6mA levels in axenic drosophila larvae are very low and not affected by TET loss. (a) The presence of bacterial contaminations in wild type (wt) and tetnull/+ adult flies was checked by PCR using universal primers against bacterial 16S rDNA and against the endosymbiotic bacteria gene wsp. PCR were performed on DNA from parental (F0) flies and after 3 generations of breeding in axenic conditions (F3). (b-d) 6mA levels were measured by LC-MS/MS in gDNA from whole larvae (b) or dissected CNS (c, d) generated from axenic flies reared on holidic medium (b,c) or conventional flies reared on classic medium (c,d). (e) 5mC levels were measured by LC-MS/MS in gDNA from dissected CNS generated flies reared on classic medium. (f, g) 6mA levels were measured by LC-MS/MS in embryos (f) and dissected adult brains (g) collected from crosses with conventional or axenic (Ax.) individuals raised on classic fly medium supplemented (Ax.) or not with antibiotics. wt: wild type (w1118); tetnull: tetnull/null; tet1/2: tetDMAD1/DMAD2. Filled circles: conventional flies; open triangles: axenic flies. Individual values, means and standard deviations are plotted. No statistically significant differences were observed between wt and tet mutant samples (Mann-Whitney test).

SMRT-seq analysis of larval CNS gDNA does not reveal an increase in 6mA in the absence of TET. (a) Percentage of adenines identified as 6mA in the wild type fusion dataset depending on the QV and coverage values used for 6mA selection. The dashed grey line indicates the level of 6mA measured by LC-MS/MS (0.0005%). (b) Influence of the QV and the coverage values on the proportion of 6mA identified in the wild type fusion dataset and in the three original samples. (c) Percentage of adenines covered at least 25× and identified as 6mA in each of the 3 wild type (wt) or tet null (tet) datasets depending on the QV. Means and standard deviations are represented. (d) Fraction of methylation in wt or tet null datasets depending on the QV (coverage≥25×). Means and standard deviations are represented.

TET does not oxidize 6mA. (a, b) In vitro assays showing TET activity profile on 5mC (a) and 6mA (b) containing double stranded oligonucleotide substrates. The levels of 5mC and its oxidised products (5hmC and 5fC) are represented relative to 5mC level at t=0. The levels of 6mA are represented relative to 6mA level at t=0. Error bars denote standard deviations from 3 independent experiments.

TET catalytic activity is largely dispensable in drosophila. (a, b) Schematic representation of tet locus (a) and main protein isoforms (b). (a) tet is transcribed from two alternative promoters giving rise to tet-long (tet-l) and tet-short (tet-s) isoforms. Filled boxes represent exons; non-coding exons (UTR) are depicted in green, coding exons in black or according to their domain-associated color. Introns are represented as gray lines (not to scale). The tet null, DMAD1, DMAD2 and catalytic dead (CD) alleles are depicted in red. The location of the GFP insertion generated by CRISRP/Cas9-mediated knock-in is also indicated. (b) The conserved domains of TET are colored; pink: CXXC DNA binding domain, orange: Cystein-rich domain, blue: double-stranded ß helix (DSBH) domain, red: HxD (iron binding motif). Amino acid positions are indicated according to the longest TET-l and TET-s isoforms. (c-h) Expression pattern of the wild type and catalytic-dead versions of TET in the larval CNS. tet-GFP (c-e) and tetCD-GFP (f-h) knock-in lines were used to detect TET proteins by confocal imaging after immunostaining against GFP (green). Nuclei were labelled with DAPI (blue). (c, f): stitched images showing dorsal views of the entire CNS. Scale bar: 100 µm. (d, e, g, h): high-magnification views of TET expression in the ventral nerve cord (d, g) or the central brain (e, h). DAPI only and GFP only channels are presented in the middle (‘) and lower (‘’) panels, respectively. Scale bar: 10 µm. (i) Percentage of adult flies of the indicated genotypes hatching from their pupal case. Means and standard deviations from 4 independent experiments. (j-l) Wing positioning (j), ovaries (k) and mushroom bodies (l) of wild type adult flies (ctr: tet-GFP) as compared to flies lacking TET expression (tet1/2: tetDMAD1/DMAD2 adult escapers) or TET enzymatic activity (tetCD). (l-l”) Immunostaining against Fas2 on adult brains was used to label mushroom body a, b and g lobes. (k-k”) Scale bar 500µm. (l-l”) Scale bar 50µm.

Detection of bacterial contamination in parental stocks and after 3 generations of breeding in axenic conditions. (a) The presence of bacterial contamination in adult flies of the indicated genotypes was checked by PCR using universal primers against bacterial 16S rDNA and against the endosymbiotic bacteria gene wsp. (b, b’) Genome-wide sequencing was used to assess the presence of contamination in gDNA from adult flies of the indicated genotypes. Between 15 and 19 million reads were analysed per sample. The absolute numbers of reads mapping to bacteria, virus or fungi genomes are represented in b, and their proportions normalized to the total number of reads are presented in b’. tet1/2: tetDMAD1/DMAD2.

Proportion of the drosophila genome covered in wild type (wt) and tetnull (tet) SMRT-seq fusion datasets according to coverage density.

Influence of the QV and the coverage values on the proportion of 6mA identified in the wt fusion dataset and in the three original samples. The percentage of 6mA identified in at least 2 out of 3 replicates is shown.

(a) Percentage of all adenines identified as 6mA by SMRT-seq in the tetnull fusion dataset depending on the QV and coverage values used for 6mA selection (log10 scale). (b, b’) Influence of the QV and the coverage values on the proportion of 6mA identified in the tetnull fusion dataset and in the three original samples (b: percentage of 6mA identified in at least 2 out of 3 samples; b’: percentage of 6mA identified in all 3 samples.

In vitro assays showing TET activity profile on double stranded oligonucleotide substrates containing or not 5mC (a) or 6mA (b). The levels of 5mC, 5hmC and 5fC normalized to dC (a) or 6mA normalized to dA (b) are represented. Error bars denote standard deviations from 3 independent experiments. Only background levels of modified nucleosides were detected when purified recombinant TET catalytic domain was incubated with unmodified (ctr) oligonucleotides.

Multiple sequence alignment of TET/JBP family members. The sequence surrounding the HxD motif in Coprinopsis cinerea TET (ccTET), Trypanosoma brucei JBP1, Drosophila melanogaster TET (dTET) and Homo sapiens TET1, TET2, TET3 is shown. Conserved amino-acids between TET homologs are boxed in yellow. The two key amino acids required for 6mA oxidation by ccTET are labelled with a star.

Survival assays showing the percentage of hatching embryos, larvae and adults of the indicated genotypes. Means and standard deviations are from at least 6 independent experiments.