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The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine

Nature Cell Biology volume 17, pages 545–557 (2015)Cite this article

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Abstract

How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.

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Figure 1: SIRT6 deficiency skews ESC differentiation towards neuroectoderm and promotes expression of Oct4, Sox2 and Nanog.
Figure 2: SIRT6-dependent regulation of core pluripotent genes.
Figure 3: OCT4:SOX2-dependent upregulation of TETs in S6KO versus WT ESCs and EBs.
Figure 4: Tet knockdown rescues the differentiation phenotype of S6KO ESCs and global levels of 5hmC.
Figure 5: Characterization of genomic regions with change of 5hmC in S6KO compared with WT ESCs.
Figure 6: Association of 5hmC with H3K4me2, but not H3K9ac and/or H3K56ac, in neuroectoderm genes upregulated in S6KO compared with WT ESCs.
Figure 7: SIRT6 deficiency triggers an in vivo differentiation defect in mouse and in human EBs, which is rescued by Tet knockdown on mouse teratomas.
Figure 8: Sirt6 deficiency triggers an in vivo differentiation defect in mouse and in human EBs.

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Acknowledgements

This work was supported in part by NIH grants GM093072-01, DK088190-01A1 (R.M.), 5R01HD058013-05 (to K.H.) and HD065812, CA151535 (to A.R.). R.M. is the Kristine and Bob Higgins MGH Research Scholar, the Warshaw Institute Fellow, and a Howard Goodman Awardee. L.C. was the recipient of a Feodor Lynen Research Fellowship from the Alexander von Humboldt Foundation. Y.H. was supported by a postdoctoral fellowship from the Leukemia and Lymphoma Society. C.A.S. is the recipient of the Evans Center Fellow Award. A.Gladden and A.Goren were the recipient of the Broad Institute SPARC (Scientific Projects to Accelerate Research and Collaboration) program. We thank O. Bar-Nur and S. Cheloufi for advice on the reprogramming of neural progenitor cells, and E. Kelliher for setting up the automation of the ChIP-Seq experiments.

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Author notes

  1. Lukas Chavez & Yun Huang

    Present address: Present addresses: Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany (L.C.); Institute of Biosciences & Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA (Y.H.).,

  2. Lukas Chavez and Yun Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts 02114, USA

    Jean-Pierre Etchegaray, Kenneth N. Ross, Jiho Choi, Barbara Martinez-Pastor, Ryan M. Walsh, Sita Kugel, Sridhar Ramaswamy, Konrad Hochedlinger & Raul Mostoslavsky

  2. The MGH Center for Regenerative Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA

    Jean-Pierre Etchegaray, Kenneth N. Ross, Jiho Choi, Barbara Martinez-Pastor, Ryan M. Walsh, Sita Kugel, Sridhar Ramaswamy, Konrad Hochedlinger & Raul Mostoslavsky

  3. UCSD Department of Pharmacology, La Jolla Institute for Allergy and Immunology, Sanford Consortium for Regenerative Medicine, UCSD Moores Cancer Center, La Jolla, California 92037, USA

    Lukas Chavez, Yun Huang, Matthias Lienhard & Anjana Rao

  4. The Center for Regenerative Medicine (CReM), Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA

    Cesar A. Sommer & Gustavo Mostoslavsky

  5. Broad Technology Labs (BTL), The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA

    Adrianne Gladden & Alon Goren

  6. Department of Human Biochemistry, Medical School, CEFyBO-UBA-CONICET, Buenos Aires, CP1121, Argentina

    Dafne M. Silberman

  7. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA

    Konrad Hochedlinger

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Contributions

J-P.E. conceived and designed most of the experiments, collected and analysed data, and wrote the manuscript; L.C., M.L. and K.N.R. performed computational analyses; Y.H. performed ChIP-Seq experiments; J.C. performed human ESC experiments; C.A.S. and G.M. performed iPSC experiments; B.M-P. performed and analysed immunofluorescence experiments; R.M.W. performed neurogenesis and immunofluorescence experiments; A.Gladden performed ChIP-Seq experiments; S.K. performed cloning of human SIRT6 expression system; D.M.S. performed in vitro experiments; S.R. supervised computational analyses; K.H. supervised the human ESC experiments; A.Goren conceived, supervised and analysed all the ChIP-Seq experiments and their computational analysis; A.R. supervised computational analyses, 5hMeC Chip-Seq experiments and edited the manuscript; R.M. conceived and supervised the study, analysed the data and wrote the manuscript.

