Identification of mouse Tet3 transcript variants encoding a CXXC domain.

<p>(A) Drawing illustrating the generation of alternative transcripts from the <i>Tet3/Cxxc10-1</i> locus. The positions of primers used in B are reported. The lower part reports a schematic representation of alternative Tet3 transcripts. The positions of the probes used for northern blotting in C are reported. (B) Amplification of fragments from NSCs cDNA identifying Tet3 transcripts that include the Cxxc10-1 ORF. (C) Northern blot detection of alternative Tet3 transcripts in ESCs and NSCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755.s001" target="_blank">Fig. S1</a> for full and additional blots).</p

Genomic arrangement of mouse <i>Tet</i> genes and adjacent <i>Cxxc</i> loci (A) and homology of CXXC domains from mouse Cxxc4, Cxxc5 and Tet homologues in various animal species (B).

<p>(A) Schematic representation of mouse <i>Tet1</i>, <i>Tet2/Cxxc4</i> and <i>Tet3/Cxxc10</i> loci. Exons are depicted as blue rectangles. Annotated transcriptional start sites and transcription orientation are indicated with half arrows. (B) Alignment of CXXC domains from mouse Cxxc4, Cxxc5 and Tet homologues in various animal species (Mm, <i>Mus Musculus</i>; Hg, <i>Heterocephalus glaber</i>; Xt, <i>Xenopus tropicalis</i>; Hm, <i>Hydra mangipallata</i>). The alignment was generated with Unipro UGENE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755-Okonechnikov1" target="_blank">[64]</a>. Numbers on the right side indicate the position of the last amino acid in the corresponding protein. The KTXXXI motif, previously identified as determinant for the interaction of Cxxc4 with Dvl <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755-London1" target="_blank">[54]</a>, is boxed (see Discussion). The scale at the bottom indicates the upper limit of percent identity represented by each color. GenBank accession numbers: MmCxxc10, JX946278; XtTet3, NP_001090656.1; HgTet3, EHB01729.1; MmTet1, NP_081660.1; MmCxxc4, NP_001004367; MmCxxc5, NP_598448; HmTet, XP_002161163.1.</p

Tet3 and Cxxc4 interact <i>in vivo</i>.

<p>The interaction was detected by the F3H assay in BHK cells harboring a <i>lac</i> operator array (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755.s002" target="_blank">Fig. S2</a> for explanations). (A) An N-terminal fusion of Tet 3 with Cherry (Ch) was used as prey and GFP-Cxxc4 (upper row) or GFP (as control; lower row) as baits. Localization patterns are representative of 8 (upper row) and 9 (lower row) out of 10 imaged cells. (B) Ch-Cxxc4 was used as prey and GFP-Tet3 (upper row) or GFP (as control; lower row) as baits. Localization patterns are representative of 4 out of 5 (upper row) and 6 out of 7 (lower row) imaged cells. Arrowheads indicate the position of the <i>lac</i> operator array as identified by bait signals (GFP channel). Scale bars: 5 µm.</p

Levels of Tet3, Cxxc4 and Cxx5 transcripts in mouse adult tissues, NSCs and ESCs.

<p>Transcript levels were determined by qPCR analysis of total cDNA. (A) Amplfied fragments identify the Tet3 mRNA refseq NM_183138 (Tet3), the alternative Tet3 transcript containing the Cxxc10-1 ORF and exon 2 of NM_183138 (Tet3^CXXCL) and all transcripts including the Cxxc10-1 ORF. (B) Cxxc4 and Cxx5 transcript levels. Data relative to kidney, liver, cerebellum and cortex samples are from three biological replicates (two 6 week old 129Sv mice and a 30 week old C57BL/6 mouse). Data relative to spleen, heart, lung and hippocampus are from two biological replicates (a 6 week old 129/Sv mouse and a 30 week old C57BL/6 mouse). Data relative to NSCs and ESCs are from three independent cultures each. Shown are mean values and standard errors of the mean (SEM).</p

<i>In vitro</i> DNA binding properties of Cxxc4 and 5, isolated CXXC domains and full length constructs of Tet1 and Tet3^CXXC.

