Transcription factor Foxp3 and its protein partners form a complex regulatory network
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1 Supplementary figures Resource Paper Transcription factor Foxp3 and its protein partners form a complex regulatory network Dipayan Rudra 1, Paul deroos 1, Ashutosh Chaudhry 1, Rachel Niec 1, Aaron Arvey 1,2, Robert M. Samstein 1, Christina Leslie 2, Scott A. Shaffer 3,4, David R. Goodlett 3 & Alexander Y. Rudensky 1 1 Howard Hughes Medical Institute and Immunology Program and 2 Computational Biology Program, Sloan-Kettering Institute, New York, NY 10065; 3 Department of Medicinal Chemistry, University of Washington, Seattle, WA 9819; 4 Current address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA
2 Supplementary Fig.1 Comparable expression of Foxp3 in retrovirally transduced TCli cell line and primary Treg cells. a) Flow cytometric analysis of Foxp3 expression in lymph node (LN) Treg cells and AVI or AVI-Foxp3 transduced TCli cells. Numbers in bold italics over the gated populations represent mean fluorescent intensity (MFI). b) An average Foxp3 MFI from three independent experiments described in (a). c) Western blot analysis demonstrating Foxp3 expression in Treg cells compared to TCli cells expressing AVI-Foxp3. 2
3 Supplementary Fig.2 Co-immunoprecipitation and western blot analysis of Foxp3 association with transcriptional-related nuclear factors identified by mass spectrometry. Foxp3 and its co-factors were immunoprecipitated using streptavidin- and Foxp3 antibody-conjugated magnetic beads from nuclear lysates of TCli-AVI-Foxp3 or primary Treg cells and probed using corresponding antibodies. 3
4 Supplementary Fig.3 Foxp3 complexes with its transcription-related partners are independent of DNA and RNA. Co-immunoprecipitation followed by Western blot analysis of the TEV eluted Foxp3 associated proteins after indicated treatments with DNase and RNase. 4
5 Supplementary Fig.4 Map of known interactions among Foxp3-associated transcriptional controlrelated proteins generated by STRING 9.0 software package. Interactions within known protein complexes are depicted as colored circles and ellipses. 5
6 Supplementary Fig.5 A schematic depiction of regulatory relationships between Foxp3 and its partners. Parallel lines signify interactions between Foxp3 and its partners (Fp); the unidirectional arrows indicate binding to and regulation of corresponding genes. Broken arrows indicate gene expression affected in either positive or negative manner. Examples of Fp: Nfat, Runx/Cbfβ, GATA-3, Bcl-11b, Ets, Stat3. 6
7 Supplementary Fig.6 Gata3 binding to Foxp3 CNS2 and Gata3-dependent potentiation of Foxp3 expression. a) ChIP-qPCR analysis of Gata3 binding to Foxp3 regulatory elements in purified CD4 + CD25 + Treg cells using Gata3 antibody and normal IgG as control. b) Mean fluorescence intensity (MFI) of Foxp3 expression assessed by flow cytometric analysis of CD4 + CD8 T cells from spleen, lymph nodes (LN) and mesenteric lymph nodes (mln) of Gata3 fl/fl Foxp3 YFP-Cre (red bars) and Gata3 fl/+ Foxp3 YFP-Cre (blue bars) littermate control mice (n=5 mice per group). 7