Corresponding authors

Correspondence to Jean-Pierre Etchegaray, Alon Goren or Raul Mostoslavsky.

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Integrated supplementary information

Supplementary Figure 1 Gene expression linked to the skewed phenotype towards neuroectoderm in S6KO EBs, Related to Fig. 1.

(A) Immunofluorescence of EBs derived from WT and S6KO iPSCs (129 mouse strain) for Gfap (green). Scale bar, 500 μm. (B) Gene expression of trophectoderm and neuroectoderm genes in WT versus S6KO EBs. qRT-PCR data is expressed relative to WT EBs. Data is expressed relative to WT values. The data are n = 3 experimental replicates (from independent RNA preparations). Values are mean ± s.e.m. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗∗P < 0.00001, by t-test analysis. (C) Immunofluorescence of in vitro generated neurons from WT and S6KO EBs for β-III Tubulin (red). Undifferentiated neurons are visualized with dapi staining (blue). This is a representative of n = 3 experimental replicates (independent experiments). Scale bar, 100 μm. (D) Intensity of immunofluorescence of images from n = 3 experimental replicates (independent cell preparations) was analyzed by Image J. Data was normalized to WT values and represented as mean ± s.e.m. ∗∗P < 0.01, by t-test analysis. (E) Western blots showing expression of Nestin and Tet2 in WT versus S6KO EBs cultured under regular EB-medium. Expression of Sirt6 and its target H3K56ac are also shown. (F) Gene expression of trophectoderm genes in WT versus S6KO ESCs. qRT-PCR data is expressed relative to WT EBs. Data is expressed relative to WT values. The data are n = 3 experimental replicates (from independent RNA preparations). Values are mean ± s.e.m. ∗∗∗P < 0.001, ∗∗∗∗∗P < 0.00001, by t-test analysis. (G) Inability to silence expression of Sox2 and Nanog upon retinoic acid-induced differentiation in S6KO ESCs. Western blot analysis showing the expression of core pluripotent proteins in WT versus S6KO ESCs during a time-course retinoic acid-induced differentiation assay. Molecular weight markers are indicated. (H) Levels of H3K9ac and H3K56ac in S6KO versus WT ESCs after retinoic acid (RA)-mediated differentiation. ChIP-Seq binding profiles of the histone marks H3K56ac and H3K9ac on Oct4, Sox2 and Nanog genes in WT versus S6KO ESCs after retinoic acid-mediated differentiation. Images were created with the Integrative Genomic Viewer (IGV)51. Data are normalized to total counts, and the scale range is 0.0–1.0. The data on panels (B) and (F) are n = 3 experimental replicates, values are mean ± s.e.m. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗∗P < 0.00001 by t-test throughout the figure.

Supplementary Figure 2 The upregulated expression of neuronal differentiation related-genes exhibiting a 5hmC gain in S6KO over WT ESCs is rescued upon Tet knockdown, Related to Figures 5 and 6.

Gene expression analysis by qRT-PCR is expressed relative to WT ESCs. Data are n = 3 experimental replicates (independent RNA preparations), values are mean ± s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001,∗∗∗∗∗∗∗∗∗P < 0.00000001, by t-test throughout the figure.

Supplementary Figure 3 The upregulated expression of neuronal differentiation related-genes exhibiting a 5hmC gain in S6KO over WT ESCs is rescued upon Tet knockdown, Related to Figures 5 and 6.

Gene expression analysis by qRT-PCR is expressed relative to WT ESCs. Data are n = 3 experimental replicates (independent RNA preparations), values are mean ± s.e.m. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001,∗∗∗∗∗∗∗∗∗P < 0.00000001, by t-test throughout the figure.

Supplementary Figure 4

(A) The upregulated expression of neuronal differentiation related-genes exhibiting a 5hmC gain in S6KO over WT ESCs is rescued upon Tet knockdown, Related to Figures 5 and 6. Gene expression analysis by qRT-PCR is expressed relative to WT ESCs. Data are n = 3 experimental replicates (independent RNA preparations), values are mean ± s.e.m. ∗P < 0.05,∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, by t-test throughout the figure. (B) Sirt6 is recruited to the core pluripotent factors in human ESCs, Related to Figure 8. Genome wide Sirt6 ChIP-Seq data originated by Ram and colleagues30 was used to determine recruitment of human Sirt6 (hSirt6) to Oct4 and Sox2. (C) A small but significant peak for hSirt6 binding is apparent in Tet1, but not Tet2 gene. Statistically enriched hSirt6 peaks are shown as red boxes. Histone marks (H3K4me1, H3K4me2 and H3K36me3) and p300 signals are also shown for reference. Supplementary Table S10 shows all the genomic regions targeted by hSirt6 analysed using the same approach as in Ram and colleagues30. The accession number for these data can be found at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32509.

