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. 2012 Apr;32(8):1354-62.
doi: 10.1128/MCB.06359-11. Epub 2012 Feb 6.

Unmodified histone H3K4 and DNA-dependent protein kinase recruit autoimmune regulator to target genes

Kristina Žumer  1 , Audrey K Low, Huimin Jiang, Kalle Saksela, B Matija Peterlin
Affiliations

Unmodified histone H3K4 and DNA-dependent protein kinase recruit autoimmune regulator to target genes

Kristina Žumer et al. Mol Cell Biol. 2012 Apr.

Abstract

Autoimmune regulator (AIRE) directs the expression of otherwise tissue-restricted antigens (TRAs) in medullary thymic epithelial cells, allowing their presentation to developing T cells, which leads to central tolerance. We addressed the conundrum of how AIRE is recruited to these otherwise silent genes in cells. Our studies confirmed that interactions between AIRE and the unmodified histone H3K4 (H3K4me0) are important for targeting AIRE to the mouse insulin promoter in chromatin. By replacing its H3K4me0-binding module with one that binds to the methylated H3K4me3, we redirected the mutant AIRE.ING protein to an actively transcribed gene. Nevertheless, the mutant AIRE D297A protein, which could not bind to H3K4me0, still activated the human insulin promoter on an episomal plasmid target. This targeting was due to DNA-dependent protein kinase (DNA-PK). Thus, in cells that lacked the catalytic subunit of DNA-PK (DNA-PKcs), the assembly and activity of AIRE on DNA, whether in chromatin or on episomal plasmids, was abrogated. However, by the heterologous tethering of AIRE to DNA, we could restore its activity on a plasmid target in DNA-PKcs-negative cells. Importantly, mutations in the putative DNA-binding residues in its SAND domain had no effect on the transcriptional effects of AIRE. Thus, AIRE is recruited to TRA genes in chromatin via cooperative interactions with H3K4me0 and DNA-PK.

