Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages (original) (raw)

Accession codes

Primary accessions

BioProject

References

  1. Gialitakis, M., Sellars, M. & Littman, D.R. The epigenetic landscape of lineage choice: lessons from the heritability of CD4 and CD8 expression. Curr. Top. Microbiol. Immunol. 356, 165–188 (2012).
    CAS PubMed PubMed Central Google Scholar
  2. Taniuchi, I., Ellmeier, W. & Littman, D.R. The CD4/CD8 lineage choice: new insights into epigenetic regulation during T cell development. Adv. Immunol. 83, 55–89 (2004).
    CAS PubMed Google Scholar
  3. Taniuchi, I. et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 111, 621–633 (2002).
    CAS PubMed Google Scholar
  4. Taniuchi, I., Sunshine, M.J., Festenstein, R. & Littman, D.R. Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation. Mol. Cell 10, 1083–1096 (2002).
    CAS PubMed Google Scholar
  5. Zou, Y.R. et al. Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage. Nat. Genet. 29, 332–336 (2001).
    CAS PubMed Google Scholar
  6. Sawada, S., Scarborough, J.D., Killeen, N. & Littman, D.R. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell 77, 917–929 (1994).
    CAS PubMed Google Scholar
  7. Chong, M.M. et al. Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells. Genes Dev. 24, 659–669 (2010).
    CAS PubMed PubMed Central Google Scholar
  8. Lee, P.P. et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15, 763–774 (2001).
    CAS PubMed Google Scholar
  9. Tucker, K.L. et al. Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 10, 1008–1020 (1996).
    CAS PubMed Google Scholar
  10. Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat. Neurosci. 13, 423–430 (2010).
    CAS PubMed PubMed Central Google Scholar
  11. Jeong, M. et al. Large conserved domains of low DNA methylation maintained by Dnmt3a. Nat. Genet. 46, 17–23 (2014).
    CAS PubMed Google Scholar
  12. Day, K., Song, J. & Absher, D. Targeted sequencing of large genomic regions with CATCH-Seq. PLoS ONE 9, e111756 (2014).
    PubMed PubMed Central Google Scholar
  13. Henson, D.M., Chou, C., Sakurai, N. & Egawa, T. A silencer-proximal intronic region is required for sustained CD4 expression in postselection thymocytes. J. Immunol. 192, 4620–4627 (2014).
    CAS PubMed Google Scholar
  14. Collings, C.K., Waddell, P.J. & Anderson, J.N. Effects of DNA methylation on nucleosome stability. Nucleic Acids Res. 41, 2918–2931 (2013).
    CAS PubMed PubMed Central Google Scholar
  15. Jimenez-Useche, I. et al. DNA methylation regulated nucleosome dynamics. Sci. Rep. 3, 2121 (2013).
    PubMed PubMed Central Google Scholar
  16. Egawa, T. & Littman, D.R. ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 9, 1131–1139 (2008).
    CAS PubMed PubMed Central Google Scholar
  17. Sun, G. et al. The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 6, 373–381 (2005).
    CAS PubMed Google Scholar
  18. He, X. et al. The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826–833 (2005).
    CAS PubMed Google Scholar
  19. Egerton, M., Scollay, R. & Shortman, K. Kinetics of mature T-cell development in the thymus. Proc. Natl. Acad. Sci. USA 87, 2579–2582 (1990).
    CAS PubMed PubMed Central Google Scholar
  20. Pénit, C. & Vasseur, F. Expansion of mature thymocyte subsets before emigration to the periphery. J. Immunol. 159, 4848–4856 (1997).
    PubMed Google Scholar
  21. Ernst, B., Surh, C.D. & Sprent, J. Thymic selection and cell division. J. Exp. Med. 182, 961–971 (1995).
    CAS PubMed Google Scholar
  22. Schübeler, D. Function and information content of DNA methylation. Nature 517, 321–326 (2015).
    PubMed Google Scholar
  23. Booth, M.J. et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012).
    CAS PubMed Google Scholar
  24. Sato, T. et al. Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes. Immunity 22, 317–328 (2005).
    CAS PubMed Google Scholar
  25. He, Y.F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).
    CAS PubMed PubMed Central Google Scholar
  26. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).
    CAS PubMed PubMed Central Google Scholar
  27. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. 50, 7008–7012 (2011).
    CAS Google Scholar
  28. Egawa, T., Tillman, R.E., Naoe, Y., Taniuchi, I. & Littman, D.R. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells. J. Exp. Med. 204, 1945–1957 (2007).
    CAS PubMed PubMed Central Google Scholar
  29. Liu, S. et al. Interplay of RUNX1/MTG8 and DNA methyltransferase 1 in acute myeloid leukemia. Cancer Res. 65, 1277–1284 (2005).
    CAS PubMed Google Scholar
  30. Cheng, C.K. et al. Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia. Blood 118, 6638–6648 (2011).
    CAS PubMed Google Scholar
  31. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).
    CAS PubMed Google Scholar
  32. Nguyen, S., Meletis, K., Fu, D., Jhaveri, S. & Jaenisch, R. Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan. Dev. Dyn. 236, 1663–1676 (2007).
    CAS PubMed Google Scholar
  33. Okano, M., Bell, D.W., Haber, D.A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999).
    CAS PubMed Google Scholar
  34. Ruzankina, Y. et al. Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1, 113–126 (2007).
    CAS PubMed PubMed Central Google Scholar
  35. Grusby, M.J., Johnson, R.S., Papaioannou, V.E. & Glimcher, L.H. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253, 1417–1420 (1991).
    CAS PubMed Google Scholar
  36. Zijlstra, M. et al. Beta 2-microglobulin deficient mice lack CD4−8+ cytolytic T cells. Nature 344, 742–746 (1990).
    CAS PubMed Google Scholar
  37. Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37, 1281–1288 (2005).
    CAS PubMed Google Scholar
  38. Gobeil, S., Zhu, X., Doillon, C.J. & Green, M.R. A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene. Genes Dev. 22, 2932–2940 (2008).
    CAS PubMed PubMed Central Google Scholar
  39. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).
    CAS PubMed Google Scholar
  40. Kumaki, Y., Oda, M. & Okano, M. QUMA: quantification tool for methylation analysis. Nucleic Acids Res. 36, W170–W175 (2008).
    CAS PubMed PubMed Central Google Scholar
  41. Booth, M.J. et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8, 1841–1851 (2013).
    CAS PubMed PubMed Central Google Scholar
  42. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
    CAS PubMed Google Scholar

