Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

An Nfil3–Zeb2–Id2 pathway imposes Irf8 enhancer switching during cDC1 development

Nature Immunology volume 20, pages 1174–1185 (2019)Cite this article

Subjects

Abstract

Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Zbtb46-GFP expression in CDPs identifies the earliest committed cDC1 progenitor.
Fig. 2: Single-cell RNA transcriptome analysis of CDPs.
Fig. 3: Zeb2 and Id2 heterogeneity identifies cDC1 specification in CDPs.
Fig. 4: Nfil3 is required for cDC1 specification.
Fig. 5: Zeb2 is downstream of Nfil3 in cDC1 development.
Fig. 6: Expression of Id2 and Zeb2 is mutually repressive in the CDP.
Fig. 7: Id2 imposes a switch from the +41-kb Irf8 enhancer to the +32-kb Irf8 enhancer by reducing E-protein activity.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Microarrays are available on the Gene Expression Omnibus (GEO) database with the SuperSeries accession no. GSE123800. Data from Fig. 1 are available with accession number GSE123747, from Fig. 3 with accession numbers GSE123794 and GSE123796, and from Fig. 6 with accession numbers GSE123797 and GSE123799. Data from Supplementary Fig. 4 are available with accession number GSE123797 and from Supplementary Fig. 5 with accession numbers GSE123798 and GSE123799. The scRNA-seq data are available with the accession number GSE132770, and are used in Fig. 2 and Supplementary Fig. 1b. The ATAC-seq data of DC progenitors are available with the accession number GSE132240 and are used in Fig. 7. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, K.M.M.

References

  1. Saxena, M. & Bhardwaj, N. Re-emergence of dendritic cell vaccines for cancer treatment. Trends Cancer 4, 119–137 (2018).

    Article  CAS  Google Scholar 

  2. Steinman, R. M. & Cohn, Z. A. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J. Exp. Med. 137, 1142–1162 (1973).

    Article  CAS  Google Scholar 

  3. Cella, M. et al. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5, 919–923 (1999).

    Article  CAS  Google Scholar 

  4. Murphy, T. L. et al. Transcriptional control of dendritic cell development. Annu. Rev. Immunol. 34, 93–119 (2016).

    Article  CAS  Google Scholar 

  5. Naik, S. H. et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat. Immunol. 8, 1217–1226 (2007).

    Article  CAS  Google Scholar 

  6. Onai, N. et al. Identification of clonogenic common Flt3+ M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat. Immunol. 8, 1207–1216 (2007).

    Article  CAS  Google Scholar 

  7. Liu, K. et al. In vivo analysis of dendritic cell development and homeostasis. Science 324, 392–397 (2009).

    Article  CAS  Google Scholar 

  8. Schlitzer, A. et al. Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow. Nat. Immunol. 16, 718–728 (2015).

    Article  CAS  Google Scholar 

  9. Grajales-Reyes, G. E. et al. Batf3 maintains autoactivation of Irf8 for commitment of a CD8alpha+ conventional DC clonogenic progenitor. Nat. Immunol. 16, 708–717 (2015).

    Article  CAS  Google Scholar 

  10. Lee, J. et al. Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow. J. Exp. Med. 212, 385–399 (2015).

    Article  CAS  Google Scholar 

  11. Breton, G. et al. Human dendritic cells (DCs) are derived from distinct circulating precursors that are precommitted to become CD1c+ or CD141+DCs. J. Exp. Med 213, 2861–2870 (2016).

    Article  CAS  Google Scholar 

  12. See, P. et al. Mapping the human DC lineage through the integration of high-dimensional techniques. Science 356, 1044–1044 (2017).

    Article  CAS  Google Scholar 

  13. Schiavoni, G. et al. ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8alpha+ dendritic cells. J. Exp. Med. 196, 1415–1425 (2002).

    Article  CAS  Google Scholar 

  14. Tamura, T. et al. IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J. Immunol. 174, 2573–2581 (2005).

    Article  CAS  Google Scholar 

  15. Durai, V. et al. Cryptic activation of an Irf8 enhancer governs cDC1 fate specification. Nat. Immunol. https://doi.org/10.1038/s41590-019-0450-x (2019).

