2011 Apr 29
Hydroxylation of 5-methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain
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Hydroxylation of 5-methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain
Cytosine methylation is the major covalent modification of mammalian genomic DNA and plays important roles in transcriptional regulation. The molecular mechanism underlying the enzymatic removal of this epigenetic mark, however, remains elusive. Here, we show that 5-methylcytosine (5mC) hydroxylase TET1, by converting 5mCs to 5-hydroxymethylcytosines (5hmCs), promotes DNA demethylation in mammalian cells through a process that requires the base excision repair pathway. Though expression of the 12 known human DNA glycosylases individually did not enhance removal of 5hmCs in mammalian cells, demethylation of both exogenously introduced and endogenous 5hmCs is promoted by the AID (activation-induced deaminase)/APOBEC (apolipoprotein B mRNA-editing enzyme complex) family of cytidine deaminases. Furthermore, Tet1 and Apobec1 are involved in neuronal activity-induced, region-specific, active DNA demethylation and subsequent gene expression in the dentate gyrus of the adult mouse brain in vivo. Our study suggests a TET1-induced oxidation-deamination mechanism for active DNA demethylation in mammals.
Copyright © 2011 Elsevier Inc. All rights reserved.
Figure 1. TET1 Catalyses 5mC Hydroxylation and Promotes Demethylation of Exogenous SssI-Methylated Reporter Plasmids and Endogenous Genomic Loci
(A) Reactivation of methylation-silenced reporter plasmids by TET1. Shown on the left are sample images of immunostaining of HEK293 cells co-transfected with SssI-methylated GFP expression plasmids and expression constructs for TET1 or TET1m. Scale bar: 10 μm. Shown on the right is a summary of quantification of GFP
+ cells as measured by FACS. Values represent mean ± SEM (n = 3; **: P < 0.01; Student’s t-test). (B) Bisulfite sequencing analysis of reporter plasmids. Shown on the top is a schematic diagram of the region around GFP transcription start site (TSS) for bisulfite sequencing (indicated by the red line). Shown on the left are illustrations of methylation status of CpGs within the sequenced region. Black: methylated; yellow: unmethylated. Note the much higher level of demethylation in GFP + sorted cells. Shown on the right is a summary of mean numbers of CpGs that are demethylated within each clone. Values represent mean ± SEM (n = 3; *: P < 0.05; Student’s t-test). (C) Effects of TET1 and TET1m overexpression on the HpaII sensitivity of the GFP TSS region and several endogenous genomic loci in HEK293 cells. Open circles represent data from individual experiments and lines represent mean values (**: P < 0.01; *: P < 0.05; #: P > 0.1; Student’s t-test).
Figure 2. 5hmC-containing DNA Are Demethylated in HEK293 Cells
(A) A schematic diagram of a PCR-based approach to generate 5mC-, or 5hmC-containing DNA fragments, that contain an Ubiquitin promoter (pUbC) followed by the GFP open reading frame. (B) Summary of CpG methylation levels of GFP TSS regions under different conditions as quantified by the HpaII sensitivity. Open circles represent data from individual experiments and lines represent mean values (**:
P < 0.01; *: P < 0.05; #: P > 0.1; Student’s t-test). (C and D) Bisulfite sequencing analysis of 5mC-/5hmC-GFP DNA fragments under different conditions. Shown in (C) are sample traces from bisulfite sequencing of 5hmC-GFP DNA before or after transfection. Open and filled triangles indicate cytosines in CpG and CpH contexts, respectively. Arrows indicate demethylated 5hmCs. Shown in (D) are quantifications of demethylated CpGs (left) and CpHs (right) on 5mC-/5hmC-GFP DNA. Values represent mean ± SEM (n = 3; **: P < 0.01; *: P < 0.05; #: P > 0.1; Student’s t-test).