Download references

Acknowledgements

We thank R. Jaenisch (Whitehead Institute for Biomedical Research) for _Dnmt1_L2 and _Dnmt1_chip mouse strains; the University of Massachusetts Medical School RNAi Core Facility for shRNAs; A. Cuesta and A. Chen for technical help; and members of the Littman laboratory for discussion. Supported by the US National Institutes of Health (R00DK091508 to J.R.H., 5 T32 CA009161-36 to M.S., and GM033977 to M.R.G.), the Jane Coffin Childs Fund (J.R.H.), the Cancer Research Institute (M.S. and P.D.I.) and the Howard Hughes Medical Institute (M.R.G. and D.R.L.).

Author information

Author notes

  1. Stephane Gobeil
    Present address: Present Address: Centre Hospitalier de l'Université Laval, Québec, Canada.,
  2. MacLean Sellars and Jun R Huh: These authors contributed equally to this work.

Authors and Affiliations

  1. The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York, USA
    MacLean Sellars, Jun R Huh, Priya D Issuree, Carolina Galan & Dan R Littman
  2. Howard Hughes Medical Institute, New York University School of Medicine, New York, New York, USA
    MacLean Sellars, Jun R Huh, Priya D Issuree, Carolina Galan & Dan R Littman
  3. Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA
    Jun R Huh
  4. HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA
    Kenneth Day & Devin Absher
  5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
    Stephane Gobeil & Michael R Green
  6. Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA
    Stephane Gobeil & Michael R Green

Authors

  1. MacLean Sellars
    You can also search for this author inPubMed Google Scholar
  2. Jun R Huh
    You can also search for this author inPubMed Google Scholar
  3. Kenneth Day
    You can also search for this author inPubMed Google Scholar
  4. Priya D Issuree
    You can also search for this author inPubMed Google Scholar
  5. Carolina Galan
    You can also search for this author inPubMed Google Scholar
  6. Stephane Gobeil
    You can also search for this author inPubMed Google Scholar
  7. Devin Absher
    You can also search for this author inPubMed Google Scholar
  8. Michael R Green
    You can also search for this author inPubMed Google Scholar
  9. Dan R Littman
    You can also search for this author inPubMed Google Scholar