8 Supplementary Fig.7 Heightened spontaneous Th2 cytokine production in Gata3 f/f Foxp3 YFP-Cre mice. a) Flow cytometric analysis of Gata3 expression in lymph node Treg and Foxp3 - CD4 + T cells from mice of indicated genotypes (at least 8 mice per group were analyzed). b) Body weights and c) lymph node (LN), spleen and mesenteric lymph nodes (mln) cellularity of 5-6 months old Gata3 fl/fl Foxp3 YFP- Cre (KO) mice and Gata3 fl/+ Foxp3 YFP-Cre (WT) control littermates. d) Expression of the indicated activation markers and e) cytokines by splenic, LN, and mln CD4 + Foxp3 T cells in 5-6 months old WT and KO mice. Data are representative of 2-3 independent experiments. f) Gata3 and T-bet expression in CD4 + Foxp3 T cells from WT and KO mice. g) IL-17 production by splenic, LN, and mln CD4 + Foxp3 + and CD4 + Foxp3 T cells in WT and KO mice. h) Percentages of Foxp3 + cells within CD4 + T cell populations in LN, spleen, and mln of WT and KO mice. 8
9 Supplementary Fig.8 Increased Th2 cytokine production by the large intestine lamina propria lymphocytes (LI-LPL) in Gata3 f/f Foxp3 YFP-Cre mice. a) Representative FACS plots demonstrating the expression of Gata3 in LI-LPL in mice of indicated genotypes. b) Representative FACS plots and frequencies of indicated cytokines produced by LI-LPL in mice of indicated genotypes. The data are representative of six mice analyzed in two independent experiments. 9
10 Page 1 of 3 Rudra et al. Supplementary table 5 Primers used to make plasmid constructs Primer name Sequence Primer 1 Primer 2 Primer 3 Primer 4 Primer 5 Primer 6 Primer 7 Primer 8 Primer 9 Primer name Gmpr-forward Gmpr-reverse Pde3b-forward Pde3b-reverse Helios-forward Helios-reverse Prdm1-forward Prdm1-reverse Runx1-forward Runx1-reverse Ikzf1-forward Ikzf1-reverse Stat3-forward Stat3-reverse Bcl11b-forward Bcl11b-reverse Nfatc2-forward Nfatc2-reverse Foxp1-forward Foxp1-reverse Chd4-forward Chd4-reverse Rcor1-forward Rcor1-reverse Arid1a-forward Arid1a-reverse Ptpn22-forward Ptpn22-reverse Pdk1-forward Pdk1-reverse Stx11-forward Stx11-reverse GCAGATCTATGGCCAGCAGCCTGAGGCAGATCCTGGACAGCCA GAAGATGGAGTGGAGGAGCAACGCCGGCGGCAGCGTTAACAT GCCCAACCCTAGGCCAGC GGCAGATCTATGGGCCTGAATGACATCTTTGAGGCCCAGAAGAT CGAGTGGCATGAGAACCTGTACTTCCAGGGAGCCATGCCCAACC CTAGGCCAGC GCCGAATTCTCAGTTAACCTCAAGGGCAGGGATTGGAGCACTTG GAGCTGGAAAAGAAGCTGGGAGCTATG CATAGCTCCCAGCTTCTTTTCCAGCTC GCACGCCATGGTGAAGGACAACACCGTGCCCCTG GGCGGATCCCTTCTCTGCGCTTCTCAGGGAG GGCGGATCCCATGCTCGAGGGCTCTGGAGAGG ACGCGTCGACTCACAGAGAAATGAAGTCCAGGGC Primers used for ChIP-qPCR Sequence CAGCTGGAACAGCCTTGGAA AAATGTCAAGGCCCCTGTGA TTTGGGCCGCATAGAGAAAA CAGTGAATCATCAGCAGCACAA TCAGTTTACTGTTCTTGGGTGAGAA AAAGTACAGAAATGGTTAATGTGAACAGA TTGTTTACTCTGACGCGCAAA GATCGGCACACCCTCTGCTA CTGCGGTTTTCTCGCTCTTG GGGATGCTGACAGCCTCAGA CTGGCTTCAAGTACGGATGTGA TCGCTTCAGAAGAGATGCATTTC ACCTCCCTGAGTTGGCTGTTC CCCGGGCTTTTTTGTAAGCT CAGAGCCTGTTGGCCAAGAC GGCCAGACGTACATCTTCAGTTC GGGAAGTTCCAGTTTCACATTTG GCTGTAGCTCGATGGCTCCTA CCCCGTGAAGCTGATGGT ATGGCAGATAGGGCAGGTAAAC TTGCCACTTCCTGTCTGTCCTA TCACCCACTGAGGAGAAGGAAA GGGCTAACTCCTCGCTGTTG ACGCGTATCGCCCACTGTAC TGGCAAATATGGACTGAATCGT CCCGGCTTGGTGATAAGAAC TGGAAGAACTGAGTGCTGTCTGA AACAAGAGGCTAACCAGAACACAA TGCACCACACCCCACAAAG TGGTTAAAGACACGCCCATGT GTCCCCGATGTCGAAAGAGTT GTCCGGACGCTGGATCAG
11 Page 2 of 3 Sytl2-forward Sytl2-reverse Klf3-forward Klf3-reverse Satb1-forward Satb1-reverse Ets2-forward Ets2-reverse Prodh-forward Prodh-reverse Gata3 Pro1-forward Gata3 Pro1-reverse Gata3 Pro2-forward Gata3 Pro2-reverse Gata3 Intron1-1- forward Gata3 Intron1-1-reverse Gata3 Intron1-2- forward Gata3 Intron1-2-reverse Gata3 Intron2-1- forward Gata3 Intron2-1-reverse Gata3 Intron2-2- forward Gata3 Intron2-2-reverse Primer name Prdm1 RT-forward Prdm1 RT-reverse Ptpn22 RT-forward Ptpn22 RT-reverse Helios RT-forward Helios RT-reverse Pdk1 RT-forward Pdk1 RT-reverse Stx11 RT-forward Stx11 RT-reverse Pde3b RT-forward Pde3b RT-reverse Sytl2 RT-forward Sytl2 RT-reverse Klf3 RT-forward Klf3 RT-reverse Satb1 RT-forward Satb1 RT-reverse Ets2 RT-forward Ets2 RT-reverse Prodh RT-forward Prodh RT-reverse Gapdh RT-forward Gapdh RT-reverse