  16. Kashiwada, M. et al. NFIL3/E4BP4 is a key transcription factor for CD8alpha+ dendritic cell development. Blood 117, 6193–6197 (2011).

    Article  CAS  Google Scholar 

  17. Kusunoki, T. et al. TH2 dominance and defective development of a CD8+ dendritic cell subset in Id2-deficient mice. J. Allergy Clin. Immunol. 111, 136–142 (2003).

    Article  CAS  Google Scholar 

  18. Hacker, C. et al. Transcriptional profiling identifies Id2 function in dendritic cell development. Nat. Immunol. 4, 380–386 (2003).

    Article  CAS  Google Scholar 

  19. Scott, C. L. et al. The transcription factor Zeb2 regulates development of conventional and plasmacytoid DCs by repressing Id2. J. Exp. Med. 213, 897–911 (2016).

    Article  CAS  Google Scholar 

  20. Wu, X. et al. Transcription factor Zeb2 regulates commitment to plasmacytoid dendritic cell and monocyte fate. Proc. Natl Acad. Sci. USA 113, 14775–14780 (2016).

    Article  CAS  Google Scholar 

  21. Cowell, I. G., Skinner, A. & Hurst, H. C. Transcriptional repression by a novel member of the bZIP family of transcription factors. Mol. Cell Biol. 12, 3070–3077 (1992).

    Article  CAS  Google Scholar 

  22. Seillet, C. et al. CD8alpha+ DCs can be induced in the absence of transcription factors Id2, Nfil3, and Batf3. Blood 121, 1574–1583 (2013).

    Article  CAS  Google Scholar 

  23. Ghosh, H. S. et al. Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells. Immunity 33, 905–916 (2010).

    Article  CAS  Google Scholar 

  24. Watowich, S. S. & Liu, Y. J. Mechanisms regulating dendritic cell specification and development. Immunol. Rev. 238, 76–92 (2010).

    Article  CAS  Google Scholar 

  25. Grajkowska, L. T. et al. Isoform-specific expression and feedback regulation of E protein TCF4 control dendritic cell lineage specification. Immunity 46, 65–77 (2017).

    Article  CAS  Google Scholar 

  26. Satpathy, A. T. et al. Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J. Exp. Med. 209, 1135–1152 (2012).

    Article  CAS  Google Scholar 

  27. McInnes, L., Healy, J. & Melville, J. UMAP: Uniform Manifold Approximation and Projection for dimension reduction. Preprint at https://arxiv.org/abs/1802.03426 (2018).

  28. McInnes, L. et al. UMAP: Uniform Manifold Approximation and Projection. Journal of Open Source Software https://doi.org/10.21105/JOSS.00861 (2018).

    Article  Google Scholar 

  29. Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38–44 (2019).

    Article  CAS  Google Scholar 

  30. Wu, X. et al. Bcl11a controls Flt3 expression in early hematopoietic progenitors and is required for pDC development in vivo. PLoS ONE 8, e64800 (2013).

    Article  CAS  Google Scholar 

  31. Chopin, M. et al. Transcription factor PU.1 promotes conventional dendritic cell identity and function via induction of transcriptional regulator DC-SCRIPT. Immunity 50, 77–90 (2019).

    Article  CAS  Google Scholar 

  32. Tussiwand, R. et al. Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 42, 916–928 (2015).

    Article  CAS  Google Scholar 

  33. Briseno, C. G. et al. Notch2-dependent DC2s mediate splenic germinal center responses. Proc. Natl Acad. Sci. USA 115, 10726–10731 (2018).

    Article  CAS  Google Scholar 

  34. Nishizaki, Y. et al. SIP1 expression patterns in brain investigated by generating a SIP1-EGFP reporter knock-in mouse. Genesis 52, 56–67 (2014).

    Article  CAS  Google Scholar 

  35. Jackson, J. T. et al. Id2 expression delineates differential checkpoints in the genetic program of CD8alpha+ and CD103+ dendritic cell lineages. EMBO J. 30, 2690–2704 (2011).