Contributions

M.S. performed E4P rescue experiments, proliferation assays in thymus and T4-βGT analysis; J.R.H. did the genetic screen and follow-up analyses; M.S. and K.D. did the analyses of Cd4 locus-wide methylation and nucleosome sequencing, with bioinformatics support from D.A.; P.D.I. performed oxidative bisulfite analysis; M.S. and C.G. performed amplicon bisulfite sequencing; S.G. and M.R.G. provided the mouse shRNA retroviral pools; and M.S., J.R.H. and D.R.L. designed the experiments and wrote the manuscript with input from the other authors.

Corresponding author

Correspondence toDan R Littman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DNA-methylation machinery is essential for silencing of Cd4 in cytotoxic T cells.

(a) Scheme for the retroviral shRNA screen. (b) Histogram showing CD4 expression (MFI) in WT cytotoxic T cells infected with a Dnmt1 shRNA-GFP retrovirus (shaded area) or sham shRNA-GFP retrovirus (open area). Representative of 2 independent experiments. (c) Cytotoxic CD4-8+ cells from _Dnmt1_Chip/Chip animals were loaded with e670 and cultured in vitro for 4 days after infection with Dnmt1 shRNA retroviruses. Virus-infected cells were selected with puromycin. Gates define non-, low-, medium- and highly-cycled cells (i.e. e670 dilution), and the percentage of cells de-repressing CD4 in each gate is indicated. The red line shows the CD4 staining level used to determine de-repression. Representative of 2 independent experiments.

Supplementary Figure 2 Reproducibility and coverage of locus-wide bisulfite sequencing.

Genomic DNA was prepared from biological replicates of the indicated samples, and then subjected to bisulfite CATCH-seq (Cd4 TSS +/- ~75kb). (a) For each replicate, the fraction methylation at CpG dinucleotides with at least 30x coverage is graphed. Linear regressions were performed (red lines) and R2 calculated. (b) The median CpG sequencing coverage for the indicated samples is graphed. Error bars represent the 5th and 95th percentiles for CpG sequencing coverage.

Supplementary Figure 3 Silencer-dependent DMR in the first intron of Cd4.

Naïve (Thy1.2+CD44loCD62L+CD25-) WT CD4+, WT CD8+ and Cd4S4Δ/S4Δ CD4+8+ cells were isolated from LNs. Their genomic DNA was either isolated immediately or after 5 days of in vitro population expansion, using CFSE-labeling and dilution to identify and sort cells that had completed at least 5 divisions. Genomic DNA was then subjected to CATCH-seq. Percent CpG methylation was graphed on the UCSC genome browser for (a) Chromosome 6, positions 124,746,000-124,909,000 and (b) Chromosome 6, positions124,814,000-124,855,000 (UCSC Mus musculus genome assembly mm9). UCSC genes are indicated below the graphs. The samples correspond to those Fig. 2. Biological replicates were derived from two experiments.

Supplementary Figure 4 Hypermethylation of the Cd4 locus in the cytotoxic lineage and immature T cell progenitors.

