Figure 9.
Bcl2A1 is up-regulated by NFATc1/ΔS and repressed by Blimp-1.(A) Heatmap of 200 genes whose expression changed in CD4+ CD90.1+ WT versus NFATc1/ΔS+ T cells (signal to noise, SD adjusted) regained from aGvHD-induced mice on day 4. Shown is the relative difference in RPKM (Morpheus; Broad Institute). (B)Bcl2a1 RNA expression in Th1-skewed cells, measured by qRT-PCR. n = 6, statistical evaluation by Student’s t test; *, P < 0.05. (C) Frequency of 7-AAD+ Annexin+ Th1 cells; n = 4. (D) Detection of Bcl2a1 mRNA in relation to other members of the Bcl2 family and control genes L32 and Gapdh by RNase protection assay using the mApo2 template set (5 µg RNA/lane). EL-4 cells had been retrovirally infected with NFATc1/C-ER and its ΔSUMO and N-terminal SUMO fusion mutants and stimulated with 5-hydroxytamoxifen and T/I for 24 h. 1–6 below phosphoImager values correspond to the lanes of the autoradiography. Shown is one representative from three independent experiments. (E) EMSA with NFAT binding sites-containing probes from the Nfatc1 promoter P1 (tandem site) and Bcl2a1 promoters. Nuclear extracts were prepared from EL-4 cells, stimulated with T/I for 4 h. NFATc1 and NFATc2-specific antibodies supershift the respective proteins (c1ss and c2ss). Consensus nucleotides for NFAT-binding are indicated in red, for AP1 in bold only. (F)Bcl2a1 (n = 9) and Prdm1 (n = 6) RNA expression in CD4+ T cells, collected on day 4 of spleen and intestine of mice induced for aGvHD by WT versus NFATc1/ΔS CD3+ T cells, measured by qRT-PCR. Student’s t test *, P < 0.05. (G)Bcl2a1 promoter activity in response to Blimp-1 was evaluated after transfection with pEYZ/MCS or pEYZ/Blimp-1F along with Bcl2a1 promoter (length indicated)-driven luciferase reporter plasmids in HEK 293T cells. After 36 h, luciferase activity was measured. Data are represented as the mean ± SE. Statistical significance was calculated using Student’s t test; *, P < 0.05. (H) EMSA with oligonucleotides containing Blimp-1 binding from the Myc (PRF) and Bcl2a1 (−50) promoters, using nuclear extracts from HEK 293T cells transiently transfected with Flag-tagged Blimp-1. Blimp-1 was supershifted by either anti-Blimp (α-B) or its tag Flag (α-F). Consensus nucleotides for Blimp-1 binding are indicated in blue. Right: Summary of the hypothesized sequential events downstream of endogenous NFATc1/ΔS expression.

Bcl2A1 is up-regulated by NFATc1/ΔS and repressed by Blimp-1. (A) Heatmap of 200 genes whose expression changed in CD4+ CD90.1+ WT versus NFATc1/ΔS+ T cells (signal to noise, SD adjusted) regained from aGvHD-induced mice on day 4. Shown is the relative difference in RPKM (Morpheus; Broad Institute). (B)Bcl2a1 RNA expression in Th1-skewed cells, measured by qRT-PCR. n = 6, statistical evaluation by Student’s t test; *, P < 0.05. (C) Frequency of 7-AAD+ Annexin+ Th1 cells; n = 4. (D) Detection of Bcl2a1 mRNA in relation to other members of the Bcl2 family and control genes L32 and Gapdh by RNase protection assay using the mApo2 template set (5 µg RNA/lane). EL-4 cells had been retrovirally infected with NFATc1/C-ER and its ΔSUMO and N-terminal SUMO fusion mutants and stimulated with 5-hydroxytamoxifen and T/I for 24 h. 1–6 below phosphoImager values correspond to the lanes of the autoradiography. Shown is one representative from three independent experiments. (E) EMSA with NFAT binding sites-containing probes from the Nfatc1 promoter P1 (tandem site) and Bcl2a1 promoters. Nuclear extracts were prepared from EL-4 cells, stimulated with T/I for 4 h. NFATc1 and NFATc2-specific antibodies supershift the respective proteins (c1ss and c2ss). Consensus nucleotides for NFAT-binding are indicated in red, for AP1 in bold only. (F)Bcl2a1 (n = 9) and Prdm1 (n = 6) RNA expression in CD4+ T cells, collected on day 4 of spleen and intestine of mice induced for aGvHD by WT versus NFATc1/ΔS CD3+ T cells, measured by qRT-PCR. Student’s t test *, P < 0.05. (G)Bcl2a1 promoter activity in response to Blimp-1 was evaluated after transfection with pEYZ/MCS or pEYZ/Blimp-1F along with Bcl2a1 promoter (length indicated)-driven luciferase reporter plasmids in HEK 293T cells. After 36 h, luciferase activity was measured. Data are represented as the mean ± SE. Statistical significance was calculated using Student’s t test; *, P < 0.05. (H) EMSA with oligonucleotides containing Blimp-1 binding from the Myc (PRF) and Bcl2a1 (−50) promoters, using nuclear extracts from HEK 293T cells transiently transfected with Flag-tagged Blimp-1. Blimp-1 was supershifted by either anti-Blimp (α-B) or its tag Flag (α-F). Consensus nucleotides for Blimp-1 binding are indicated in blue. Right: Summary of the hypothesized sequential events downstream of endogenous NFATc1/ΔS expression.

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