Mina haplotype predicts T_H2-bias phenotype. (a) Map of the promoter- proximal end of the Mina genomic locus. Indicated are the 21 SNPs identified across this interval, along with exons (red), coding region (yellow) and conserved non-coding sequences (CNS; cyan). (b) Position of each SNP (relative to the translational start site in exon2) and its allelic identity for 3 low (red) and 2 high (green) T_H2-bias strains.

Transient enforced Mina elevation impairs Il4 expression in CD4⁺ T cells. CD4⁺T cells isolated from three independent Mina transgenic lines (Lines #6, #38 and #21; closed symbols, 2 mice each) and their corresponding littermate controls (open symbols, 2 mice each) were stimulated with antibodies to the TCR and CD28. RNA harvested at the indicated times post-stimulation was analyzed for Mina, Il4, Il2 and Ifng by quantitative real-time RT-PCR. Data, expressed as relative expression (Actb normalized, arbitrary units), are from two independent experiments. Each trace represents an individual mouse.

Il4 expression in CD4⁺ T cells is constrained by Mina-dependent repression. (left) Protein immunoblots of Mina and Actin expression in total cellular extracts from 4–6 × 10⁶ CD4⁺ T cells activated for 24 h following 16 h mock, CoMO or MinaMO pre-treatment. (middle) Quantification of Mina expression from the protein blot. (right) Relative Il4 and Ifng expression (Hprt1-normalized, arbitrary units) in the same cells shown in the previous two panels. Shown is the mean of duplicate PCR reactions. Data are representative of 2 independent experiments.

Mina is transcriptionally regulated. (a) Mina and Actin immunoblots from cytosolic and nuclear extracts of BALB/c and C57BL/6 naive T_H cells activated by PMA-ionomycin over the indicated time course. The upper band in the nuclear Mina immunoblot is non-specific. Actin-normalized data are plotted to the right of each set of blots. Data are representative of two independent experiments. (b) Mina pre-mRNA expression (Hprt1-normalized, arbitrary units) time course from TCR-crosslinking activated naïve T_H cells isolated from pools of BALB/c (open squares, n = 5) and C57BL/6 (closed squares, n = 20) mice. Expression was measured by quantitative PCR using primers targeting sequences from Mina intron 1. Data are representative of 2 independent experiments and error bars indicate SEM for triplicate PCR measurements.

Mina is a Dice1.2 candidate gene. (a) Location of chromosome 16 congenic intervals in C16D2/8 and C16D2/8D. Shown for each strain are the chromosomal regions inherited from B10.D2 (grey), BALB/c (white) or undetermined (striped). Genotyped locations are indicated with cyan dots. Genotyping markers are indicated on the right axis, with Mina in red. Long and short vertical bars indicate the original and new Dice1.2 genetic intervals, respectively. (b) Relative Il4 expression (Hprt1-normalized, arbitrary units) in TCR-activated CD4⁺ T cells from BALB/c (BALB), B10.D2 (D2) and C16D2/8D mice (n = 2–5; each symbol represents an individual mouse and the mean is indicated by a horizontal line). * Statistical significance was determined using a t-test; P = 0.0317. Data are representative of 2 experiments with similar results.

Inverse correlation in Mina and Il4 expression in naïve T_H cells. (a) Mina (left) and Il4 (right) mRNA expression (Actb-normalized, arbitrary units) time course from TCR cross-linking-activated CD4⁺ T cells isolated from low (B10.D2, C57BL/6, and C3H/HeN; filled symbols) and high (BALB/c, DBA/1J, DBA/2J, and DBA/2N; open symbols) T_H2-biased strains. Data are the mean of three independent experiments and error bars indicate SEM. (b) Mina, Il4 and Ifng mRNA expression (Hprt1-normalized, arbitrary units) time course from (top) TCR cross-linking-activated BALB/c (open symbol) and C57BL/6 (closed symbol) naive T_H cells or from (bottom) T_H1- (closed symbol) and T_H2- (open symbol) primed C57BL/6 naive T_H cells. Data are representative of two independent experiments and error bars indicate SEM for triplicate PCR measurements. (c) Mina expression (Actb-normalized, arbitrary units) in CD4⁺ T cells stimulated with TCR cross-linking for 24 h in the presence of graded doses of IL-4 (B10.D2) or anti-IL-4 (BALB/c). Data are the mean of three independent experiments and error bars indicate SEM. (d) Mina expression (Actb-normalized, arbitrary units) in CD4⁺ T cells from B10.D2 mice stimulated for 24 h in the presence of anti-IL12. Data are the mean of three independent experiments and error bars indicate SEM.

Mina recruitment to the Il4 promoter requires NFAT. (a) Nucleotide sequence of oligonucleotide probes from the Il4 (B and B^NM) and Il2 (IL2) promoters used in EMSA and DNA pull down experiments in b and c. NFAT and Oct binding sites are labeled. Probe B^NM is identical to probe B except for a dinucleotide mutation in the NFAT-binding site, indicated in bold italicized type. (b) The Mina nucleoprotein complex with Probe B requires NFATc1 and NFATc2 binding. Left, nuclear extracts from 4 h PMA-ionomycin induced EL4 cells were incubated with end-labeled probes B and B_NM in the presence or absence of 100, 33, or 11 ng of ‘cold’ Probe B or B_NM. Right, nuclear extracts from 4 h PMA- ionomycin induced EL4 cells were incubated with end-labeled Probe B and rabbit serum, 3 μg anti-Mina, 2 μg anti-NFATc1 or 2 μg of anti-NFATc2. Thick and thin arrows indicate the locations of the Mina complex and the anti-NFAT ‘supershifted’ complex, respectively. Data are representative of 3 independent experiments. (c) NFATc2 and Mina protein blots of ‘pull-down’ products formed by combining nuclear extracts (input) from 4 h PMA-ionomycin-induced EL4 cells with biotin-labeled probes (IL2, B and B_NM). DNA-protein complexes were isolated using streptavidin-agarose beads. Data are representative of 2 independent experiments.

Mina can bind to and repress transcription from the Il4 promoter. (a) Identification of a Mina-responsive region in the Il4 promoter. The transcriptional response to Mina of the Il4 promoter (left; varying in length from 766 to 140 bp, indicated below the graph) and the Il2 promoter (right) is shown. Reporter activity was measured from transductants stimulated (open bars) or not (filled bars) with anti-TCR for 12 h. Data are the average of three independent experiments and error bars indicate SEM (* P < 0.05; t test). (b) Nucleotide sequence of the proximal Il4 promoter with regions (indicated as horizontal lines) used as oligonucleotide probes (A, B, C, and D) in electrophoretic mobility shift assays (EMSAs). (c) Mina interacts with the proximal Il4 promoter in EMSAs. Nuclear extracts from 24 h TCR-stimulated B10.D2 CD4⁺ T cells were incubated with end-labeled probes A-D in the absence (left) or presence (right, probes A and B only) of 2 μg anti-Mina. Arrows indicate the location of the Mina-containing nucleoprotein complex. Data are representative of 3 independent experiments. (d) Differential binding of Mina53 to the Il4 promoter. ChIP plot depicting fold-enrichment for chromatin-bound Mina53 at five sites (numbered relative to the transcriptional start site) across the Il4 promoter and at the Cd3e locus (as a negative control) in 24 h TCR-stimulated CD4⁺ T cells from strains BALB/c (open bars) and B10.D2 (filled bars). Data are the mean of five independent experiments and error bars indicate SEM (* P < 0.05, t test).