CCTGTTACCTGCCCCTCCTT TGGTCCACACTGCATTGGTT TCAGGGAGGGCTGGCTAAAT AACCCCAGCCTTGAAACAAA GTGGGTGCGACTCATCAAGA TCCCCTTATGTTCTCCCAAAGA AAGGGCTCAGCCTCTCTTCCT GGTGTGTGCATGTGTGTTTACACT AGCACAGCAGCAGAGACCATT CAGTGGCACGCCTGTTCTT GGAAAACGGTCCCCTCTATAATATC CCTGTCCCAGAGACCCAAAA GTCACAATACCAACCTGAGTAGCAA GAGACATAGAGAGCTACGCAATCTGA CGCCAAGGGTTAAGGTTCTTT CAGTTAAGTGGAAATGAGGTAGATCAGA GAGGCGGGCGGCATA TCACTGCGGTATTCTTCTTTTTTTT TGACTCTACCAACGAGGCTACCT CGGTTAGGCCGACAGAAAAA GGCTACATGCTCACTCCCTGTAC GGTCAGGGTGCCTAGGTATCTCT Primers used for cdna real-time PCR Sequence AAACTCCATGACCTCGCTATGAC CACCCTCACCTCTGCACTGA CCGTTAAACCAAGAGACAGCTGTAG AGACTCGGGTGTCCGTTCAG GCCCCCAAGGGCTCTCT GACTCGGCAGTGCTCACACTT TACGGGACAGATGCGGTTATC ATGCTTCCAGGCGGCTTTAT CATTGTCCCCATCTGGGAAT CCTAGAAGCCTGGCAAAATGA TTGGTTCTGGACAGATTGCTTACA ATGCAGGGATGTTTGAAGATAGG GATCGGCTTTGGAACAGGAA ACCATCTTCTCCCAGAGAGCAA GCCCCTGGCGAGAAACTT AGCCAAATGACCCAGCAGTTA CTGGATTCCACTTCCCAACCT TGATAGAGATGGCGTTGCTGTCT AGGACTTCCCCAGCAGCAA AGCTGTCCCCACCGTTCTCT TCAATGTGGATAAGCCGTTCAT GAGCCAGTTCCATATCCAAGGT GTGGAGATTGTTGCCATCAACG CAGTGGATGCAGGGATGATGTTCTG
12 Page 3 of 3 Ikzf1 RT-forward Ikzf1 RT-reverse Stat3 RT-forward Stat3 RT-reverse Bcl11b RT-forward Bcl11b RT-reverse Foxp1 RT-forward Foxp1 RT-reverse Runx1 RT-forward Runx1 RT-reverse Cnot3 RT-forward Cnot3 RT-reverse Gata3 RT-forward Gata3 RT-reverse β2m RT-forward β2m RT-reverse GGAAGAAACTAACCACAACGAGA AGCTCTTACGTTTGGCGACAT CACCTTGGATTGAGAGTCAAGAC AGGAATCGGCTATATTGCTGGT ATCTGAGAACGTGTACTCGCA ATGTGGCGAAAGGCGACTG GGTCTGAGACAAAAAGTAACGGA TGCCCCGCACTCTAGTAAGT ACCAGCCTCTCTGCAGAACTTT ATGGACGGCAGAGTAGGGAACT AGCCAACGCGAACCAGAAA CTGCCTTTTGTCCTTGATCTCA GTCATCCCTGAGCCACATCT AGGGCTCTGCCTCTCTAACC CCTGGTCTTTCTGGTGCTTG TTCAGTATGTTCGGCTTCCC
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Supplementary Figure 1 Efficiency of Mll4 deletion and its effect on T cell populations in the periphery. Expression of Mll4 floxed alleles (16-19) in naive CD4 + T cells isolated from lymph nodes and
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Supplementary Fig. 1. Delivery of mirnas via Red Fluorescent Protein.
prfp-vector RFP Exon1 Intron RFP Exon2 prfp-mir-124 mir-93/124 RFP Exon1 Intron RFP Exon2 Untransfected prfp-vector prfp-mir-93 prfp-mir-124 Supplementary Fig. 1. Delivery of mirnas via Red Fluorescent
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SUPPLEMENTARY INFORMATION
-. -. SUPPLEMENTARY INFORMATION DOI: 1.1/ncb86 a WAT-1 WAT- BAT-1 BAT- sk-muscle-1 sk-muscle- mir-133b mir-133a mir-6 mir-378 mir-1 mir-85 mir-378 mir-6a mir-18 mir-133a mir- mir- mir-341 mir-196a mir-17
pplementary Figur Supplementary Figure 1. a.
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Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
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E10.5 E18.5 P2 10w 83w NF1 HF1. Sham ISO. Bmi1. H3K9me3. Lung weight (g)
Myociyte cross-sectional Relative mrna levels Relative levels Relative mrna levels Supplementary Figures and Legends a 8 6 4 2 Ezh2 E1.5 E18.5 P2 1w 83w b Ezh2 p16 amhc b-actin P2 43w kd 37 86 16 wt mouse
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IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
Supplementary Figure 1
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Supplementary Materials for
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University of Miami Miller School of Medicine, Miami, FL 33136, USA, 3 State Key Laboratory
Supplementary File ASXL1 plays an important role in erythropoiesis Hui Shi 1,2,3, Shohei Yamamoto 1,2,4, Mengyao Sheng 3, Jie Bai 3, Peng Zhang 1,2, Runze Chen 1,2, Shi Chen 1,2, Lihong Shi 3, Omar Abdel-Wahab