The eGFP-tagged Blimp1-fusion protein is efficiently expressed in the developing placenta and embryonic small intestine, but fails to rescue germ cell defects.

<p>(A) Immunohistochemical staining of E9.5 placentae demonstrates the eGFP-tagged Blimp1-fusion protein is correctly expressed in the spongiotrophoblast layer. The position of the central maternal artery is outlined. Bar, 200 μm. SpT, spongiotrophoblast; dec, maternal decidua; lab, labyrinth trophoblast. (B) Western blot analysis demonstrates robust expression in E16.5 small intestine. RIPA lysates of STO fibroblasts are included as a negative control. Levels of Blimp1 expression were quantified relative to the first wild type littermate. (C) Immunohistochemical staining demonstrates nuclear expression of both endogenous and eGFP-tagged Blimp1 protein in E16.5 villus epithelium. Bar, 200 μm. (D) The eGFP-tagged Blimp1 knock-in allele fails to reconstitute germ cell formation. Fast red alkaline phosphatase staining at E7.5 demonstrates markedly reduced numbers of PGCs (black arrows). Hematoxylin- and eosin-stained sections confirm that adult homozygous BEG/BEG testes lack spermatocytes. Bar, 200 μm.</p

The eGFP-tagged Blimp1 knock-in allele efficiently rescues plasma cell differentiation.

<p>(A) Schematic representation of the targeting vector, wild type locus, and Southern blot screening strategy. (B) Intercross matings of heterozygous BEG animals generate Mendelian ratios of wild type, heterozygous and homozygous mutant progeny. (C) Western blot analysis demonstrates the eGFP-Blimp1 fusion protein is robustly induced in LPS-treated splenocytes. Levels of Blimp1 protein were quantified relative to the 129/SvEv wild type sample. Additional positive controls were wild type C57BL/6 and a BEG intercross littermate (+/+), heterozygous Blimp1-Venus BAC transgene (Blimp1-Venus) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref025" target="_blank">25</a>] or Blimp1-IRES-GFP (Blimp1-GFP/+) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref026" target="_blank">26</a>] reporter strains. Prdm1ΔEx1A splenocytes lacking the ability to generate plasma cells were included as a negative control [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref027" target="_blank">27</a>]. (D) Imagestream analysis of LPS-treated splenocytes reveals nuclear localization of the Blimp1-eGFP fusion protein in contrast to the cytoplasmic/plasma membrane localization of Venus expressed under the control of the Blimp1 BAC-transgene. (E) Homozygous mutant B cells that exclusively express the eGFP-tagged Blimp1-fusion protein efficiently undergo plasma cell (CD138+B220+) terminal differentiation.</p

BLIMP1 and IRF1 are co-expressed in fetal human intestine.

<p>Nuclear BLIMP1 staining detected in the pseudostratified epithelium of the developing gut tube at (A) 9 weeks of gestation persists in (B) the villus epithelium including the immature crypts of Lieberkuhn at 15 weeks of gestation. (C) Although not detected in the gut tube prior to villus formation nuclear IRF1 staining is present in the villus epithelium at (D) 15 weeks of gestation. (A, C) Bar, 800 μm, (B, D) Bar 200 μm.</p

The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition.

<p>(A) Western Blot analysis reveals down-regulated Blimp1 and slightly increased Irf1 co-incident with the onset MHC class I expression detectable at post weaning stages. As a positive control Irf1 and MHC class I were strongly induced in IFNγ-treated MEFs. (B) Immunohistochemical analysis demonstrates MHC class I expression initially appears in immature Peyer’s patches (PP). Bar, 200 μm. (C) MHC class I is robustly expressed in post-natal villus epithelium (P28), as well as mature Peyer’s patches and villus mesenchyme. Bar, 200 μm.</p

ChIP-seq analysis of genome-wide Irf1 binding sites in E18.5 small intestine.