To confirm locus-wide bisulfite sequencing, two amplicons were chosen for targeted bisulfite sequencing (a-b, d-h) and four amplicons were chosen for methylation-sensitive restriction enzyme digest analysis (c) (locus (not drawn to scale) and CpG analysis map at top: S4: Silencer; black arrow: TSS; lollipops: CpG dinucleotides; blue bars: bisulfite sequencing amplicons; purple arrows: HpaII sites with positions relative to TSS). (a-b) Naïve WT CD4+ and CD8+ T cells were sorted, genomic DNA was prepared and bisulfite treated, and amplicons were cloned and sequenced. Filled circles indicate methylated CpG dinucleotides and empty circles indicate unmethylated CpG dinucleotides. Colored bars correspond to amplicons in map. The 5’ and 3’ amplicon methylation patterns are shown in (a) and (b), respectively. Data are from 3 mice from two experiments. (c) HpaII digestion of genomic DNA from naïve WT CD4+, WT CD8+ and _Cd4_S4Δ/S4Δ CD8+ T cells was assessed by qPCR at the indicated CpG dinucleotides. HpaII digests only unmethylated-CCGG motifs; thus, percent-undigested DNA corresponds to percent methylation. All samples were normalized to an HpaII-insensitive loading control amplicon in the Cd4 locus. Graphs represent the average (± s.d.) (n=2 for WT CD4+ and WT CD8+) or amount of undigested DNA (n=1 for Cd4S4Δ/S4Δ) are shown. Data are representative of at least two 2 experiments. Average (± s.d.) of percent methylation from locus-wide bisulfite sequencing of biological replicates is presented in the graphs on the left for each CpG (n=2; samples correspond to those in Fig. 2 ). (d) CFSE-labeled naïve WT CD4+ and CD8+ T cell populations were expanded for 5 d in vitro with anti-CD3, anti-CD28 and IL-2, and cells that had undergone at least 6 divisions were sorted and subjected to bisulfite sequencing of the 3’ amplicon (n = 1, one experiment). (e) CFSE-labeled naïve WT CD4+ and CD8+ T cells were injected into Rag2-/- mice, and 20 days later CFSE-negative cells were sorted (>10 divisions) and subjected to amplicon bisulfite sequencing of the 3’ amplicon (n = 1, one experiment). (f) DN3 and WT DP T cells were sorted and the 5’ amplicon was sequenced as in (a). (g-h) Bisulfite analysis of the 3’ intronic amplicon from WT (g) and _Cd4_S4Δ/S4Δ (h) DP cells. (f-h) Data are from 1 (DN3), 2 (_Cd4_S4Δ/S4Δ DP) or 3 (DP) mice from three experiments.

Supplementary Figure 5 Nucleosome positioning correlates with CD4 expression rather than with DNA methylation.

Nuclei from the indicated samples were isolated and treated with micrococcal nuclease, and mono-nucleosome fragments from the Cd4 locus ~ +/-75kb were analyzed by CATCH-seq (without bisulfite treatment). The upper density graphs show nucleosome occupancy (blue = high nucleosome density, white: naked DNA). The lower graphs show coordinate-specific CpG methylation (data from samples in Fig. 2-4; red = hyper-methylation, green = hypo-methylation). Tracks were graphed with the IGV browser platform (Chromosome 6, positions 124,831,500-124,838,486 (UCSC Mus musculus genome assembly mm9)). For clarity, yellow lines separate CD4 high- and low-expressing samples (above and below, respectively). The red arrowhead indicates a region in which nucleosome paucity is highly correlated with CD4 expression. Replicate samples are from two experiments.

Supplementary Figure 6 E4P controls proximal demethylation events early in T cell development.

CATCH-seq was performed on sorted populations of WT DN3 (Thy1.2+Lin-CD25+CD44-), WT and _Cd4_E4PΔ/E4PΔ DP (TCRβloCD24+CD69-CD4+CD8+), naïve (Thy1.2+CD25-CD44loCD62L+) WT CD4+, WT CD8+, _Cd4_E4PΔ/E4PΔ CD4+ T, and _Cd4_S4Δ/S4Δ CD8+ T cells. The heat map depicts percentage CpG methylation from -9270bp to -15869bp relative to the Cd4 TSS (Chromosome 6, positions 124847307-124853906; UCSC Mus musculus genome assembly mm9). The approximate location of the region within the Cd4 locus is indicated above (genes, S4 and E4P are noted), and the lone CpG within the proximal enhancer is indicated below the heat map (green arrow head). Replicates are from 2 independent mice or pools of mice from two independent experiments. 7 CpG dinucleotides in this region experienced complete or partial demethylation at the DN3 to DP transition, and this hypo-methylated state was preserved in mature T cell lineages. E4P is responsible for de-methylation before the DN3 stage (note _Cd4_E4PΔ/E4PΔ DP hyper-methylation compared to DN3 cells), as well as at the DN3 to DP transition (note that DN3 vs. DP differentially methylated CpG dinucleotides are all highly methylated in _Cd4_E4PΔ/E4PΔ DP cells).

Supplementary Figure 7 Diminished Dnmt1 activity ‘rescues’ CD4 expression in _Cd4_E4PΔ/E4PΔ helper T cells.