    Article  CAS  Google Scholar 

  36. Calero-Nieto, F. J. et al. Key regulators control distinct transcriptional programmes in blood progenitor and mast cells. EMBO J. 33, 1212–1226 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).

    Article  CAS  Google Scholar 

  38. Cisse, B. et al. Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development. Cell 135, 37–48 (2008).

    Article  CAS  Google Scholar 

  39. Cowell, I. G. & Hurst, H. C. Transcriptional repression by the human bZIP factor E4BP4: definition of a minimal repression domain. Nucleic Acids Res. 22, 59–65 (1994).

    Article  CAS  Google Scholar 

  40. Zhang, W. et al. Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter. Mol. Cell Biol. 15, 6055–6063 (1995).

    Article  CAS  Google Scholar 

  41. Ishizuka, I. E. et al. The innate lymphoid cell precursor. Annu. Rev. Immunol. 34, 299–316 (2016).

    Article  CAS  Google Scholar 

  42. Lee, J. et al. Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors. Nat. Immunol. 18, 877–888 (2017).

    Article  CAS  Google Scholar 

  43. Sathe, P. et al. Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells. Blood 121, 11–19 (2013).

    Article  CAS  Google Scholar 

  44. Rodrigues, P. F. et al. Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells. Nat. Immunol. 19, 711–722 (2018).

    Article  CAS  Google Scholar 

  45. Herman, J. S., Sagar & Grun, D. Fate ID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data. Nat. Methods 15, 379–386 (2018).

    Article  CAS  Google Scholar 

  46. Ghosh, H. S. et al. ETO family protein Mtg16 regulates the balance of dendritic cell subsets by repressing Id2. J. Exp. Med. 211, 1623–1635 (2014).

    Article  CAS  Google Scholar 

  47. Heng, T. S. & Painter, M. W. Immunological genome project consortium the immunological genome project: networks of gene expression in immune cells. Nat. Immunol. 9, 1091–1094 (2008).

    Article  CAS  Google Scholar 

  48. Kamizono, S. et al. Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo. J. Exp. Med. 206, 2977–2986 (2009).

    Article  CAS  Google Scholar 

  49. Higashi, Y. et al. Generation of the floxed allele of the SIP1 (Smad-interacting protein 1) gene for Cre-mediated conditional knockout in the mouse. Genesis 32, 82–84 (2002).

    Article  CAS  Google Scholar 

  50. Rawlins, E. L. et al. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development 136, 3741–3745 (2009).

    Article  CAS  Google Scholar 

  51. Butler, A. et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).

    Article  CAS  Google Scholar 

  52. Estrada, J. et al. SiteOut: an online tool to design binding site-free DNA sequences. PLoS ONE 11, e0151740 (2016).

    Article  Google Scholar 

  53. Shooshtari, P. et al. Correlation analysis of intracellular and secreted cytokines via the generalized integrated mean fluorescence intensity. Cytometry A 77, 873–880 (2010).

    Article  Google Scholar 

  54. Darrah, P. A. et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat. Med. 13, 843–850 (2007).

    Article  CAS  Google Scholar 

  55. Bailey, T. L. et al. MEME suite: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Chen and L. Goldstein for technical assistance. We thank the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine for help with genomic analysis. The Center is partially supported by the National Cancer Institute’s Cancer Center Support grant no. P30 CA91842 to the Siteman Cancer Center and by the Institute of Clinical and Translational Sciences/Clinical and Translational Science Award grant no. UL1TR000448 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of the NCRR or NIH. This work benefitted from data assembled by the ImmGen consortium47. This work was supported by the Howard Hughes Medical Institute (K.M.M. and H.Y.C.), the National Science Foundation (grant no. DGE-1745038 to P.B.), the US NIH (grant nos. F30DK108498 to V.D., K08 CA23188-01 to A.T.S. and P50-HG007735 to H.Y.C.) and the Parker Institute for Cancer Immunotherapy (A.T.S. and H.Y.C.). A.T.S. was supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund.

