Supporting InformationKoh et al. 10.1073/pnas.0808470105SI MethodsPlasmids, Protein Expression, and Purification. GST-tagged mouseAire-PHD1 cloned in pGEX-4T-1 (GE Healthcare) was ex-pressed and purified as previously described (1). His-taggedmouse Aire domain(s) for ITC were cloned in pET28a vector(Novagen); transformed into Escherichia coli BL21 (DE3);expressed in LB supplemented with 10 �M ZnCl2 at 18°Covernight; pelleted, and resuspended in 25 mM Hepes, pH 7.5,500 mM NaCl, and 5 mM imidazole; sonicated; and centrifuged,and supernatants were subjected to Ni-NTA-agarose. Columnwas washed in 25 mM Hepes, pH 7.5, 500 mM NaCl, and 20 mMimidazole, and Aire was eluted with 20–200 mM gradient ofimidazole in 50 mM Hepes, pH 7.4, and 500 mM NaCl. Eluateswere purified on a Sephacryl S-200 column (GE Healthcare LifeSciences) in 50 mM Tris, pH 7.5, and 150 mM NaCl andconcentrated in a Centricon (Millipore) with a 3-kDa membranecutoff. Full-length Flag-tagged Aire cloned between BglII andNotI sites (excising EGFP) in pEGFP-N1 (Clontech) was trans-fected into 293FT (American Type Culture Collection) cells byusing Mirus TransIT293 and expressed for 42–48 h at 37°C. Cellswere resuspended in 10 mM Tris, pH 7.4, 10 mM NaCl, 5 mMMgCl2, 0.5% Nonidet P-40, and Complete Protease Inhibitortablet (Roche) and lysed with 20 mM Tris, pH 7.4, 1 M NaCl,0.2% Nonidet P-40, 0.2 mM MgCl2, and Protease InhibitorTablet for 2 h at 4°C. Centrifuged lysates were subjected to M2anti-Flag resin (Sigma) for 3 h at 4°C. Resulting resin was loadedonto a column (Bio-Rad), washed in 20 mM Tris, pH 7.9, 0.5 MNaCl, 20% glycerol, and 0.1% Triton X-100, and eluted in washbuffer containing 0.8 mg/ml Flag peptide (Biosynthesis). Elutedfull-length Aire was subjected to overnight dialysis at 4°C in 20mM Hepes, pH 7.9, 100 mM KCl, 20% glycerol, 2 mM DTT, and10 �M ZnCl2. Mutations were introduced by using QuikChange(Stratagene) and verified by sequencing.

Peptide Microarray. Peptide microarray experiments were per-formed as described previously (2). Briefly, biotinylated histonepeptides were printed in six replicates onto a streptavidin-coatedslide (ArrayIt) using a VersArray Compact Microarrayer (Bio-Rad). After a short blocking incubation with biotin (Sigma), theslides were incubated with GST-fused Aire-PHD1 in peptide-binding buffer (50 mM Tris�HCl, pH 7.5, 150 mM NaCl, 0.1%Nonidet P-40, and 20% FBS) overnight at 4°C with gentleagitation. After being washed with the same buffer, slides wereprobed first with anti-GST antibody and then fluorescein-conjugated secondary antibody and visualized with a GenePix4000 scanner (Molecular Devices).

Biotinylated Peptide-Binding Assays. Biotinylated peptide pull-down assays were performed as described previously (1). Briefly,1 �g of biotinylated peptides was incubated with 1 �g ofGST-AirePHD1 or full-length Aire-Flag in peptide-bindingbuffer (50 mM Tris, pH 7.5, 300 mM NaCl, and 0.1% NonidetP-40) overnight at 4°C. After 1 h of incubation with streptavidinbeads (Amersham), complexes were washed three times with thebinding buffer, and the bound proteins were subjected to West-ern blotting.

