1. Essential Roles of SATB1 in Specifying T Lymphocyte Subsets
Kiyokazu Kakugawa, Satoshi Kojo, Hirokazu Tanaka, Wooseok Seo, Takaho A. Endo, Yohko Kitagawa, Sawako Muroi, Mari Tenno, Nighat Yasmin, Yoshinori Kohwi, Shimon Sakaguchi, Terumi Kowhi-Shigematsu, Ichiro Taniuchi 
Cell Reports 
Volume 19, Issue 6, Pages (May 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 1176-1188DOI: (10.1016/j.celrep.2017.04.038)
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3. Figure 1
Identification of SATB1 as a Binding Factor to Regulatory Regions in the Thpok Gene
(A) Gel shift assay showing protein complexes that specifically recognize wild-type (w), but not mutant (m), Thpok silencer sequence. Arrowhead and asterisk designate protein complexes and a band super-shifted by anti-Runx1 antibody, respectively. Comp, competitor; NE, nuclear extract.
(B) Western blot showing enrichment of SATB1 by pull-down with wild-type (WT) or mutant (mut.) Thpok silencer sequence in the absence or presence of pre-depletion (PD) or poly-Lysine (PL) treatment.
(C) Immunoprecipitation assay with anti-Bcl11b antibody showing the association of SATB1 with Bcl11b and Runx1 in total thymocytes. Results shown in (A)–(C) are one representative of two experiments.
(D) Summary of three independent SATB1 ChIP-seq tracks at the Thpok locus in total thymocytes (bottom, gene structure and transcriptional orientation). Arrowhead indicates the position of the Sth silencer.
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4. Figure 2
Re-directed Differentiation of MHC-II-Specific Thymocytes in the Absence of SATB1
(A) Flow cytometry analyzing the expression of CD4, CD8, CD24, and TCRβ on thymocytes and lymph node (LN) T cells from Satb1+/+:Cd4-Cre and Satb1F/F:Cd4-Cre mice. Numbers in quadrants indicate percentage cells in each.
(B) Dot plots showing CD4 and CD8 expression on lymph node T cells differentiated in an MHC class I-deficient (MHC-Io) background. Numbers in quadrants indicate percentage cells in each.
(C) Flow cytometry analyzing the expression of CD4, CD8, Thpok-gfp, and Runx3-tdTomato in LN cells expressing OT-II TCR defined as TCRVα2+ cells. Loss of SATB1 resulted in low Thpok expression and re-directed differentiation of OT-II cells, as manifested by the emergence of CD8+ subset (highlighted with red gates). Numbers in quadrants indicate percentage cells in each.
(D) Relative expression level of P1- and P2-Thpok mRNA in indicated spleen T cell subset of control and Satb1F/F:Cd4-Cre (Satb1 cKO) mice. One representative of two independent measurements is shown.
(E) Histograms showing the effect of SATB1 deficiency on Thpok-gfp expression from reporter alleles harboring specific mutations in the indicated LN T cell subsets of Satb1+/+ (filled square) and Satb1−/− (open square) mice. Schematic structures of mutant reporter alleles are shown at the left with regulatory regions highlighted. One representative of at least three mice is shown.
(F) Summary of three ChIP-qPCRs showing kinetic changes of SATB1 bindings to the Sth (open circle) and PE (filled circle) regions during development into CD4 lineage. SATB1-binding signals to each region relative to those in DP thymocytes are shown (means ± SD).
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5. Figure 3 Involvement of SATB1 in Cd4 Gene Regulation
(A) Dot plots and histograms showing the effects of transgenic ThPOK expression on T cell differentiation, CD40L induction, and IL-4 production. Dotted lines indicate CD40L expression from activated CD8+ T cells as a negative control.
(B) Percentage of methylated CpG islands at intronic regions in the CD4+CD8− T cells analyzed by bislufite-converted PCR. SATB1 is essential for DNA demethylation at the Cd4 locus. ∗∗p < 0.01 and ∗∗∗p <
(C) Summary of three independent SATB1 ChIP-seq tracks at the Cd4 locus in total thymocytes (bottom, gene structure and transcriptional orientation) along with a Runx ChIP-seq (GEO: GSE90794) track for reference. Significant binding peaks are marked with triangles. Arrowheads indicate positions of cis-regulatory regions in the Cd4 locus.
(D) Dot plots showing the effects of conditional Satb1 inactivation and germline SATB1 defciency on CD4 expression in pre-selection (CD24hiTCRlo) and mature (CD24loTCRhi) thymocytes in the E4p enhancer-deficient (Cd4ΔE4p/ΔE4p) background. Schematic structures of Cd4, Cd4ΔE4p, and Cd4ΔE4p: ΔE4m alleles are indicated at the top. CD4 expression from the Cd4ΔE4p: ΔE4m allele lacking both E4p and E4m enhancers is shown as reference. Decreased CD4 expression in CD4+CD8− subsets is highlighted with red gates. NA, not analyzed.