Author information

Author notes

  1. These authors contributed equally: Prachi Bagadia, Xiao Huang, Tian-Tian Liu.

Authors and Affiliations

  1. Department of Pathology and Immunology, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

    Prachi Bagadia, Xiao Huang, Tian-Tian Liu, Vivek Durai, Gary E. Grajales-Reyes, Sunkyung Kim, Theresa L. Murphy & Kenneth M. Murphy

  2. Howard Hughes Medical Institute, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

    Tian-Tian Liu & Kenneth M. Murphy

  3. Research Biology, Genentech, South San Francisco, CA, USA

    Maximilian Nitschké & Andrey S. Shaw

  4. Department of Molecular Biology, Genentech, South San Francisco, CA, USA

    Zora Modrusan

  5. Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA

    Jeffrey M. Granja, Ansuman T. Satpathy & Howard Y. Chang

  6. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Jeffrey M. Granja

  7. Biophysics Program, Stanford University School of Medicine, Stanford, CA, USA

    Jeffrey M. Granja

  8. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Ansuman T. Satpathy

  9. Department of Oncology, Amgen Inc., South San Francisco, CA, USA

    Carlos G. Briseño

  10. Department of Experimental Medicine, University of Perugia, Perugia, Italy

    Marco Gargaro

  11. Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

    Arifumi Iwata

  12. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

    Howard Y. Chang

Authors
  1. Prachi Bagadia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tian-Tian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vivek Durai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gary E. Grajales-Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maximilian Nitschké
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zora Modrusan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeffrey M. Granja
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ansuman T. Satpathy
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Carlos G. Briseño
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Marco Gargaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Arifumi Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Sunkyung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Howard Y. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Andrey S. Shaw
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Theresa L. Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Kenneth M. Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.B., X.H., T.T.L., T.L.M. and K.M.M. designed the study. P.B., X.H. and T.T.L. performed experiments related to analysis of immune populations, cell sorting and culture, gene microarray and generation of mice, with advice from C.G.B., G.E.G.-R., M.G. and S.K. P.B., M.N., Z.M. and A.S.S. performed and analyzed scRNA-seq data. V.D., J.M.G., A.T.S. and H.Y.C. performed ATAC-seq of DC progenitors. J.M.G. and A.T.S. performed computational analysis of ATAC-seq data. A.I. assisted with analysis of E-box motifs. P.B. performed all retroviral and reporter assays. P.B., X.H., T.T.L. and K.M.M. wrote the manuscript with advice from all authors.

Corresponding author

Correspondence to Kenneth M. Murphy.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Z. Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 cDC1 specification occurs in the CDP.

a, Zbtb46–GFPpos CDPs, Zbtb46–GFPneg CDPs, pre-cDC1 and pre-cDC2 were sort purified from Zbtb46gfp/+ mice, cultured for 5 d in Flt3L, and analyzed by flow cytometry for development of pDCs and cDC1 (data presented for three independent experiments, n = 4 for Zbtb46–GFPpos, Zbtb46–GFPneg CDPs, pre-cDC1 and n = 3 for pre-cDC2). Small horizontal lines indicate the mean. b, Violin plots depicting cluster identity and expression level for the indicated genes.

Supplementary Figure 2 ZEB2-EGFP and Id2-GFP Expression in BM and Spleen.

