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Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

Abstract

DNA 5-methylcytosine is a dynamic epigenetic mark with important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases, and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic-site processing during TET-TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG-substrate turnover. In early Xenopus embryos, Neil2 cooperates with Tdg in removing oxidized methylcytosines and specifying neural-crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP-site handover during oxidative DNA demethylation.

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Figure 1: 5fC and 5caC are demodified by short-patch BER in HeLa cell extracts.
Figure 2: NEIL1 and NEIL2 are required for removal of 5fC and 5caC in HeLa cells.
Figure 3: NEIL2 is required for demethylation of TCF21 in HNO387 cells.
Figure 4: NEIL1 and NEIL2 neither process nor specifically bind 5fC and 5caC in vitro.
Figure 5: NEIL1 and 2 promote TDG-mediated 5fC and 5caC excision.
Figure 6: Neil2 is required for neural-crest development in X. laevis.
Figure 7: Neil2 is required for 5fC and 5caC removal in Xenopus embryos and cooperates with Tet3 and Tdg.

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Acknowledgements

We thank U. Stapf (IMB) for technical assistance, A. Rao (Division of Signaling and Gene Expression, La Jolla Institute for Allergy and Immunology), P. Schär (Department of Biomedicine, University of Basel) and Y. Zheng (New England BioLabs) for reagents and the IMB core facilities for technical support. This work was supported by an European Research Council Senior Investigator Grant to C.N. ('DNAdemethylase'). M.U.M. was supported by a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (NSERC-PDF 403829-2011).

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L.S. and A.v.S. carried out biochemical assays. D.H. conducted Xenopus experiments. M.U.M. performed LC-MS/MS measurements. K.A. performed experiments on HNO387 cells. L.S. and S.K. carried out protein-protein interaction assays. All authors analyzed and discussed the data. L.S. and C.N. conceived the study, designed experiments and wrote the paper.

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Correspondence to Lars Schomacher or Christof Niehrs.

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Integrated supplementary information

Supplementary Figure 1 DNA demodification and glycosylase activities are sequence-context independent.

(a) Scheme of the substrates used for the assays. Left, sequence context 1, right, sequence context 2. The sequence context difference flanking the HpaII recognition site is shown. (b, c) DNA demodification assay (b) and DNA glycosylase assay (c) using HeLa cell extract (50 µg in a 50 µl assay) and 20 nM 5mC, 5hmC, 5fC and 5caC containing 160 bp dsDNA of the respective sequence context (compare also Figure 1b and Figure 2b). Product peaks of demodification- (+ HpaII treatment) and glycosylase activities (79mers; arrows) are shown with efficiencies in % of 79 nt peak integral relative to the total fluorescent signal per electropherogram. Note the similar glycosylase and demodification activities of the extract toward both oligos.

Supplementary Figure 2 Representative electropherograms of DNA glycosylase and DNA demodification assays.

(a,b) Primary data electropherograms from DNA glycosylase assays from (a) Figure 2c and (b) Figure 2d. Arrows indicate cleavage products (79 nt) with amounts of products shown as percentages above. (c) Primary data electropherograms from DNA demodification assay of Figure 2e. Arrows indicate HpaII cleavage products (79 nt) with amounts of products shown as percentages (arrow).

Supplementary Figure 3 Recombinant NEIL1 and NEIL2 possess 5hU glycosylase and AP lyase activity.

(a) Coomassie-stained SDS-PAGE of the indicated purified recombinant proteins used in this study. NgTet1: Naegleria gruberi Tet1. His6: N- or C-terminal hexahistidin tag. Relative molecular weights of marker proteins (M) [x103] are indicated on the left. (b-d) Electropherograms of reaction products from DNA glycosylase assays with ds (5hU:G; AP:G) and ss (5hU) oligonucleotide substrates. (b) NEIL1 and NEIL2 process ds and ssDNA containing 5-hydroxyuracil. Arrows indicate reaction products with efficiencies (%). Note: N-terminally hexahistidin-tagged NEIL1 or NEIL2 (H6-NEIL1, H6-NEIL2) are inactive and serve as negative controls. (c) NEIL1 and NEIL2 process AP sites. Arrows indicate reaction products with efficiencies (%). Note that spontaneous AP site hydrolysis under mock conditions is 25% (background). Synthesis of AP site containing oligonucleotides is described in ‘Online Methods’ section. (d) Recombinant NEIL1 and NEIL2 harbor AP lyase activity. A 79mer marker oligonucleotide was mixed with cleavage products shown in (c). The APEX1 cleavage product containing a 3’-hydroxyl co-migrated with the 79mer marker (blue peak). The cleavage products of NEIL1 or NEIL2 migrate slightly faster (arrows), indicative of a 3’-phosphate residue (containing two additional negative charges) after AP lyase reaction (β,δ-elimination).

