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

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.

SUBMITTER: Waterfield M

PROVIDER: S-EPMC4172453 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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

Waterfield Michael M Khan Imran S IS Cortez Jessica T JT Fan Una U Metzger Todd T Greer Alexandra A Fasano Kayla K Martinez-Llordella Marc M Pollack Joshua L JL Erle David J DJ Su Maureen M Anderson Mark S MS

Nature immunology 20140126 3

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 ...[more]

PMID: 24464130

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Project description:X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi.We found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs.Together, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells.

Project description:BackgroundThe autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus.Method and resultsTherefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase.ConclusionAIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions.

Project description:BACKGROUND:The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. RESULTS:We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. CONCLUSIONS:Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

Dataset Information

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

Publications

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

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