a, BM from Zeb2egfp/egfp (left) and Id2gfp/+ (right) mice were analyzed by FACS to identify GFP expression in the indicated progenitors. Numbers are the MFI (data representative of three independent experiments, n = 3 mice). b, Spleen from Zeb2gfp/gfp (left) and Id2gfp/+ (right) mice was analyzed by FACS for GFP expression in T cells (CD3+CD45R–), B cells (CD45R+CD3–), NK cells (NK1.1+CD3–), monocytes (Ly6C+CD115+), and DCs as gated in the Methods. Numbers are the MFI (data representative of three independent experiments, n =3 mice). c, Id2-GFPlo or Id2-GFPhi CDPs were sort purified from Id2gfp/+ mice, cultured for 5 or 7 d in Flt3L, and analyzed by FACS for development of cDC1 (red) or cDC2 (blue). Number indicates percentage of cells in the gate (data representative of two independent experiments, n = 2 mice). d, Percentages of cDC1s from in vitro Flt3L cultures as described in (c). Small horizontal lines indicate the mean. e, Zbtb46–GFPpos or Zbtb46–GFPneg CDPs were sort purified from Zbtb46gfp/+ mice, cultured for 5 or 7 d in Flt3L, and analyzed by FACS for development of pDCs (brown). Number indicate percentage of cells in the gate (data representative of two independent experiments, n = 2 mice). f, Percentages of pDCs from in vitro cultures as described in (e). Small horizontal lines indicate the mean. g, ZEB2–EGFPlo or ZEB2–EGFPhi CDPs were sort purified from Zeb2egfp/egfp mice, cultured for 5 or 7 d in Flt3L, and analyzed by FACS for development of pDCs (brown). Number indicates percentage of cells in gate (data representative of two independent experiments, n = 2 mice). h, Percentages of pDCs from in vitro Flt3L cultures as described in (g). Small horizontal lines indicate the mean. i, Id2–GFPlo or Id2–GFPhi CDPs were sort purified from Id2gfp/+ mice, cultured for 5 or 7 d in Flt3L, and analyzed by FACS for development of pDCs (brown). Number indicates percentage of cells in the gate (data representative of two independent experiments, n = 2 mice). j, Percentages of pDCs from in vitro Flt3L cultures as described in (i). Small horizontal lines indicate the mean.

Supplementary Figure 3 Nfil3 is Required for cDC1 Specification.

a, BM from Zbtb46gfp/+ (WT), Nfil3–/–Zbtb46gfp/+ and Batf3–/–Zbtb46gfp/+ mice was analyzed by FACS for development of pre-cDC1 (left), Lin–CD135+CD117intZbtb46–GFPpos (middle) and Zbtb46–GFPpos CDPs (right) (data representative of three independent experiments, n = 3 mice).

Supplementary Figure 4 Id2-Zeb2 Loop Regulates cDC1 Fate.

a, cDCs derived from in vitro Flt3L BM cultures from WT, Zeb2–/–, Id2–/–, and Zeb2–/– Id2–/– mice were analyzed for cDC1 (red) and cDC2 (blue) frequency. Numbers are the percent of cells in the indicated gates (data representative of two independent experiments, n = 2 for Id2–/– mice, n = 3 for Zeb2–/– Id2–/– mice, n = 4 for Zeb2–/– mice, and n = 5 for WT mice). b, cDC1 frequency is shown for individual mice in (a) as a percentage of total cDCs. Small horizontal lines indicate the mean. c, Splenic cDC1 from WT, Zeb2–/–, Id2–/–, and Zeb2–/– Id2–/– mice were purified by sorting and analyzed by gene expression microarrays. Shown are expression for the indicated genes for each genotype. Numbers are the average gene expression of three biological replicates.

Supplementary Figure 5 Id2 Does Not Regulate cDC2 Development or Transcriptome, But May Indirectly Repress Zeb2 through E proteins.

a, Spleens from radiation chimeras receiving either Zbtb46gfp+ (WT) or Id2–/–Zbtb46gfp/gfp (Id2–/–) BM were analyzed by FACS for the frequency of pDC (left), and cDC1 and cDC2 (middle) populations, and for Zbtb46-GFP expression on cells gated on cDC2 populations (right) (data representative of three independent experiments, n = 3 mice) b, Microarray analysis of Id2+/+ and Id2–/– cDC2. Gene expression comparing Id2+/+ cDC2 to Id2–/– cDC2 described in (a) are shown (left), or WT cDC2 compared to WT cDC1 (right) (data pooled from two independent experiments, n = 2 mice). 255 genes were at least twofold differentially expressed between Id2+/+ and Id2–/– cDC2. Green lines indicate twofold change threshold c, Gene expression microarray was performed on two CDP populations, sort purified separately as either Lin-CD117intZbtb46–GFPneg (CDP), or Lin-CD117intZbtb46-GFPpos (GFP+) cells, from each of three genotypes, Zbtb46gfp/+ (WT), Id2-/-Zbtb46gfp/gfp (Id2–/–), and Batf3-/-Zbtb46gfp/gfp (Batf3–/–) mice. Shown are averages of duplicate or triplicate gene expression values of the indicated genes in the indicated populations. d, BM from E2A-GFP reporter mice (Tcf3gfp/+) or WT (gray histograms) were analyzed for GFP expression in CDPs (left), or as an overlay (right) for pre-cDC1 (red) and CDP (blue) (data representative of four independent experiments, n = 5 mice). e, Shown are E2-A and CTCF peaks identified by ChIP-seq in HPC-7 cells at the Zeb2 locus (mm9)36. Squares represent E-box motifs (CANNTG) within the indicated enhancer regions. f, Proposed epistatic model for Nfil3, Id2, Zeb2, E protein, and cDC1 fate.

