PTA and Deaf1 isoform expression in the PLN of NOD mice. a) Expression of pancreatic PTAs in the PLN of NOD versus NOD.B10 mice, as assessed by microarray analysis. b) Deaf1 mRNA expression in the PLN of NOD mice, as detected by Probe 1 and Probe 2 using microarray analysis. Data in panels a and b represent the mean log ratio of (NOD/NOD.B10) ± s.d. (n = 10). c) Two isoforms of Deaf1 (DF1 and DF1-VAR1) were cloned from the PLN of 12 week old NOD mice. DF1 hybridizes to Probe 1 and 2, while DF1-VAR1 only hybridizes to Probe 2. DF1-VAR contains a deletion in the N-terminal alanine-rich region and a partial intronic insertion between exon 6 and 7 that introduces a premature stop codon. Abbreviations: Ala (alanine); SAND (Sp100,AIRE-1-NucP41/75,DEAF-1); NLS (nuclear localization signal); NES (nuclear export signal); Zf-MYND (zinc finger-Myeloid, Nervy, and DEAF-1).

Cellular localization, hetero-dimerization and transcriptional activity of DF1 and DF1-VAR1. a) Immunoblot of HEK 293 whole cell lysates transfected with 1 μg of DF1-Flag, DF1-VAR1-EGFP, 1 μg of both plasmids or empty control plasmid. Both DF1 (~70 kDa) and DF1-VAR1 (~50 kDa) were recognized by the Deaf1 antibody used. Cyclophilin B expression was assessed as a loading control. b) Confocal images showing the subcellular localization of EGFP-tagged DF1 and turbo-RFP-tagged DF1-VAR1 in DAPI-stained HEK 293 cells. Scale bar = 20 nm; images are representative of more than 100 similar cells. c) Immunoblot of nuclear lysates extracted from HEK 293 cells transfected with 0 or 0.5 μg of DF1-Flag and/or DF1-VAR1-EGFP. The nuclear marker histone H3 expression was assessed as a loading control. The densiometric ratio of DF1-VAR1 to histone H3 for each of these samples is shown below. Data is representative of 4 separate experiments. d) Co-immunoprecipitation assay. HEK 293 cells were transfected with the indicated Flag- or V5-tagged DF1 constructs. Lysates were subjected to immunoprecipitation and immunoblot with the indicated antibodies. e,f) Transcriptional activity of DF1 and DF1-VAR1 (or empty EGFP control vector) alone or in combination, as assessed using the 26 bp Deaf1-response element-luciferase reporter plasmid. Data in panel c–f are representative of similar results obtained in 3 separate experiments.

Deaf1 regulates gene expression in NIH 3T3 cells. NIH 3T3 cells were co-transfected with Deaf1 or nonsense siRNA and with DF1-Flag. (a) The expression of exogenous Deaf1 was assessed by immunoblot with anti-Flag. A non-specific band (N.S.) was used as a loading control. (b, c) Expression of the indicated endogenous mRNA transcripts was measured by QPCR at indicated times after transfection (b) or at 48 h after transfection (c). Cells transfected with nonsense siRNA and Deaf1 siRNA are indicated by white and grey bars, respectively. Gene expression was measured in triplicate by QPCR and normalized with 18S rRNA expression. Values represent the mean ± SEM of 3 separate experiments. *P ≤ 0.001; ^**P ≤ 0.05, Student’s unpaired t-test, two-tailed.

Identification and characterization of an alternatively spliced DEAF1 isoform in human PLN. a) Two DEAF1 isoforms were cloned from human PLN, a canonical DEAF1 transcript (Hu-DF1) and an alternatively spliced variant (Hu-DF1-VAR). b) Confocal images showing the subcellular localization of EGFP-tagged Hu-DF1 and turbo-RFP-tagged Hu-DF1-VAR in DAPI-stained HEK 293 cells. Scale bar = 30 nm; images are representative of more than 100 similar cells. c) The transcriptional activity of Hu-DF1 and Hu-DF1-VAR was assessed in HEK 293 cells using the 26 bp Deaf1-response element-luciferase reporter plasmid. Data are representative of 2 similar experiments. (d,e) HEK 293 cells were transfected with Hu-DF1-turboRFP and/or Hu-DF1-VAR-EGFP. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblot. The cytoplasmic marker GAPDH and nuclear marker histone H3 were used as loading controls. Data are representative of 3 similar experiments. f) Quantification of Hu-DF1-VAR gene expression in the PLN of healthy and T1D patients by QPCR. (n = 5; *P < 0.02 , Mann Whitney test, two-tailed).