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6. Figure 4
Distinct Regulation of Thpok Enhancer Activity by SATB1 in the Cd4 Gene
(A) SATB1 ChIP-seq tracks at the Cd4 locus during differentiation into CD4 lineage. Arrowheads indicate positions of cis-regulatory regions in the Cd4 locus.
(B) Summary of three ChIP-qPCRs analyzing SATB1 bindings to two Cd4 enhancers, E4p and E4m. Relative SATB1 bindings to its to Sth in the Thpok gene (left) and relative SATB1 binding to E4m to E4p (right) in the indicated cell subsets are shown. ∗p < 0.05 and ∗∗p < 0.01.
(C) Summary of three ChIP-qPCRs showing SATB1 binding to E4m in the Cd4ΔE4p allele in total thymocytes.
(D) Schematic structure of the Cd4Eth allele is shown at the top with regulatory regions highlighted. Representative dot plots show the CD4 and CD8 expression in indicated thymocyte subsets of mice with the indicated genotypes.
(E) Summary of three ChIP-qPCRs showing SATB1 binding to the Eth sequences in the Cd4 locus.
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7. Figure 5 SATB1 Is Dispensable for Maintenance of Thpok Expression
(A) Relative Thpok mRNA amount in CD4+ cells 4 days after transduction with empty (GFP) and Cre (CRE)-encoding retroviral vector into CD4+ T cells to remove Satb1 (A) and proximal enhancer (PE).
(B) Summary of three independent experiments (means ± SD).
(C) Effect of Satb1 inactivation during maturation toward the CD4SP thymocyte by Thpok-Cre on T cell development (left dot plot) and Thpok expression (right graph). Numbers indicate percentage cells in the shown gate. One representative dot plot of at least five mice and summary of five measurements of Thpok amounts are shown (means ± SD).
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8. Figure 6
Impaired Differentiation of OT-I Cells and Attenuated Runx3/Cd8 Expression upon the Loss of SATB1
(A) Flow cytometry analyzing the expression of CD4, CD8, Thpok-gfp, and Runx3-tdTomato in LN cells expressing OT-I TCR defined as TCRVα2+ cells.
(B) Dot plots showing unstable CD8 expression in OT-I cells lacking SATB1 3 days after TCR stimulation.
(C) Dot plots showing expression of CD8 and Runx3-tdTomato 3 days after TCR stimulation. Histogram shows the CD8 expression after retroviral Runx3 transduction into control (shaded) and SATB1-defcient (open) OT-I cells.
(D) Increase of the CD4+CD8− SP subset expressing TCRβ at low levels in the thymus of mice harboring Thpok-Tg over SATB1-deficiency, showing a variegated CD8 expression.
(E) Percentage of methylated CpG islands at specified regions in the Cd8a locus of indicated cell subsets. ∗∗p < 0.01 and ∗∗∗p <
(F) Summary of three independent SATB1 ChIP-seq tracks at the Cd8 and Runx3 loci in total thymocytes along with a Runx ChIP-seq (GEO: GSE90794) track for reference. Significant binding peaks are marked with triangles. Arrowheads indicated positions of cis-regulatory regions in these loci.
(G) Histograms showing the effects of Satb1 inactivation on Runx3-tdTomato expression in the −21E enhancer-deficient background. Runx3-tdTomato expression from the Runx3tdTom:Δ21/39E allele lacking both −39E and −21E enhancers is shown as a reference.
(H) Representative dot plots showing the effect of Satb1 inactivation on CD8 expression in the E8I enhancer-deficient (Cd8ΔE8I/ΔE8I) background. Numbers in quadrants shown in this figure indicate percentage cells in each.
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9. Figure 7
Defective Development of iNKT and Treg Subsets in the Thymus upon the Loss of SATB1
(A) Dot plots showing CD25 and Foxp3 expression in CD4+CD8− SP thymocyte population from 9-day-old SATB1-sufficent and -deficient mice. Numbers in quadrants indicate percentage cells in each. Right graph shows a statistical summary. ∗∗p < 0.01.
(B) Summary of three independent SATB1 ChIP-seq tracks at the Foxp3 locus in total thymocytes (bottom, gene structure and transcriptional orientation). Significant binding peaks are marked with triangles. Arrowhead indicates the position of CNS3 enhancer.
(C) Left dot plots showing the iNKT cell subset in total thymocytes from 4-week-old Satb1+/+:Cd4-Cre and Satb1F/F:Cd4-Cre mice. Numbers indicate percentage cells in the shown gate. Right dot plots show CD4 and CD8 expression in iNKT cell subset. Numbers in quadrants indicate percentage cells in each. Graph at the right shows a statistical summary of percentages of iNKT cells in total thymocytes. ∗p < 0.05.
(D) Dot plots showing biphasic expression of surface αβTCR complexes on total thymocytes from Satb1F/F:Cd4-Cre mice.
(E) Relative Cα mRNA amounts to Gapdh in the indicated subsets from mice with the indicated genotype showing decreased Tcra mRNA in surface TCRβlo cells.
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