d5mCTP were used for this experiment. About 0.25–2ng of PCR product with dCTP swapped for d5hmCTP was used as a positive control. Signal obtained from cells expressing wild-type TETs is stronger than from cells expressing GFP or a catalytically inactivated honeybee TET. (c) Data obtained from three independent dot-blots were quantified by densitometry. Results were normalized with DNA obtained from cells expressing GFP (set as 1). (d) TET localization and 5hmC presence in transfected HEK293T cells was analysed via immunofluorescence. HA-tagged TET proteins (orange) localize mainly in the nuclei (blue). Cells that express catalytically competent TETs also have more 5hmC (red). The increase of hydroxymethylation in transfected cells is statistically significant (the p-value of the hypothesis is 0.054). Scale bar, 20 µm. The arrows point to cells with TETs. The image represents a single slice of a confocal stack. The ring for 5hmC staining is expected because of the penetration depth of denaturation affecting the 5hmC detection. In contrast, both TET detection and DAPI staining do not require denaturation of the DNA, and hence do not show the same ring feature. Although HEK293 cells have very low endogenous TET and 5hmC, their residual amounts result in a small background.

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)

In vitro expression of AmTET in human embryonic kidney (HEK293T) cells. (a) Western blot analysis of the expression levels of TET-HA proteins were analysed 48 h after transfection. GAPDH level is a protein loading control. (b) Genomic DNA from these cells was isolated and dot-blotted with specific anti-5hmC antibody. About 24–190 ng of each genomic DNA and the same amounts of PCR product with dCTP swapped for d5mCTP were used for this experiment. About 0.25–2ng of PCR product with dCTP swapped for d5hmCTP was used as a positive control. Signal obtained from cells expressing wild-type TETs is stronger than from cells expressing GFP or a catalytically inactivated honeybee TET. (c) Data obtained from three independent dot-blots were quantified by densitometry. Results were normalized with DNA obtained from cells expressing GFP (set as 1). (d) TET localization and 5hmC presence in transfected HEK293T cells was analysed via immunofluorescence. HA-tagged TET proteins (orange) localize mainly in the nuclei (blue). Cells that express catalytically competent TETs also have more 5hmC (red). The increase of hydroxymethylation in transfected cells is statistically significant (the p-value of the  hypothesis is 0.054). Scale bar, 20 µm. The arrows point to cells with TETs. The image represents a single slice of a confocal stack. The ring for 5hmC staining is expected because of the penetration depth of denaturation affecting the 5hmC detection. In contrast, both TET detection and DAPI staining do not require denaturation of the DNA, and hence do not show the same ring feature. Although HEK293 cells have very low endogenous TET and 5hmC, their residual amounts result in a small background.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140110F3: In vitro expression of AmTET in human embryonic kidney (HEK293T) cells. (a) Western blot analysis of the expression levels of TET-HA proteins were analysed 48 h after transfection. GAPDH level is a protein loading control. (b) Genomic DNA from these cells was isolated and dot-blotted with specific anti-5hmC antibody. About 24–190 ng of each genomic DNA and the same amounts of PCR product with dCTP swapped for d5mCTP were used for this experiment. About 0.25–2ng of PCR product with dCTP swapped for d5hmCTP was used as a positive control. Signal obtained from cells expressing wild-type TETs is stronger than from cells expressing GFP or a catalytically inactivated honeybee TET. (c) Data obtained from three independent dot-blots were quantified by densitometry. Results were normalized with DNA obtained from cells expressing GFP (set as 1). (d) TET localization and 5hmC presence in transfected HEK293T cells was analysed via immunofluorescence. HA-tagged TET proteins (orange) localize mainly in the nuclei (blue). Cells that express catalytically competent TETs also have more 5hmC (red). The increase of hydroxymethylation in transfected cells is statistically significant (the p-value of the hypothesis is 0.054). Scale bar, 20 µm. The arrows point to cells with TETs. The image represents a single slice of a confocal stack. The ring for 5hmC staining is expected because of the penetration depth of denaturation affecting the 5hmC detection. In contrast, both TET detection and DAPI staining do not require denaturation of the DNA, and hence do not show the same ring feature. Although HEK293 cells have very low endogenous TET and 5hmC, their residual amounts result in a small background.
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