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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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In vivo expression of AmTET. (a) In situ hybridization showing the localization of AmTET transcripts in eggs and during embryogenesis. (b) In situ hybridization showing the localization of AmTET transcripts in adult brain (nurse bee): (i) whole brain; (ii) high magnification of one calyx. The red arrow indicates the area occupied by large Kenyon cells. Control hybridizations with sense probe detect no signal (not shown). (c) qPCR analysis of AmTET and AmDNMT3 expression in eggs, embryos and adult brains, relative to ovarian expression of both genes.
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RSOB140110F4: In vivo expression of AmTET. (a) In situ hybridization showing the localization of AmTET transcripts in eggs and during embryogenesis. (b) In situ hybridization showing the localization of AmTET transcripts in adult brain (nurse bee): (i) whole brain; (ii) high magnification of one calyx. The red arrow indicates the area occupied by large Kenyon cells. Control hybridizations with sense probe detect no signal (not shown). (c) qPCR analysis of AmTET and AmDNMT3 expression in eggs, embryos and adult brains, relative to ovarian expression of both genes.

Mentions: Using both qPCR and in situ hybridization, we have examined the expression of AmTET during early development and in adult tissues. AmTET transcripts are relatively abundant in 0–5 h eggs, but scarce during early/mid-blastoderm formation (14–20 h; figure 4a,c). Because 0–5 h eggs contain only four to seven nuclei, a relatively high level of any transcript at this stage is considered to be of maternal origin. The maternal transcripts are eliminated from the embryo at the midblastula transition [20,21] and are replaced by zygotic transcription that already is detectable at late blastoderm formation phase (25–30 h, figure 4a). From the germ band stage (approx. 40–48 h) until the completion of larval body at the pre-hatching phase (69–72 h), the levels of AmTET are relatively high especially in the nervous system that begins to form around 40 h (figure 4a). The expression levels in adult brains are comparable to those in late embryos and are very similar in foragers, nurses and mated egg-laying queens (figure 4c). Although AmTET appears to be ubiquitously expressed in most or all brain cells (figure 4b(i)), a higher magnification of the mushroom body calyces reveals a distinct pattern indicative of a preferential expression of this gene in large Kenyon cells (red arrow in figure 4b(ii)) whose somata are located at the inside edges of the calyces [21].Figure 4.


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 vivo expression of AmTET. (a) In situ hybridization showing the localization of AmTET transcripts in eggs and during embryogenesis. (b) In situ hybridization showing the localization of AmTET transcripts in adult brain (nurse bee): (i) whole brain; (ii) high magnification of one calyx. The red arrow indicates the area occupied by large Kenyon cells. Control hybridizations with sense probe detect no signal (not shown). (c) qPCR analysis of AmTET and AmDNMT3 expression in eggs, embryos and adult brains, relative to ovarian expression of both genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140110F4: In vivo expression of AmTET. (a) In situ hybridization showing the localization of AmTET transcripts in eggs and during embryogenesis. (b) In situ hybridization showing the localization of AmTET transcripts in adult brain (nurse bee): (i) whole brain; (ii) high magnification of one calyx. The red arrow indicates the area occupied by large Kenyon cells. Control hybridizations with sense probe detect no signal (not shown). (c) qPCR analysis of AmTET and AmDNMT3 expression in eggs, embryos and adult brains, relative to ovarian expression of both genes.
Mentions: Using both qPCR and in situ hybridization, we have examined the expression of AmTET during early development and in adult tissues. AmTET transcripts are relatively abundant in 0–5 h eggs, but scarce during early/mid-blastoderm formation (14–20 h; figure 4a,c). Because 0–5 h eggs contain only four to seven nuclei, a relatively high level of any transcript at this stage is considered to be of maternal origin. The maternal transcripts are eliminated from the embryo at the midblastula transition [20,21] and are replaced by zygotic transcription that already is detectable at late blastoderm formation phase (25–30 h, figure 4a). From the germ band stage (approx. 40–48 h) until the completion of larval body at the pre-hatching phase (69–72 h), the levels of AmTET are relatively high especially in the nervous system that begins to form around 40 h (figure 4a). The expression levels in adult brains are comparable to those in late embryos and are very similar in foragers, nurses and mated egg-laying queens (figure 4c). Although AmTET appears to be ubiquitously expressed in most or all brain cells (figure 4b(i)), a higher magnification of the mushroom body calyces reveals a distinct pattern indicative of a preferential expression of this gene in large Kenyon cells (red arrow in figure 4b(ii)) whose somata are located at the inside edges of the calyces [21].Figure 4.

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