(a-c) Naïve CD4+ T cells (Thy1.2+CD25-CD8-CD44loCD62L+) from DNMT1-deficient and control mice, both with deletions of E4P, were CFSE labeled and stimulated in vitro with anti-CD3, anti-CD28 and IL-2. Analysis was performed at 96 h and 120 h, to determine (a) the percentage of CD4+ cells, (b) the MFI of the CD4+ cells, and (c) the percentage of CD4+ cells at each cell division as measured by CFSE dilution. Representative of at least 4 independent experiments. (d-e) CFSE stained Cd4 E4PΔ/E4PΔ CD4+ T cells were stimulated for 24 h with anti-CD3 and anti-CD28, infected with retroviral vectors expressing Puro, RFP and either Dnmt1 shRNA in a mir30 context (red, sh_Dnmt1_) or an empty mir30 (blue, vector). Transduced cells were maintained with puromycin selection and analyzed by gating for RFP+ cells. (a) The percentage of CD4+ cells, as well as the CD4 MFI of CD4+ cells, was measured by flow cytometry at 72 h, 96 h and 120 h. (b) CFSE dilution was used to measure the percentage CD4+ cells in each generation at 72 h, 96 h and 120 h. Representative of at least 4 independent experiments.

Supplementary Figure 8 ThPOK expression and cell division during helper T lineage differentiation.

(a) Helper and cytotoxic T cell differentiation can be traced by CD4, CD8 and GFP (from the _Zbtb7b_GFP allele) expression (Egawa, T. & Littman, D.R. ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 9, 1131–1139 10.1038/ni.1652 (2008)). Upon positive selection, DP T cells (A gate) up-regulate CD69 and TCRβ (not shown) and down-regulate CD8 expression, becoming CD4+CD8lo (B gate). MHCI-selected cells then up-regulate CD8 expression (D gate) before finally down-regulating HSA (not shown) and silencing CD4 expression (E gate). MHCII-selected _Zbtb7b_GFP/+ cells begin to express GFP at the CD4+CD8lo stage (green filled circles, B gate), before fully down regulating CD8 and HSA (C gate and not shown). In _Zbtb7b_GFP/GFP MHCII-selected cells, GFP expression is still induced at the CD4+CD8lo stage (green filled circles, B gate), but cells then up-regulate CD8 (D gate) before extinguishing CD4 expression to become GFP+ cytotoxic T cells (E gate). (b) To sort CD4+CD8lo cells at different stages of helper cell differentiation, we enriched _Zbtb7b_GFP/+ and _Zbtb7b_GFP/GFP thymocytes for TCRβ expression using MACS beads, before sorting HSA+CD69+CD4+CD8lo cells (middle panel) based on Zbtb7b expression: GFP- (MHCI- or early MHCII-selected), GFPmid (MHCII-selected, initiating commitment) and GFP+ (MHCII-selected, late commitment). (c) MHCII-selected CD4SP (_Zbtb7b_GFP/+) and CD8SP (_Zbtb7b_GFP/GFP) thymocytes were MACS enriched for TCRβ+ expression, before gating of HSA-TCRβhiCD69-GFP+ cells and sorting of CD4+CD8- and CD4-CD8+ cells, respectively. (b-c) Stainings are representative of at least 3 experiments. (d) Scheme for thymic injection to assess cell division during helper T cell development. (e-f) 106 Tg(TcraTcrb)425Cbn(OT-IItg)+Δ/Δ_H2-AbI_Δ/Δ CD45.2+ and 107 CD45.2- DP cells (HSA+CD69-CD4+CD8+) were CFSE-labeled and injected intra-thymically into CD45.2- recipients. Tg(TcraTcrb)425Cbn+Δ/Δ_H2-AbI_Δ/Δ were used to ensure a homogenous population of unselected DP cells that would differentiate into the helper lineage. After four days, mice were sacrificed and thymocytes were analyzed for phenotype and CFSE dilution. (e) Gating of host and donor-derived cells in recipient thymus at day 4 after injection. (f) CFSE levels in cells from gates indicated in (e). Note that injected DP thymocytes do not dilute CFSE following positive selection. Representative of at least 2 experiments.

Supplementary information

Rights and permissions

About this article

Cite this article

Sellars, M., Huh, J., Day, K. et al. Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages.Nat Immunol 16, 746–754 (2015). https://doi.org/10.1038/ni.3198

Download citation