Supplementary Figure 4 TDG stimulation controls with SMUG1 and APEX1 and effects of APEX1 on 5fC and 5caC processing in HeLa cells.

(a) DNA glycosylase assay of TDG and SMUG1 alone or in combination (100-fold molar excess SMUG1 (total protein) over TDG) on 5fC and 5caC containing ds oligonucleotides. Product peaks of glycosylase activities (79mers) are highlighted in red with efficiencies shown in % and -fold stimulations. Note: SMUG1 had no detectable glycosylase activity toward 5fC and 5caC substrates and was not able to stimulate base excision of TDG. S1, SMUG1. (b) DNA glycosylase assay as in (a) but with purified APEX1 instead of SMUG1 using a 5-fold molar excess (total protein) over TDG. A1, APEX1. (c) LC-MS/MS quantification of genomic cytosine modifications as in Figure 2f from HeLa cells siRNA depleted of the indicated genes. (d) DNA glycosylase assay on 5fC and 5caC containing oligonucleotides using HeLa extracts as in c. (e) Quantification of DNA glycosylase activities shown in d. Error bars, s.d. (n = 3 assay repetitions). **P < 0.01, ***P < 0.005 by two-tailed unpaired Student’s t-test. (f) Demodification assay on 5fC and 5caC containing oligonucleotides using HeLa extracts siRNA depleted of the indicated genes. Repair efficiencies are shown as %.

Supplementary Figure 5 NEIL1 and NEIL2 interact with TDG.

(a,b) Microscale thermophoresis binding assays. (a) Binding of fluorescently-labeled NEIL1 and NEIL2 to non-labeled TDG (reverse labeling as compared to binding assay shown in Figure 5c). (b) Left: Fluorescently-labeled TDG and SMUG1 do not interact in vitro. Right, positive control: High affinity binding of GFP to αGFP antibody. Fitted curves for each binding experiment (n = 3 binding assays), normalized fluorescence timetraces and calculated Kd-values are shown. Kd errors are calculated technical errors derived from the curve fittings. Fnorm (‰), normalized fluorescence per mill.

Supplementary Figure 6 Characterization of neil1, neil2, tdg and tet3 in X. laevis embryogenesis.

(a) qPCR expression profiles of neil1, neil2, tdg and tet3 during different stages of X. laevis development. Highest expression values per profiled gene were arbitrarily set to 100. Error bars, s.d. (n = 3 embryo batches each consisting of at least 5 embryos). (b) TUNEL (apoptosis) assay of unilaterally morpholino injected embryos (lineage traced by lacZ, light blue speckles) and quantification of TUNEL signal (dark blue speckles; n = 25, 32, 36, 36 embryos from left to right). Note that only tdg MO induces substantive apoptosis. (c) Phospho-histone H3 (cell proliferation) assay of unilaterally morpholino injected embryos as in (b). Note: No obvious differences in proliferation were detected (n >30 embryos each). (d) Phenotype of stage 34 embryos resulting from neil2 MO and neil2 MO2 injections. Note: Both morpholinos induced the same phenotype. (e), Relative expression levels of neil2 (normalized to h4) in control MO and neil2 MO injected embryos at stage 16 and stage 23. Scale bars, 200 µm.

Supplementary Figure 7 Relative abundance of X. laevis genomic DNA modifications.

(a) Quantification of total genomic 5mC, 5hmC, 5fC and 5caC levels in X. laevis whole embryos at stage 32, human HEK293T cells and mouse ES cells (mESCs) by LC-MS/MS. Error bars, s.d. (n = 3 X. laevis embryo batches consisting each of at least 5 embryos and 3 cell cultures of HEK293T and mESCs, respectively). (b) LC-MS/MS quantification as in (a) but in control and neil2 morphant Xenopus animal cap explants including measurements of 8oxoG. Error bars, s.d. (n = 3 explant batches each consisting of 20 animal cap explants). n.s., not significant. *P < 0.05, ***P < 0.005 by two-tailed unpaired Student’s t-test.

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Schomacher, L., Han, D., Musheev, M. et al. Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation. Nat Struct Mol Biol 23, 116–124 (2016). https://doi.org/10.1038/nsmb.3151

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