<p>All proteins were expressed as GFP fusion constructs in HEK293T cells and affinity purified using a GFP-trap. Fluorescently labeled DNA substrates with the same sequence and a single CpG site either unmethylated, symmetrically methylated or symmetrically hydroxymethylated were incubated in direct competition. Shown are mean values of bound substrate/protein ratios and SEM from n independent replicate experiments: Tet1, n = 10; Tet3, CXXC^Tet3, n = 6; Tet3^CXXCL, n = 7; CXXC^Tet1, Cxxc4 and GFP, n = 3; Cxxc5, n = 2.</p

5‑Formyl- and 5‑Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates

Uniform downregulation of Oct4 protein levels during EB differentiation.

<p>Undifferentiated ESCs colonies (day 0) and EBs at indicated stages of differentiation (day 4–12) were dissociated into single cells, stained with an Oct4 specific antibody and subjected to FACS analysis. Shaded curves depict Oct4 intracellular staining. Dashed lines indicate isotype controls and horizontal bars represent individual gates for positive Oct4 signals. The overlay shows exclusively Oct4 positive signals for each cell line. Numbers in red, blue and green indicate the percentages of Oct4 positive cells in wt, <i>Dnmt1^−/−</i> and TKO ESCs/EBs, respectively, after subtraction of background signal from isotype controls.</p

CpG island methylation and disregulated expression of selected bivalent genes in wt and mutant EBs.

<p>Transcript levels and CpG island methylation of bivalent genes <i>Nestin</i> (A,B) and <i>Sox1</i> (C,D) as well as the non-bivalent gene <i>Tet1</i> (E,F) in undifferentiated ESCs (day 0) and at day 4 and 16 of EB culture. (A,C,E) Transcript levels were measured by RT-qPCR in wt, <i>Dnmt1^−/−</i> and TKO ESCs/EBs as indicated. Mean values and standard errors from three independent biological replicates are shown. All values are normalized to undifferentiated wt ESCs. Asterisks indicates the significance level p<0.05 (Student t-test). (B,D,F) DNA methylation analysis by bisulfite sequencing in wt ESCs/EBs. In the gene cartoons large arrows indicate transcriptional start sites (TSS), open rectangles represent exons, grey shaded rectangles represent CpG islands and numbers indicate the positions of their borders with respect to the TSS. Small arrows indicate the analyzed regions. In the panels grey and black squares indicate unmethylated and methylated CpG sites, respectively. Percentages of total CpG methylation within the analyzed regions/clones are reported at the bottom of each panel.</p

Genome-wide expression changes in early wt, <i>Dnmt1^−/−</i> and TKO EBs.

<p>Scatter plots of genome-wide expression changes between day 0 and 4 in TKO (A) and <i>Dnmt1^−/−</i> EBs (B) relative to wt EBs. Data points are heat-colored according to their local density. About 17% of 25528 analyzed genes changed expression level in wt EBs according to the thresholds indicated by the vertical dotted lines. Genes whose expression levels increased and decreased in wt but not in TKO EBs (“TKO non-responders”, parts of the corridor between the horizontal dotted lines to the left and right of the vertical dotted lines in A) are marked in green in both A and B and their numbers are reported in green in the respective sectors.</p

LIF signaling induces reversion of cells from <i>Dnmt1^−/−</i> and TKO EBs to the ESC state.

<p>(A) Overview of experimental set up. Day 12 EBs were dissociated and their cells were plated and further cultured for three days (R1–3) in the presence or absence of LIF. (B) Transcript levels of pluripotency associated genes <i>Oct4</i>, <i>Nanog</i> and <i>Tet1</i>, as well as differentiation marker genes <i>Fgf5</i>, <i>Brachyury</i> and <i>Eomes</i> were determined by RT-qPCR in undifferentiated ESCs (day 0), day 12 EBs and 1, 2 and 3 days after replating (R1–3). Wild type, <i>Dnmt1^−/−</i> and TKO samples are represented in shades of red, blue and green, respectively, as indicated in the box at the upper right corner. Mean values relative to wt ESCs (day 0) and standard errors are from three independent biological replicates. Asterisks indicate significance levels: * p<0.05; ** p<0.001; *** p<0.0001 (Student t-test).</p