Identification Of The Foxp3-interacting Proteins In Mice Treg Cells

Background and aimsRegulatory T cells(Treg) is a new CD4+ T cell subtype. The signature transcription factor(TF) Foxp3 has been verified to substantially contribute to the differentiation and function of Treg cells. Treg cells involve in many kinds of diseases through their effector cytokines, of which Foxp3 is the key transcription factor for the expressoin. However, the detailed mechanisms for how Foxp3 regulates the expression of these cytokines have not been completely clarified yet. In addition, it has been well known that TFs usually function in cooperation with other trans-factors by forming a protein complex, and it is probable that Foxp3 should also function is such manner. However, whether and how Foxp3 interact with its in vivo partners remains unclear yet. Therefore, we established a Tags knock-in mouse model, wherein the fused tags sequence was inserted just before the stop codon of Foxp3 gene. By using the affinity purification technique, we will isolate the Foxp3 protein complex from the purified Treg cells of the KI mice, dissecting the components of the complex, and finally verify the mutual interaction between Foxp3 and its putative partners by co-IP method.Methods1. Preparation of transgenic miceWe got the plasmid pCe MM-CTAP(SG) from the Frankfurt university EUROSCARF institutions, and sent it to ingenious Targeting laboratory, Inc.,(http://client.genetargeting. com) to prepare the tags-IRES-GFP knock-in(KI) mice. The tandem tags contain Myc, SBP and Protein G binding domain, and there is a TEV restriction site between SBP and Protein G binding domain. The tags-IRES-GFP fusion sequence was inserted just before the stop codon(TAG) of Foxp3 gene. Therefore, Foxp3 interacting proteins could be isolated by affinity purification strategy from Treg cells in KI mice by using these tags that were fused with the C-terminus Foxp3 protein.2. Identification of transgenic mice2.1 Identification of correct intertion of GFP into the genomeThe genome DNA was isolated from the spleen of KI mice. Using the genome DNA as the template, PCR was conducted to verify whether GFP gene has been inserted into the mouse genome correctly, with primers: 5’-TGAAGG ATGCCCAGAAGGTA-3’(sense) and 5’-CTGCTTGTCGGCCATGATAT-3’(anti-sense), which were designed by using Premier 5.0 software and synthesized by Invitrogen company in Shanghai. The PCR primer pair located within GFP gene with the expected product size 635 bp.2.2 Identification of the Neo gene deletion from the genome of KI miceAfter crossing with Cre recombinase-expressing mice, the offsprings of KI mice were confirmed for the deletion of Neo gene from the mice genome by using PCR with primers: 5’-GGCGATACCGTAAAGCAC-3’(sense) and 5’-CTATGACTGGGCACAACAGA-3’(anti- sense). The primer pair located within Neo gene and the PCR producted was expected as 679 bp.2.3 Identification of Foxp3 homozygous mice by PCRThe genome DNA was exacted from KI mice after Neo gene deletion and confirmed for Foxp3 homozygote by PCR with primers: 5’-GTGCTTTGTGCGAGTGGAGA- 3’(sense) and 5’-GCTGTGTCTGACTTGTATTTTGGGC-3’(anti-sense). The primer pair was located flanking GFP gene with the expected 2,697 bp product. The wild type(WT) mice and the heterozygous mice would present a 724 bp band on agarose gel electrophoresis, however, the homozygous mice would neither present this band nor present the 2,697 bp band(because it is too long to be amplified by routine PCR).2.4 mRNA expression of GFP and Foxp3 gene in KI miceTotal RNA was extracted from the spleen of KI mice or WT mice, and the mRNA expression of GFP and Foxp3 gene was determined by quantitiative reverse transcriptionpolymerase chain reaction(RT-PCR).2.5 Expression of tags fused with Foxp3 protein in KI miceTotal protein was extracted from the spleen of KI mice or WT mice, and the expression of the individual tag was verified by SDS-PAGE and Western-blot using antibodies against Foxp3(Abcam, ab54501), SBP(Santa, Sc-101595), CD3 and Myc(Abbkin, A02060).3. Isolation of Treg cellsLymphocytes were harvested from the spleen and the mesenteric lymph nodes of KI mice and WT mice using Percoll reagent, respectively. After stained with APC-anti-CD4 and PE-anti-CD25 antibodies, the CD4+CD25+GFP+ Treg and CD4+CD25+ T cells were sorted from the above KI and WT lymphocytes suspension by using BD FACSAriaTM II Special Order System(USA). The sorted Treg cells were stored at-80 oC.4. Isolation of Foxp3 protein complex by affinity purification techniqueThe lysate supernatant of CD4+CD25+GFP+ Treg or CD4+CD25+ T cells was incuabed with the pre-washed IgG magnetic beads or streptavidin beads over night with rotation at 4oC. Next day, the non-specifically absorbed proteins were washed with PBS, and the specifically bound proteins on the magnetic beads were subjected to SDS-PAGE and Western blot assays, or subjected to analysis with mass spectrometry assay.5. Mass spectrometry assayThe specifically bound proteins on the magentic beads were analyzed with LTQ orbitrap velos pro(Thermo Fisher scientific). The data were analyzed with Proteome Discoverer 1.4 software.6. Co-immunoprecipitation assayThe sorted Treg cells were lysed in 1×Lysis buffer(50mM Tris-HCl, pH 7.5, 1% NP-40, 1.5mM MgCl2, 25% Glycerol, 125 mM NaCl, 1mM sodium vanadate(adding 1mM DTT, 1mM EDTA, Complete Protease Inhibitor Cocktail tablet just before the experiment)). After centrifugation, the