Isothermal Titration Calorimetry. ITC was performed as previouslydescribed (3). Briefly, His-tagged Aire domain(s) were ex-changed into 50 mM Tris, pH 7.5, 150 mM NaCl, 50 �M ZnCl2,and 5 mM DTT after gel-filtration chromatography. LyophilizedH3 peptides (Keck Facility, New Haven, CT) were dissolved in

the same buffer. ITC measurements were carried out from 40 to80 �M protein concentration, with 1 mM peptide concentration,on a MicroCal VP-ITC instrument at 24°C. Binding constantswere calculated by fitting the data using the ITC data analysismodule of Origin 7.0 (OriginLab).

Mononucleosome Assembly EMSA. The 601 positioning DNA tem-plate was generated by PCR as described (4), except by using 5�Cy5-labeled primers (IDT) and purified with a Hi-Trap Q HP(GE Healthcare Life Sciences) column equilibrated in 10 mMTris, pH 8.0, and 0.1 mM EDTA and eluted in a gradient of0.4–1.2 M NaCl. The templates were assembled into mononu-cleosomes with bacterially expressed recombinant core histonesby step-gradient salt dialysis, followed by purification on a10–30% glycerol gradient (4). EMSA experiments were per-formed at 4°C in 20 �l of reaction buffer (10 mM Hepes, pH 7.8,50 mM KCl, 1 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, and 10%glycerol] with 10 nM mononucleosomes with or without poly(dI-dC) (Pierce). The total reaction mixtures were directly loadedonto a 4% native polyacrylamide gel (45:1) in 0.3� TBE and runat 15 mA in 4°C for 3 h.

Chromatin Immunoprecipitation. ChIP was performed as previ-ously described (5) except: 293FT or 4D6 human thymic epi-thelial cells were transfected with full-length Aire-Flag vectors byusing Mirus TransIT293 or Lipofectamine 2000 (Invitrogen),respectively. Cross-linked cells were resuspended in 0.3 Msucrose, 2 mM Mg(OAc)2, 3 mM CaCl2, 10 mM Hepes, pH 7.5,1% Triton X-100, and Protease Inhibitor tablet, and wereDounce-homogenized (Kontes, tight pestle) after 5 min ofincubation on ice. Nuclei were centrifuged, equilibrated in 5 mMMgCl2, 5 mM CaCl2, 70 mM KCl, and 20 mM Hepes, pH 7.5, andRoche Protease Inhibitor tablet, and incubated for 15 min at25°C with 450 units of micrococcal nuclease per milligram ofchromatin. After addition of EGTA to a final solution concen-tration of 20 mM, samples were washed five times with PBS-Tween (0.05%) and were sonicated at 33–35 W for 24 cycles of15-sec bursts, 45-sec pause with a Misonix Sonicator 3000 withmicrotip. Solubilized chromatin was incubated overnight withDynal Protein-G beads (Invitrogen) complexed with M2 anti-Flag antibody (Sigma), washed, and eluted in 50 mM Hepes, pH7.5, 100 mM NaCl, 1 mM EDTA, 1% SDS, and 1 mg/ml Flagpeptide (Biosynthesis) at 55°C. The eluate was precipitated bytrichloroacetic acid for 10 min on ice, washed two times inacetone, and resuspended in 250 mM Tris, pH 8.8, 2% SDS, and0.5 M 2-mercaptoethanol. Cross-linking was reversed for 30 minat 99°C before Western blotting using anti-Flag (Sigma), anti-H3(Abcam), or anti-H3K4me3 (Abcam) antibodies.

Quantitative RT-PCR. The 293FT or 4D6 human thymic epithelialcells were harvested 36–48 h after transfection, and total RNAwas isolated with TRIzol (Invitrogen) and was reverse tran-scribed using oligo(dT) and SuperScript II (Invitrogen), andquantitative PCR was performed with SYBR Green Master Mix(Applied Biosystems). PCR and signal detection used a 7700Sequence Detector (ABI Prism).

Primers used for the reactions were as follows: S100A9forward, 5�-CGT GTG CGA AAA GAT CTG CA; S100A9reverse, 5�-CCT TTT CAT TCT TAT TCT CCT TCT TGA G;KRT13 forward, 5�-AGA TCG CCA CCT ACC GCA; KRT13reverse: 5�-AAC CAA TCA TCT TGG CGT CC; KRT14forward, 5�-CAG TCC CTA CTT CAA GAC CAT TGA;

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KRT14 reverse, 5�ACT GTG GCT GTG AGA ATC TTG TTC;HPRT forward: 5�-TGA AGA GCT ATT GTA ATG ACC AGT

CAA C; and HPRT reverse, 5�-AGC AAG CTT GCG ACCTTG ACC A.