Supplementary Figure 6 Conservation of +41 kb Enhancer Between Human and Mice.

a, ChIP-seq peaks for E2-2 (E2-2) or control (input) for human IRF8 locus38. Numbers are chromosomal coordinates, human chromosome 16, draft genome hg19. Box indicates E2-2 binding peak corresponding to mouse +41 kb Irf8 enhancer. b, Consensus human E-box motif from E2-2 track. c, FIMO analysis depicting p-values of predicted E-boxes in human IRF8 chr16:85991064-85991633 (+58 kb from IRF8 TSS) d, Alignment of human, genome draft hg19, and mouse, genome draft mm10, for the +41 kb IRF8 enhancer regions. Conserved human (red box) and mouse (blue underlined) E-box motifs are indicated.

Supplementary Figure 7 Activity of +41 kb Irf8 Enhancer is E-box Dependent.

a, GFP expression in pDCs, from RV reporters without (empty) or with the entire 454 bp +41 kb enhancer region (IRF8 +41), or with single or double mutations of the indicated E-boxes (left) (data representative of at least three independent experiments, n > 3). b, Data shown in (a) shown as integrated MFI (iMFI; data pooled from at least three independent experiments, n > 3). Small horizontal lines indicate the mean. c, GFP expression in pDCs of RV reporters without (empty) or with the 454 bp +41 kb enhancer (IRF8 +41), or with intact segment A (A), or with mutations in E-box 1 (A-m1), E-box 2 (A-m2) or both (A-m1/m1), shown as histograms (data representative of at least three independent experiments, n > 3). d, GFP expression in pDCs of RV reporters without (empty) or with the 454 bp +41 kb enhancer (IRF8 +41), or with intact segment B (B), or with mutations in E-box 3 (B-m3), E-box 4 (B-m4) or both (B-m3/m4), shown as histograms (data representative of at least three independent experiments, n > 3). Data are presented as mean and one-way ANOVA was used to compare groups. *p < 0.05, **p < 0.01, ****p < 0.0001.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7

Reporting Summary

Supplementary Table 1

Expression of transcription factor-encoding genes with a difference in expression of at least four fold in the pre-cDC1 relative to their expression in Zbtb46-GFPneg CDP.

Supplementary Table 2

Expression of genes with a difference in expression of at least eightfold in the pre-cDC1 relative to their expression in Zbtb46–GFPneg CDP.

Supplementary Table 3

Expression of genes with a difference in expression of at least fivefold in the pre-cDC1 relative to ZEB2–EGFPhi CDP.

Supplementary Table 4

Expression of genes with a difference in expression of at least fivefold in the pre-cDC1 relative to Id2–GFPhi CDP.

Supplementary Table 5

Expresion of lineage-defining transcription factors in WT, Zeb2–/–, Zeb2–/–Id2–/–, and Zeb2–/–Nfil3–/– cDC1s.

Supplementary Table 6

Expression of genes with a difference in expression of at least twofold in Id2–/– cDC2s compared with Id2+/+ cDC2s.

Supplementary Table 7

Expression of genes with a difference in expression of at least threefold in Id2–/–CD117intZbtb46–GFPpos cells compared to Id2+/+CD117intZbtb46–GFPpos cells.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bagadia, P., Huang, X., Liu, TT. et al. An Nfil3–Zeb2–Id2 pathway imposes Irf8 enhancer switching during cDC1 development. Nat Immunol 20, 1174–1185 (2019). https://doi.org/10.1038/s41590-019-0449-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-019-0449-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing