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Role of Tet1 and 5-hydroxymethylcytosine in cocaine action

Abstract

Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.

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Figure 1: TET1 expression is decreased in NAc after repeated cocaine and negatively regulates behavioral responses to cocaine.
Figure 2: Repeated cocaine induces 5hmC alterations in NAc.
Figure 3: 5hmC alterations positively correlate with alternative splicing regulation.
Figure 4: Gene body 5hmC alterations correlate with gene transcription changes after repeated cocaine.
Figure 5: Tet1 knockdown in NAc of cocaine naive mice induces 5hmC at cocaine-regulated loci.
Figure 6: Long-lasting induction of 5hmC at particular loci after cocaine.

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Acknowledgements

We thank A. Chess for critical comments and O. Jabado and M. Mahajan from the Mount Sinai Genomics Core for technical support. This work was supported by grants from the National Institute on Drug Abuse (E.J.N.), the National Institutes of Health (P.J., P.C., G.F., K.F.F. and H.S.) and the Simons Foundation (H.S.).

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Authors

Contributions

The studies were conceived and designed by E.J.N., P.J. and J.F. J.F. performed RNA-seq and ChIP-seq. K.E.S., Y.L. and J.F. performed 5hmC capture and sequencing. L.S., N.S. and J.F. performed bioinformatic analyses. J.F. and J.H. performed oxBS-seq. JF., V.V., B.M.L., V.S. and I.M. performed qPCR analyses. V.V., D.E., T.C. and J.F. performed immunohistochemistry. J.F., V.V., D.F., J.K. and E.R. performed stereotaxic surgeries and behavioral assays. T.L., K.F.F. and G.F. contributed LC-ESI-MS/MS data. C.Z., G.M. and H.S. provided AAV-Tet1 shRNA and AAV-TET1 viruses. M.E.C. performed western blotting. P.C. contributed reagents. G.T. contributed human samples. D.F., B.L., B.M.L., V.S. and P.K. helped to prepare the samples and collect the data. The paper was written by J.F. and E.J.N. and was edited by the other authors.

Corresponding author

Correspondence to Eric J Nestler.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Representative immunohistochemistry images and TET western blots.

a, TET1 staining in NAc of mice treated with repeated cocaine or saline. A decrease in TET1 levels after cocaine is seen. b, Full length western blots for TET1, TET2, TET3, and β-actin. Saline (s) and cocaine (c) samples were loaded alternatively. The blue rectangles demonstrate the lanes cropped in Figure 1c. c, DAPI/GFP/DARPP-32 staining of a mouse brain section after viral injection. DAPI staining demonstrates intact brain structure of the injected hemisphere. GFP staining illustrates the spread of viral-mediated transgene expression in NAc. DARPP-32 immunohistochemistry indicates intact striatum structure after viral injection.

Supplementary Figure 2 Validation of virally mediated Tet1 shRNA knockdown and overexpression.

a, qPCR indicates Tet1 transcript levels are decreased after Tet1 viral knockdown (KD) in NAc. Student unpaired t-test P=0.0026, t(18)=3.494. N=10 per group. b, Quantification by Western blotting confirms TET1 protein levels are also decreased after Tet1 viral knockdown in this brain region. Student unpaired t-test, P=0.047, t(12)=1.805. N=8 per group. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value. * indicates P<0.05, ** indicates P<0.01. c, DNA gel electrophoresis picture demonstrates the specific amplification of human TET1 that is encoded by the viral overexpression (OE) construct in mouse NAc.

Supplementary Figure 3 Relative expression of Tet mRNAs in adult mouse NAc.

qPCR analysis demonstrates relative abundance of Tet1, Tet2, and Tet3 transcripts in adult male mouse NAc. N=9 per group. Data are presented as mean ± SEM.

Supplementary Figure 4 Distribution of 5hmC-enriched regions in NAc of saline- and cocaine-treated mice.

a, Histogram of 5hmC peak counts (in numbers per million bp sequencing reads) of each chromosome in saline and repeated cocaine samples. b, Circular layout of 5hmC peaks under saline conditions in blue bars below the black-white ideogram along chromosomal coordinates. 5hmC differential sites after repeated cocaine are shown in yellow (red/green) bars of the inside circle. c, Coverage plot of 5hmC peak regions under saline (orange color) and cocaine (green color) conditions from transcription start sites (TSSs) to transcription ending sites (TESs), as well as up- and downstream regions, of genes genome-wide. The coverage of up- and downstream interpolated 33% sequence is also shown. DNA input trace is in purple color.

Supplementary Figure 5 Selective enrichment of H3K4me1, H3K27ac and 5hmC at enhancer regions.

ChIP-seq for H3K4me1 and H3K27ac as well as 5hmC-seq are plotted over the mouse brain enhancer regions defined by the ENCODE project (http://genome.ucsc.edu/ENCODE/). Coverage plot of H3K4me1 (blue color), H3K27ac (orange color), and 5hmC (green color) from 1500 bp up- and downstream of enhancer sites reveals their enrichment at enhancer regions as demonstrated by sequencing read counts while compared to input which has similar read counts as H3K4me3 (data not shown).