Supplementary Figure 5

(A) Pdk1 knockdown does not rescue the differentiation phenotype in S6KO EBs, Related to Figure 3. EBs derived from WT and S6KO ESCs (129 mouse strain). Scale bar, 500 μm. Pictures were taken at days 4 and 6 during EB formation. Data are representative of n = 3 experimental replicates. (B) Elevated levels of 5caC in S6KO versus WT ESCs. Related to Figure 4. Global 5caC levels assayed by slot blot analysis in ESCs. (C) Graph showing densitometric quantification of 5caC levels at each concentration of genomic DNA from panel (B). Data are representative of n = 2 experimental replicates. (D) Tet downregulation rescues high levels of 5caC in S6KO versus WT ESCs. Global 5caC levels assayed by slot blot analysis in Tet knockdown ESCs. (E) Graph showing densitometric quantification of 5caC levels at each concentration of genomic DNA from panel (D). Data are representative of n = 2 experimental replicates. (F) Increase efficiency of somatic cell reprogramming in S6KO versus WT NPCs. iPSC colony formation assay measured with alkaline phosphatase. (G) Graph showing iPSC colonies from each genotype. (H) Graph showing the average of WT (n = 3) versus S6KO (n = 5) NPCs reprogrammed into iPSCs. Colonies were quantified by image J, and values are mean ± s.e.m. ∗P < 0.05, by t-test analysis.

Supplementary Figure 6 Ectopic expression of human Sirt6 rescues the differentiation phenotype of S6KO EBs.

(A) Western blot analysis showing expression of endogenous Sirt6 and ectopic hSirt6. Total histone H3 is shown as loading control. (B) Embryoid bodies grown till day 10. Genotypes are indicated. Scale bar, 500 μm. (C) Gene expression analysis by qRT-PCR is expressed relative to WT ESCs. Data are n = 3 experimental replicates (independent RNA preparations), values are mean ± s.e.m. ∗P < 0.05, ∗∗P < 0.01, by t-test analysis.

Supplementary Figure 7 Characterization of genomic regions with changes of H3K9ac and H3K56ac in S6KO versus WT ESCs, before and after retinoic acid (RA)-mediated differentiation.

MEDIPS software was used to find regions with differential histones H3K9ac and H3K56ac in 500 base windows with P < 0.001 and these regions with gains and losses upon Sirt6 KO were mapped to promoters, gene bodies, CpG islands, enhancers, and super-enhancers. (A) Genomic positions of H3K9ac gains and losses in mouse ESCs. (B) Genomic positions of H3K9ac gains and losses in RA-differentiated mouse ESCs. (C) Genomic positions of H3K56ac gains and losses in mouse ESCs. (D) Genomic positions of H3K56ac gains and losses in RA-differentiated mouse ESCs.

Supplementary Figure 8 Enrichment of 5hmC does not correlate with H3K9ac and/or H3K56ac in S6KO versus WT ESCs.

(A) Heat map plot of regions of H3K56ac gains found with MEDIPS software in S6KO over WT in mouse ESCs show average profile for a ±3kb band centered around the 18722 regions with H3K56ac gain (P < 0.001) for the factors 5hmC, H3K56ac, H3K9ac, Sirt6, and Sox2 in WT and S6KO ESCs. (B) Enrichment line plot of average profile for regions of H3K56ac gain in S6KO over WT in mouse ESCs (n = 18,722) for the data on panel (A). Semi-transparent band behind line shows standard error of the mean for each average profile. (C) Heat map plot of regions of H3K9ac gains found with MEDIPS software in S6KO over WT mouse ESCs show average profile for a ±3 kb band centered around the 39095 regions with H3K9ac gain (P < 0.001) for the factors 5hmC, H3K56ac, H3K9ac, Sirt6, and Sox2 in WT and S6KO ESCs. (D) Enrichment line plot of average profile for regions of H3K9ac gain in S6KO over WT in mouse ES cells (n = 39095) for the data on panel (C). Semi-transparent band behind line shows standard error of the mean for each average profile.

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Etchegaray, JP., Chavez, L., Huang, Y. et al. The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine. Nat Cell Biol 17, 545–557 (2015). https://doi.org/10.1038/ncb3147

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