Figure 3. BER Enzyme Activities Are Required for 5hmC Demethylation
(A and B) Effects of PARP1 inhibitor ABT-888 (50 μM) and APE inhibitor CRT0044876 (50 μM) on demethylation of transfected 5hmC-GFP DNA fragments. Shown in (A) is a summary of HpaII sensitivity assay. Open circles represent data from individual experiments and lines represent mean values (*:
P < 0.05; Student’s t-test). Shown in (B) is a summary of bisulfite sequencing analysis. Values represent mean ± SEM (n = 3; *: P < 0.05; Student’s t-test). (C) Effects of ABT and CRT on TET1-induced increases of HpaII sensitivity of FGF1 (left) and rDNA (right) promoters. Open circles represent data from individual experiments and lines represent mean values (**: P < 0.01; *: P < 0.05; #: P > 0.1; Student’s t-test). (D) Effects of overexpression of human DNA glycosylases on demethylation of 5hmC-GFP DNA fragments. Shown is a summary of bisulfite sequencing analysis of 5hmC-GFP DNA after co-transfection with one of the 12 human DNA glycosylase cDNAs. Values represent mean ± SEM (n = 3; #: P > 0.1; one-way ANOVA).
Figure 4. AID/APOBEC Deaminases Promote 5hmC Demethylation
(A and B) Overexpression of AID results in a decrease in the abundance of 5hmCs in genomic DNA samples from HEK 293 cells as monitored by immunoblotting (A) and ELISA (B) using anti-5hmC antibodies. (C) Effects of AID on demethylation of 5hmC-GFP and 5mC-GFP DNA. Values represent mean ± SEM (n = 3; *:
P < 0.05; #: P > 0.1; Student’s t-test) (D) Effects of overexpression of AID/APOBEC deaminases on 5hmC demethylation as measured by bisulfite sequencing. Values represent mean ± SEM (n = 3; **: P < 0.01; *: P < 0.05; Student’s t-test). (E) Immunoblotting analysis of 5hmU levels in HEK293 cells after TET1 expression or transfection of 5hmC-GFP DNA, with or without the expression of 5hmU glycosylase SMUG1. (F) An oxidation-deamination-BER model of TET1-induced active DNA demethylation in mammalian cells.
Figure 5. 5hmC Demethylation Recapitulates Known Properties of AID-Catalysed Deamination
(A) A histogram of clone distribution of the percentage of demethylated 5hmCs. A Poisson distribution with λ = 8.4% is shown as a black line. Arrows indicate 3 highly processively demethylated 5hmC-GFP clones. A small portion of the reference sequence and the bisulfite sequencing results for these 3 clones are shown in the inset. (B) Occurrence of single demethylated 5hmCs at WRC and SYC motifs. WRC motif: W = A/T; R = A/G; SYC motif: S = C/G; Y =C/T. Only demethylated 5hmCs that are at least 5 nucleotides away from any other demethylated 5hmCs are analyzed. Values represent mean ± SEM (n = 3; *:
P < 0.05; Student t-test). (C) Demethylation of promoter-truncated 5hmC-GFP variants. The same region in the GFP open-reading frame is bisulfite sequenced for all variants. Values represent mean ± SEM (n = 3; **: P < 0.01; Student t-test). (D) Demethylation of 5hmCs in the CpG (left) and CpH (right) contexts on two opposite strands. Demethylated 5hmCs of the forward (untranscribed) strand and the reverse (transcribed) strand are represented by C-to-T and G-to-A transitions in bisulfite sequencing (Figure S5C), respectively. Values represent mean ± SEM (n = 3; **: P < 0.01; Student’s t-test).
Figure 6. TET1 and AID Regulate Endogenous 5hmC Levels and Promote DNA Demethylation in the Adult Mouse Brain
(A and B) Effects of AAV-mediated overexpression of control YFP, TET1, TET1m (A), and AID (B) on the endogenous 5hmC levels as measured by immunoblotting (left) and ELISA (right). Engineered AAV viruses were stereotaxically injected into the dentate gyrus of adult mice and dentate gyrus tissue was micro-dissected one week later for analysis. Values represent mean ± SEM (n = 3 animals for each condition; **:
P < 0.01; Student’s t-test). (C and D) Effects of YFP, TET1, TET1m and AID overexpression on methylation levels of Bdnf IX (C), Fgf1B and Fgf1G (D) alternative promoters in the adult dentate gyrus. Shown on top are diagrams of promoters and bars indicate regions for bisulfite sequencing analysis. Values represent mean ± SEM (n = 3 animals for each condition; **: P < 0.01; *: P < 0.05; Student’s t-test). (E) Effects of YFP, TET1, TET1m and AID overexpression on specific isoform and total expression levels of Fgf1 and Bdnf in the adult dentate gyrus. Same groups of animals as in (C and D) were examined. Values represent mean ± SEM (**: P < 0.01; *: P < 0.05; Student’s t-test).