1. Matthews AG, et al. (2007) RAG2 PHD finger couples histone H3 lysine 4 trimethylationwith V(D)J recombination. Nature 450:1106–1110.

2. Shi X, et al. (2007) Proteome-wide analysis in Saccharomyces cerevisiae identifiesseveral PHD fingers as novel direct and selective binding modules of histone H3methylated at either lysine 4 or lysine 36. J Biol Chem 282:2450–2455.

3. Lan F, et al. (2007) Recognition of unmethylated histone H3 lysine 4 links BHC80 toLSD1-mediated gene repression. Nature 448:718–722.

4. Yang JG, Madrid TS, Sevastopoulos E, Narlikar GJ (2006) The chromatin-remodelingenzyme ACF is an ATP-dependent DNA length sensor that regulates nucleosomespacing. Nat Struct Mol Biol 13:1078–1083.

5. Lee TI, Johnstone SE, Young RA (2006) Chromatin immunoprecipitation and microar-ray-based analysis of protein location. Nat Protoc 1:729–748.

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H3(aa 1-21) H3R17me1 H3R17(s)me2 H3R17(a)me2 .

H3K9ac H3K14ac H3K18ac H3K27ac .

H4(1-21) H4K20me1 H4K20me2 H4K20me3 .

H4K5ac H4K8ac H4K12ac H4K16acc

H3(21-44) H3K27me1 H3K27me2 H3K27me3 .

H3(21-44) H3K36me1 H3K36me2 H3K36me3 .

H3(67-89) H3K79me1 H3K79me2 H3K79me3 .

H3(1-21) H3R2me1 H3R2(s)me2 H3R2(a)me2

H3(1-21) H3K4me1 H3K4me2 H3K4me3 .

H3(1-21) H3K9me1 H3K9me2 H3K9me3 .

H3(10-27) H3K18me1 H3K18me2 H3K18me3 .

H3(21-44) H3K23me1 H3K23me2 H3K23me3

H2A(1-21) H2AK5ac H2AK13ac . .

H2B(1-21) H2BK5me1 H2BK5me2 H2BK5me3 .

H2BK5ac H2BK12ac H2BK15ac H2BK20ac .

H2AX(121-142) H2AXS139ph H3T3ph H3S10ph

Fig. S1. A histone peptide microarray containing the indicated modifications was probed with GST-mAire294–481. Red spots indicate GST-specific FITC signals.H indicates histone; me, methylation; ac, acetylation; ph, phosphorylation; s, symmetric; and a, asymmetric.

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WT D299A C312W

P328LV303M

KD = 10.6 ± 0.5uM KD = 149 ± 12uM KD = Not detectable

KD = 14.7 ± 0.5uM KD = 38.9 ± 0.9uM

Fig. S2. Effect of mAire D299A, C312W, V303M, and P328L on H3K4me0 binding. ITC measurements of Aire/mutant’s binding to the N terminus of the H3 tail(residues 1–21).

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Fig. S3. Aire directly interacts with nucleosomes. (A) EMSA comparing full-length Aire binding to reconstituted, Cy5-conjugated mononucleosomes orCy5-conjugated DNA alone, with or without poly(dI-dC), run on a nondenaturing polyacrylamide gel. (B) EMSA comparing binding of full-length WT or deletionmutant Aire with Cy5-conjugated mononucleosomes.

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Fig. S4. Effect of transfected DNA concentration on PTA up-regulation. (A) Quantitative RT-PCR of total RNA isolated from 4D6 or 293T lines 36 h aftertransfection with WT or mutant Aire. Relative enrichment of endogenous PTA mRNA represents levels normalized to internal sample HPRT levels and externalempty vector (Mock) transfection. (B) Western blot analysis of 293T whole-cell lysates harvested 36 h after transfection.

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