Supplementary Figure 6 ChIP-qPCR validation of putative enhancers.

a, H3K4me1 ChIP followed by qPCR from a separate cohort of animals display significant enrichments of H3K4me1 at 10 putative enhancer sites as predicted by H3K4me1 and H3K27ac ChIP-seq co-binding. Student’s unpaired t-test for enhancers1 to 10: P=0.00008, t(14)=5.455; P=0.00001, t(14)=6.832; P=0.00143, t(14)=3.958; P=0.00005, t(14)=5.713; P=0.00001, t(14)=6.527; P=0.00005, t(14)=5.717; P=0.00013, t(14)=5.213; P=0.04619, t(14)=2.187; P=0.00050, t(14)=4.495; P=0.00004, t(14)=5.842. N=8/group. b, similar validation is observed for H3K27ac ChIP enrichment on the same putative enhancer sites. Student’s unpaired t-test for enhancers1 to 10: P=0.00008, t(14)=5.507; P=0.00008, t(14)=5.481; P=0.00255, t(14)=3.665; P=2.94444E-08, t(14)=10.97; P=3.7615831E-09, t(14)=12.88; P=2.9862057E-08, t(14)=10.95; P=5.1980284E-04, t(14)=4.479; P=0.00624, t(14)=3.214; P=0.00042, t(14)=4.586; P=0.00007, t(14)=5.566. N=8/group. c, P300 ChIP demonstrates an enrichment at most of the 10 putative enhancer sites. Student’s unpaired t-test for enhancers1 to 10: P=0.06760, t(11)=2.01; P=0.01612, t(11)=2.839; P=0.08876, t(11)=1.881; P=0.00036, t(11)=5.071; P=0.00026, t(11)=5.241; P=0.00123, t(11)=4.312; P=0.00091, t(11)=4.477; P=0.00155, t(11)=4.177; P=0.00820, t(11)=3.212; P=0.02076, t(11)=2.748. All putative enhancer bindings are compared to a non-putative enhancer control region at OCT4 gene promoter (-400bp to -550bp). All experiments are done under saline basal codition. The 10 randomly selected putative enhancer sites are chr8:124192000-124192600, chr12:32746400-32746800, chr13:49223800-49225000, chrX:53603400-53603600, chr13:74702000-74702200, chr18:38527400-38527600, chr12:118582600-118583200, chr17:25690800-25691000, chr9:61420400-61421200, chr3:90251000-90251400. Conrol n=6/group, enhancer n=7/group. Data are presented as mean ± SEM. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

Supplementary Figure 7 Gene ontology of genes that show cocaine-induced changes by both 5hmC-seq and RNA-seq.

a, Gene ontology of the most significant enriched functional annotation groups of genes that display both mRNA induction (24 hr) and gene body 5hmC induction. b, Gene ontology of the most significant enriched annotation groups of genes that display both mRNA induction (4 hr after a cocaine challenge) and gene body 5hmC induction prior to the challenge dose of cocaine.

Supplementary Figure 8 oxBS-seq methodology validation.

Two loci with a significant increase (4932411E22Rik) or decrease (Rsbn1l) of 5hmC in NAc after repeated cocaine were selected from 5hmC-seq analysis. a, oxBS-seq confirmed that Rsbn1l shows decreased 5hmC frequency at all seven CpG sites. This decrease is statistically significant across the Rsbn1l locus. Student paired t-test, P=3.12E-08, t(6)=9.501. b, oxBS-seq confirmed that 4932411E22Rik displays increased 5hmC frequency at most CpG sites tested. This increase is statistically significant across the 4932411E22Rik locus. Student paired t-test, P=0.005, t(9)=3.687. N=1-2 biological replicates/condition. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value. ** indicates P<0.01, *** indicates P<0.001.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1, 5 and 7 (PDF 1343 kb)

Supplementary Methods Checklist (PDF 588 kb)

Supplementary Table 2

Gene regions that display cocaine-induced changes in 5hmC (XLSX 595 kb)

Supplementary Table 3

Genes whose putative nearby enhancers show cocaine-induced switches to chromatin state 4 or 5 (XLS 1616 kb)

Supplementary Table 4

Gene ontology of genes whose putative nearby enhancers show cocaine-induced switches to chromatin states 4 or 5 (XLSX 22 kb)

Supplementary Table 6

Genes that show overlap in cocaine regulation of 5hmC and alternative splicing (XLSX 14 kb)

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Feng, J., Shao, N., Szulwach, K. et al. Role of Tet1 and 5-hydroxymethylcytosine in cocaine action. Nat Neurosci 18, 536–544 (2015). https://doi.org/10.1038/nn.3976

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