Quantification of DF1 and DF1-VAR1 gene expression in NOD PLN. DF1 (a), DF1-VAR1 (b) and Gapdh (c) gene expression was measured in 4, 12, and 20 week NOD and NOD.B10 PLN samples. (d) DF1, DF1-VAR1 and Gapdh mRNA was measured in 12 week NOD and NOD.B10 spleen samples. Gene expression was normalized to Actb mRNA. Values represent the mean ± SEM. *P < 0.05. P-values were determined using the Student’s unpaired t-test, two-tailed. n ≥ 5 for all groups.

Analysis of Deaf1-KO mice. (a) Microarray analysis was used to identify genes that are upregulated and downregulated in the PLN of Deaf1-KO mice. Graph shows fold change in gene expression in PLN of BALB/c Deaf1-KO compared to BALB/c wild-type mice. (n = 2) (b,c). Quantitative PCR (QPCR) was used to measure gene expression differences in BALB/c Deaf1-KO (filled bars) versus BALB/c wild-type PLN (white bars; b) and thymus (c). Gene expression was measured in triplicate by QPCR and normalized with 18S rRNA expression. Values represent the mean ± SEM, n = 4 in each group. *P ≤ 0.001; ^**P ≤ 0.01, Student’s unpaired t-test, two-tailed. d) Serum of 30 wk old Deaf1-KO or wild-type BALB/c mice was used for immunoblotting of mouse eye lysates. Data are representative of 3 similar experiments. e) Confocal images of indirect immunofluorescence experiments performed in the mouse eye. Tissue sections (5 μm) were incubated with diluted serum (1:100) from Deaf1-KO or wild-type littermate controls or with no serum (negative control), followed by Alexa-488 conjugated anti-mouse IgG (1:2000). A high magnification image of the boxed region is shown in the top right panel. (Abbreviations: OS = outer segment; OP = outer plexiform; C = choroid; ON = outer nuclear layer; IN = inner nuclear layer). Data is representative of 12 sections per group.

Regulation of PTA expression in NOD PLN and LN CD45⁻ cells by Deaf1 and DF1-VAR1. a) Gene expression of PTAs in the PLN of NOD versus NOD.B10 mice at various ages (n ≥ 4 per group). Data are normalized with 18S rRNA expression and presented as a percent of age-matched NOD.B10 controls. b) Relative expression of DF1-VAR1 mRNA in various cells isolated from the 12-week NOD PLN (n = 3). Expression was normalized to Actb. c) DF1 mRNA expression in LN CD45⁻ cells transfected with Deaf1 or nonsense siRNA (n = 3) was measured by QPCR at the indicated times after transfection. d) LN CD45⁻ cells were transfected with Deaf1 or nonsense siRNA and expression of the indicated mRNA transcripts was measured by QPCR 48 h after transfection (n ≥ 4). For c and d, cells transfected with nonsense siRNA and Deaf1 siRNA are indicated by white and grey bars, respectively. e) LN CD45⁻ cells were infected with viral particles carrying DF1-VAR1 (grey bars) or an empty control plasmid (white bars), and mRNA expression was measured 72 h later (n = 3). Gene expression was measured in triplicate and normalized with 18S rRNA expression. Values represent the mean ± SEM. ***P ≤ 0.001; ^**P ≤ 0.01; ^*P ≤ 0.05, Student’s unpaired t-test, two-tailed. f) HEK 293 cells were transfected with DF1-Flag and/or DF1-VAR1-EGFP. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblot. The cytoplasmic marker Gapdh and nuclear marker histone H3 were used as loading controls. Data are representative of 4 similar experiments.