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Figures

Fig 1
Fig 1
AIRE must bind histone H3 for its recruitment to a TRA gene in chromatin. (a) AIRE PHD1 binds to histone H3. WT and mutant GST.PHD1 fusion proteins were incubated with purified histones. Immunoprecipitated proteins were analyzed by Western blotting with the indicated antibodies. Input GST fusion proteins (10%) are presented in the lower panel. Western blotting of histone H3 is presented in the upper panel. (b) AIRE must bind H3 for its recruitment to an endogenous TRA gene. ChIPs were performed with anti-AIRE antibodies from 1C6 mTECs expressing the empty plasmid vector (C), WT AIRE, or mutant AIRE D297A (D297A) proteins. Binding to the mIns2 promoter is presented as fold enrichment above the level of the IgG control (AIRE/IgG). Expression levels for AIRE proteins are presented below the bar graph. (c) AIRE must bind histone H3 to induce the expression of an endogenous TRA gene. mIns2 RNA levels from WT AIRE and mutant AIRE D297A protein-expressing 1C6 mTECs are presented as fold activation above the level of the empty plasmid vector (C). Levels of Ins2 RNA were normalized to those for GAPDH. (d) H3K4me3 is enriched at the GAPDH but not the mIns2 promoter. ChIPs were performed with anti-H3 (αH3) and anti-H3K4me3 (αH3K4me3) antibodies from 1C6 mTECs. Binding to promoter regions is presented as fold enrichment for specific antibodies above the level of the IgG control (H3*/IgG). (e) Only the mutant AIRE.ING protein with PHD1 replaced with the PHD from ING2 is recruited to the GAPDH promoter. ChIPs were performed with anti-AIRE antibodies (αAIRE) from 1C6 mTECs expressing the empty plasmid vector (C), WT AIRE, or mutant AIRE.ING proteins. Binding to the GAPDH promoter is presented as fold enrichment above the level of the IgG control (AIRE/IgG). Expression levels for AIRE proteins are presented below the bar graph. (f) The mutant AIRE.ING protein cannot induce the expression of an endogenous TRA gene. mIns2 RNA levels from WT AIRE and mutant AIRE.ING protein-expressing 1C6 mTECs are presented as fold activation above the level of the empty plasmid vector (C). Levels of mIns2 RNA were normalized to those of GAPDH.
Fig 2
Fig 2
Mutant AIRE protein, which does not bind histone H3, still activates a TRA promoter on a plasmid target. (a) The episomal, nonreplicating human insulin plasmid target does not recruit histone H3. ChIPs were performed with anti-H3 (αH3) and anti-H3K4me3 (αH3K4me3) antibodies on the expressed hInsLuc plasmid from 1C6 mTECs. The binding of histones to endogenous (mIns2) and plasmid (hInsLuc) promoter regions is presented as fold enrichment above the level of the IgG control (specific antibodies/IgG). (b) The mutant AIRE protein, which does not bind histone H3, still binds a TRA promoter on a plasmid target. ChIPs were performed with anti-AIRE antibodies (αAIRE) on the expressed hInsLuc plasmid from 1C6 mTECs that coexpressed the empty plasmid vector (C), WT AIRE, or mutant AIRE D297A (D297A) protein. Binding to the hInsLuc promoter is presented as fold enrichment above the level of the IgG control (AIRE/IgG). (c) AIRE does not need to bind to histone H3 to induce expression from a TRA promoter on a plasmid target. The relative luciferase activity of hINSLuc for WT AIRE and mutant AIRE D297A proteins is presented as fold activation above the level of the empty plasmid vector (C).
Fig 3
Fig 3
Putative DNA-binding residues in the SAND domain are not required for the recruitment of AIRE to a TRA on a plasmid target. (a) Alignment of SAND domain residues. Numbers above the sequences point to the KDWK DNA-binding motif (positions 243 to 247). (b) Residues required for the DNA binding of other SAND domains are not required for AIRE to activate a TRA promoter on a plasmid target. Relative luciferase activity from hInsLuc for WT AIRE (WT) and mutant AIRE (K243A, K245A, and KKR-A) proteins is presented as fold activation above the level of the empty plasmid vector (C). Expression levels for AIRE proteins are presented below the bar graph.
Fig 4
Fig 4
DNA-PKcs is required to recruit AIRE to a TRA gene in chromatin and on a plasmid target. (a) AIRE does not activate mIns in cells lacking DNA-PKcs. mIns2 RNA levels from WT AIRE protein-expressing WT or DNA-PKcs−/− MEFs are presented as fold activation above the level of the empty plasmid vector (C). Levels of Ins2 RNA were normalized to those for GAPDH. (b) AIRE does not activate a TRA promoter on a plasmid target in cells lacking DNA-PKcs. The relative luciferase activity of hInsLuc for WT AIRE protein-expressing WT or DNA-PKcs−/− MEFs is presented as fold activation above the level of the empty plasmid vector (C). (c) AIRE requires DNA-PKcs to interact with a TRA promoter on a plasmid target. ChIPs were performed with anti-AIRE antibodies on hInsLuc from WT or DNA-PKcs−/− MEFs coexpressing the WT AIRE protein or empty plasmid vector (C). Binding to hInsLuc is presented as fold enrichment above the level of IgG. The expression of AIRE and DNA-PKcs proteins was confirmed by Western blotting and is presented below the bar graph.
Fig 5
Fig 5
Kinase activity of DNA-PKcs is not required for effects of AIRE in cells. (a) Catalytic activity of DNA-PKcs is dispensable for the AIRE-induced expression of the KRT14 gene. KRT14 mRNA levels were analyzed in 293T cells, which transiently expressed AIRE or the empty plasmid vector in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). (b) Inhibition of DNA-PKcs prevents its autophosphorylation after the induction of double-strand DNA breaks. After the standard incubation period, etoposide (ETO) was added for 1 h to 293T cells in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). Levels of DNA-PKcs autophosphorylated on serine at position 2056 (S2056P) were quantified relative to those of total DNA-PKcs and are presented in numbers below the panels.
Fig 6
Fig 6
When AIRE is tethered heterologously to DNA, DNA-PKcs is not required for its activation of transcription. (a) Schematic representation of plasmid effectors and targets. Plasmid effectors are given on the left. From the N terminus, domains of AIRE are the homogenously staining region (HSR); Sp100, Aire-1, NucP41/75 and DEAF-1 (SAND) domain; and plant homeodomains 1 and 2 (PHD). The hybrid Gal.AIRE protein additionally contains the heterologous Gal4 DNA-binding domain (Gal). The plasmid target is presented on the right. G5Luc contains five GAL binding sites (UAS) and the TATA box (T) upstream of the luciferase gene (Luc) and polyadenylation signal (pA). (b) Via heterologous DNA tethering, the Gal.AIRE chimera activates a plasmid reporter. The bar graph presents relative luciferase activities of G5Luc in WT or DNA-PKcs−/− MEFs expressing WT AIRE or hybrid GAL.AIRE proteins as fold activation above the level of the empty plasmid vector (C). The expression of AIRE and DNA-PKcs proteins was confirmed by Western blotting and is presented below the bar graph.
Fig 7
Fig 7
AIRE interacts with DNA-PKcs, histone H3, and γH2AX in cells. WT AIRE protein interacts with DNA-PKcs, histone H3, and γH2AX. AIRE was immunoprecipitated with anti-AIRE antibodies from cells expressing the empty plasmid vector (C) or the WT AIRE protein (top 4 panels). DNA-PKcs interacts with the WT AIRE protein in cells. DNA-PKcs was immunoprecipitated with anti-DNA-PKcs antibodies. Western blotting was performed with specific antibodies (bottom 2 panels). Analyzed proteins are indicated by arrows. The Western blottings of inputs (2% cell lysate) are presented in panels on the right, and immunoprecipitations (IP) are presented in the panels on the left.
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