supernatant was incubated with protein G beads that were coated with the antibody against the target protein overnight at 4oC. The specifically bound proteins were then detected by Western blot assays with antibodies against the candidate Foxp3 interacting proteins, respectively.Results1. Preparation of Tags-IRES-GFP knock-in miceThe genome DNA from the Tags-IRES-GFP KI mice tails was amplified by PCR to determine whether tags-GFP fusion gene was inserted into the genome correctly. In addition, the genome DNA was extracted from the F1 generation of Cre+ mice crossed with KI mice and amplified by PCR to determine whether the Neo gene was deleted by Cre recombinase. Results showed that the Tags-IRES-GFP sequence was correctly inserted into the mice genome, and Neo gene was successfully deleted from KI mice after crossing with Cre+ mice. Results also demonstrated that Foxp3 mRNA level in KI mice was similar with that in WT mice deterimed by quantitative RT-PCR analysis, indicating the insertion of Tags-IRES-GFP sequence did not affect the endogenous Foxp3 gene expression.2. Isolation of Treg by FACSThe lymphocytes were isolated from the spleen and mesenteric lymph nodes of homozygous KI mice or WT mice with the Percoll reagent. The CD4+CD25+GFP+ Treg and CD4+CD25+ T cells were further sorted by FACS, with purity 99.3% and 96.8%, respectively.3. Identification of Foxp3 and tags proteins in Treg of KI miceWe further determined whether the component of Tags-IRES-GFP fusion insert was expressed correctly in the Treg cells of KI mice by Western blot assays. Results showed that the anti-Foxp3, anti-Myc, anti-SBP and anti-Protein G binding peptide could detect a 68 kDa(47kDa Foxp3 + 21 kDa tags) blot in the Western blot assays, respectively, indicating the correct protein expression of Foxp3 and its fusion components. In contrast, though we observed the 47 kDa Foxp3 band, we did not observe any tags bands in WT Treg in the Western blot assay. Furthermore, we found that the Myc tag protein could be detected in the pulled-down product from the lysate supernatant of KI Treg by IgG and anti-SBP antibody. Collectively, these results demonstrated the correct expression of the foreign inserts in KI Treg.4. Mass spectrometry analysis of candidate Foxp3 interaction proteinsWe further isolated the Foxp3 protein complex from KI Treg by using the affinity purification with streptavidin beads, IgG beads or anti-Myc magnetic beads, to analyze the interacting proteins of Foxp3 protein, respectively. Mass spectrometry analysis showed that there were more than 100 kinds of candidate Foxp3 interacting proteins, including the reported GATA3, Foxp1, Hdac2, Nfatc2 and Cbfβ. These results indicate the mice Foxp3 protein does interact with a large number of partner protein in vivo.5. Validation of Foxp3 interaction proteins by immune coprecipitation assayTo verify whether Foxp3 protein interact with the above candidate interacting proteins identified by the mass spectrometry analysis, the lysate supernatant of CD4+CD25+GFP+ Treg from KI mice and CD4+CD25+T cells from WT mice were incubated with various anti-tags antibodies or antibodies against the candidate interacting proteins by immune magnetic beads strategy. The proteins bound to the beads were then subjected to Western blot assay using corresponding antibodies as the detecting antibodies, respectively. Results showed that the pulled-down produce with anti-HDAC2, anti-Cbfβ and anti-Myc antibodies could be detected the Cbfβ(21kDa), NFATC2(120kDa), GATA3(50kDa), Foxp1(90kDa) and My-labelled Foxp3(68kDa) proteins, respectively, in KI Treg and WT Treg. These results indicated that at least partial screened candidate interacting proteins do interact with Foxp3 protein mutually.Conclusions1. We successfully established the Tags-IRES-GFP KI mice, which was evidenced by the correct insertion of the foreign sequence, successful deletion of Neo gene from the genome, the establishment of the homozygous KI mice, and the correct mRNA expression of the endogenous Foxp3 gene, the inserted GFP gene and the various tags.2. We isolated enough CD4+CD25+GFP+ Treg with high purity from KI mice. The purity of the isolated CD4+CD25+GFP+ Treg cells reached 96.8%. Furthermore, the tags were verified to stably express in KI Treg as confirmed by Western blot assay.3. We acquired Foxp3 protein complex using affinity magnetic beads purification method, which included over 100 candidate Foxp3 interacting proteins as determined by mass spectrometry.4. We confirmed partial potential interacting proteins of Foxp3 by co-precipitation assay. We found that Nfatc2, GATA3, Foxp1, Hdac2 and Cbfβ might interact with Foxp3 mutually.Collectively, in this study, we had successfully prepared Tags-IRES-GFP KI mouse model, which is evidenced by the correct expression of the inserted tags and Foxp3 itself by PCR and Western blot assays. Following isolation of Foxp3 protein complex, the mass spectrometry showed that there were over 100 candidate interacting proteins with Foxp3 in Treg cells, among of which Nfatc2, Foxp1, Hdac2, GATA3 and Cbfβ proteins were verified to interact with Foxp3 proteins directly as confirmed by co-precipitation. The results from this study suggest there were several interacting partners of Foxp3 proteins in Treg cells, and support further study on the effects and the underlying mechanisms of the Foxp3 interacting proteins on biology and function of Treg cells in the future.