Figure 7. Tet1 and Apobec1 are Involved in Neuronal Activity-Induced Region-Specific Demethylation in the Adult Mouse Brain
(A to C) Effects of AAV-mediated shRNA knock-down of endogenous Tet1 and Apobec1 on ECS-induced CpG demethylation of
Bdnf IX (A), Fgf1B (B) and Fgf1G (C) promoters. Engineered AAV viruses expressing control shRNA (CTL), two shRNAs against mouse Tet1 (Tet1-1 and Tet1-2), and one shRNA against mouse Apobec1 (Ap1-5), were stereotaxically injected into the dentate gyrus of adult mice. One week after viral injection, animals were subjected to ECS or sham treatment. Dentate gyrus tissue was micro-dissected 4 hrs later for analysis. Values represent mean ± SEM (n = 3–6 animals for each condition; **: P < 0.01; *: P < 0.05; Student’s t-test). (D and E) Effects of Tet1 and Apobec1 knock-down on ECS-induced isoform-specific expression of Bdnf and Fgf1 in the adult dentate gyrus. Same groups of animals as in (A to C) were examined. Values represent mean ± SEM (*: P < 0.05; Student’s t-test). (F) Demethylation of 5hmC-GFP DNA fragments after transfection into primary hippocampal neurons in culture. Shown is a histogram of clone distribution of the percentage of demethylated 5hmCs in 5hmC-GFP DNA fragments retrieved 7 days after transfection. A distributive Poisson model (λ = 6.2%) is shown by a black curve.
All figures (7)
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DNA demethylation plays a central role during development and in adult physiology. Different mechanisms of active DNA demethylation have been established. For example, Gr …
DNA Demethylation Pathways: Additional Players and Regulators
M Bochtler et al.
Bioessays 39 (1), 1-13.
DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-meth …
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M Tahiliani et al.
Science 324 (5929), 930-5.
DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC …
Active DNA Demethylation by DNA Repair: Facts and Uncertainties
D Schuermann et al.
DNA Repair (Amst) 44, 92-102.
Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and mai …
PubMed Central articles
Site- And Degree-Specific C-H Oxidation on 5-methylcytosine Homologues for Probing Active DNA Demethylation
S Kavoosi et al.
Chem Sci 10 (45), 10550-10555.
Ten-eleven translocation (TET) enzymes oxidize C-H bonds in 5-methylcytosine (5mC) to hydroxyl (5hmC), formyl (5fC) and carboxyl (5caC) intermediates en route to DNA deme …
A Review of Epigenetic Contributions to Post-Traumatic Stress Disorder
H Howie et al.
Dialogues Clin Neurosci 21 (4), 417-428.
Post-traumatic stress disorder (PTSD) is a syndrome which serves as a classic example of psychiatric disorders that result from the intersection of nature and nurture, or …
MicroRNA-21 Promotes Cell Proliferation by Targeting Tumor Suppressor TET1 in Colorectal Cancer
Y Ma et al.
Int J Clin Exp Pathol 11 (3), 1439-1445.
MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. TET1 acts as tumor-suppressor, …
Aberrant DNA Methylation Profile Exacerbates Inflammation and Neurodegeneration in Multiple Sclerosis Patients
N Celarain et al.
J Neuroinflammation 17 (1), 21.
Multiple sclerosis (MS) is an autoimmune and demyelinating disease of the central nervous system characterised by incoordination, sensory loss, weakness, changes in bladd …
Dead Cas Systems: Types, Principles, and Applications
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Int J Mol Sci 20 (23).
The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particula …
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
5-Methylcytosine / metabolism
Cytidine Deaminase / metabolism
DNA-Binding Proteins / metabolism
Mixed Function Oxygenases
Proto-Oncogene Proteins / metabolism
Mixed Function Oxygenases
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