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The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance

Nature Immunology volume 15, pages 258–265 (2014)Cite this article

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A Corrigendum to this article was published on 19 August 2014

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Abstract

The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1−/− mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.

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Figure 1: Aire interacts with ATF7ip and MBD1.
Figure 2: ATF7ip and MBD1 are required for Aire-induced gene expression, and Aire and MBD1-VP16 globally upregulate a similar set of genes.
Figure 3: Aire and MBD1 are present at the promoters of Aire-dependent genes.
Figure 4: MBD1 is required for the prevention of autoimmunity.
Figure 5: MBD1 regulates the Aire-dependent expression of TSA-encoding genes in vivo.

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Change history

  • 24 February 2014

    In the version of this article initially published, the blot in the third row on the right in Figure 1c (Flag-Aire, 3% input) was incorrect. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank J. Abramson (Weizmann Institute of Science) for Flag-tagged Aire; J. Bluestone, A. Abbas and members of the Bluestone and Abbas laboratories for discussions; and M. Cheng and T. LaFlam for feedback on the manuscript. Supported by the Arthritis Foundation (M.W.), the Rheumatology Research Foundation (M.W.), the Burroughs Welcome Fund (M.S.A.), the US National Institutes of Health (M.S.A.) and the Diabetes and Endocrinology Research Center (program project grant DK59958 for microarray and microscopy experiments).

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Authors and Affiliations

  1. Diabetes Center, University of California San Francisco, San Francisco, California, USA

    Michael Waterfield, Imran S Khan, Jessica T Cortez, Una Fan, Todd Metzger, Kayla Fasano & Mark S Anderson

  2. Department of Pediatrics, University of California San Francisco, San Francisco, California, USA

    Michael Waterfield

  3. Department of Microbiology & Immunology, University of California San Francisco, San Francisco, California, USA

    Alexandra Greer

  4. Department of Liver Sciences, Division of Transplantation Immunology & Mucosal Biology, Institute of Liver Studies, King's College London, London, UK

    Marc Martinez-Llordella

  5. Department of Medicine, University of California San Francisco, San Francisco, California, USA

    Joshua L Pollack, David J Erle & Mark S Anderson

  6. Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Maureen Su

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Contributions

M.W. and M.S.A. wrote the manuscript and designed the experiments; M.W., I.S.K., J.T.C., U.F., T.M., A.G. and M.S. did the experiments; K.F. did histology; and M.M.-L., J.L.P. and D.J.E. did microarray analysis.

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Correspondence to Mark S Anderson.

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Integrated supplementary information

Supplementary Figure 1 The SAND domain of Aire interacts with ATF7ip and Aire binds to the CXXC domains of MBD1.

(a) Outline of the strategy employed to generate the prey cDNA library from an Aire-GFP reporter transgenic mouse. Aire-expressing cells were purified by flow sorting MHC Class II Hi, GFP+ mTECs pregated on CD45-, EPCAM+ events from a collagenase treated thymus. (b) Structural domains of MBD1 including an N terminal MBD1 domain, a nuclear localization sequence (NLS), three CXXC domains, and a C terminal trans repression domain (TRD). Two truncation mutants are shown that are used for subsequent coimmunoprecipitation (Co-IP). (c) Representative example of yeast plates from pairwise mating using either HSR or SAND as bait and Aire or ATF7ip domain 2(D2) as prey. Each mating was performed in duplicate as indicated by arrows. (d) Plasmids expressing c-Myc-Aire and either Flag-MBD1 1-312 or Flag-MBD1 1-161 were transiently transfected into HEK 293 cells and Co-IP was performed with an irrelevant IgG or c-Myc antibody. TATA-binding protein (TBP) is included as a loading control. Data are representative of two experiments (c,d).

Supplementary Figure 2 A cell line stably expressing Aire recapitulates Aire-dependent gene expression.

(a) Immunofluorescence of HEK 293 cells stably expressing Flag tagged Aire. The left panel shows Aire staining in red, the center panel shows Flag (green), the right panel shows the overlay (yellow). Blue indicates nuclear DAPI staining. (b) Relative expression of the Aire-dependent genes S100A8, KRT14, and ALOX12 in the Aire-stable cell line compared to a cell line expressing vector only. (c) Western blot showing the efficiency of knockdown by plasmids expressing a scrambled shRNA (shNeg), an ATF7ip shRNA (shATF7ip), or an MBD1 shRNA (shMBD1). TATA-binding protein (TBP) is included as a loading control. (d) Aire and MBD1-VP16 do not induce the non Aire-regulated gene S100A10. Relative expression of S100A10 after transient transfection with plasmids expressing Flag only (Vector), Aire-Flag (Aire), Flag-MBD1-VP16 (MBD1-VP16), or Flag-MBD1-VP16-R22A (R22A). (e) Western blot showing the expression of Flag only (Vector), Aire-Flag (Aire), Flag-MBD1-VP16 (MBD1-VP16), or Flag-MBD1-VP16-R22A (R22A) in HEK 293 cells. TATA-binding protein (TBP) is included as a loading control. Data are representative of three experiments (mean and s.e.m of pooled data from three biological replicates in b,d). Data are representative of two experiments (a,c,e).

Supplementary Figure 3 Aire and MBD1-VP16 share many target genes.

HEK 293 cells were transfected in triplicate with Flag only (Vector), Aire-Flag (Aire), or Flag-MBD1-VP16 (MBD1-VP16) and microarray analysis was performed on isolated RNA. (a) Comparison of effects of Aire and MBD1-VP16 on transcript levels (each dot represents a single gene). (b) qPCR confirmation of microarray analysis on three additional transcripts induced by both Aire and MBD1-VP16 as shown in (a). Data are representative of three experiments (a,b). Fold induction is calculated relative vector only.

Supplementary Figure 4 Mbd1−/− mice have normal thymocyte populations.

(a) Representative FACS plots of CD4 and CD8 thymocytes from 4 month old Mbd1+/+ and Mbd1−/− mice. Numbers represent the percentage of cells in each gate. (b) Representative FACS plots of thymic Tregs (CD4+/CD25+/Foxp3+) from 4month old Mbd1+/+ and Mbd1−/− mice. Numbers represent the percentage of cells in each gate. (c) Graphs show the percentage of CD4, CD8, double positive (DP), and thymic Tregs from three mice of each genotype. NS = not statistically different. Each circle or square represents one mouse.

Supplementary Figure 5 Aire specifically targets repressed TSA-encoding loci by targeting the MBD1-ATF7ip-ESET protein complex.

(a) Model shows the MBD1-ATF7ip-ESET complex interacting with methylated CpGs via MBD1 and creating the repressive histone mark H3K9me3. Data presented here indicates that this complex preferentially targets TSA genes for repression. (b) Aire binding to domain 2 (D2) of ATF7ip and to the CXXC domains of MBD1 targets Aire to the repressive MBD1-ATF7ip-ESET complex. Aire's binding to this complex specifically targets Aire to repressed TSA loci and promotes Aire's exquisite specificity for distinct TSA gene loci located throughout the genome. Aire's targeting of the MBD1-ATF7ip-ESET protein complex likely works in conjunction with Aire's interaction with H3K4me0 which may also be enriched at repressed TSA loci.

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Waterfield, M., Khan, I., Cortez, J. et al. The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance. Nat Immunol 15, 258–265 (2014). https://doi.org/10.1038/ni.2820

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