<p>(A) Functional annotation analysis of genes bound by Irf1. (B) Overlapping Irf1 peaks between those identified in the present study and a mouse LPS-BMDC ChIP-seq dataset. (C) <i>De novo</i> motif analysis identifies significant enrichment of the consensus IRF1 binding motif underlying Irf1 peaks (n = 1996, MEME <i>E</i> value 1.9 x 10⁻⁸⁰²) that closely resembles the motif identified by Garber <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref042" target="_blank">42</a>] (STAMP <i>E</i> value = 0). (D) Comparison of our Irf1 and Blimp1 ChIP-seq datasets identifies a subset of overlapping peaks that selectively display the IRF1 motif. (E) Functional annotation analysis of genes bound by both Irf1 and Blimp1 demonstrates enrichment of immune response and antigen processing genes. (F) UCSC track view of ChIP (red) and input (blue) Wiggle plots. Both Irf1 and Blimp1 ChIP-seq peaks were present proximal to the promoters of <i>Psmb8</i>, <i>Psmb9</i>, <i>Tap1</i>, <i>Tap2</i>, <i>Tapbp</i> and <i>Erap1</i>. Positions of the TSS and direction of transcription are indicated by the arrows. (G) Overlapping Irf1 and Blimp1 peaks near a subset of Irf1 target genes display increased Irf1 occupancy in the absence of Blimp1 competition.</p

DM mutant NOD mice weakly express A^g7/CLIP complexes and entirely lack mature compact A^g7 dimers.

<p>(A) To evaluate occupancy by the Ii chain-derived CLIP peptide, DM mutant spleen cells expressing either the b or k haplotype, DM loss of function and wildtype NOD splenocytes were compared. Lysates were prepared from spleen cells pulsed for 40 mins with ³⁵S-methionine and chased for different times as indicated and extensively pre-cleared with Rabbit anti-mouse IgG (H+L) Abs before immunoprecipitation with beta chain-specific Rabbit Ab. Complexes were boiled for 10 mins and analyzed on 10% polyacrylamide gels. (B) Spleen cell lysates were boiled (B) or kept on ice (NB) and samples resolved on 10% gels under reducing conditions, transferred to nitrocellulose membranes, and Western blots probed with 10-2-16 or alpha chain-specific Rabbit Ab as indicated. (C) Western blot analysis of lysates prepared from LPS-stimulated BMDCs.</p

DM functional loss favours development of the CD4+ Foxp3+ T cell subset.

<p>Thymus (A), spleen (B), and pancreatic lymph node (C) cell suspensions were stained for CD4, CD8, and Foxp3 expression and analysed by flow cytometry.</p

DM functional loss results in decreased A^g7 expression within the thymic cortical epithelium and medullary regions.

<p>Acetone-fixed cryostat sections of thymic tissue were stained with the indicated mAbs.</p

DM mutants display functionally empty I- A^g7 molecules with markedly enhanced peptide-binding capabilities.

<p>DM mutant (shown in red) and control wildtype (shown in black) splenocytes from B10.BR or NOD mouse strains were cultured for 5 hrs at 37°C with biotin-conjugated peptides or medium alone as indicated, stained with FITC-labelled avidin, and analysed by FACS. OVA 323–339 preferentially binds to I- A^g7, HEL 46–61 selectively binds to A^k molecules, whereas NOD DM mutants gain reactivity towards the lambda repressor cI peptide P12–26 (IP).</p

DM loss in NOD mice results in decreased I-A^g7 surface expression.

<p>(A) Saponin-permeabilized splenocytes from wildtype (1) or DM mutant (2) NOD mice were incubated with anti-DM or anti-Ii chain mAb and FITC-conjugated anti-rat IgG (H+L) and analyzed by FACS. The DMα mutation eliminates DM and has no effect on Ii chain expression. (B) Splenocytes from (1) wildtype, (2) DM-deficient, or (3) Ii chain mutant NOD mice were stained with biotin-conjugated mAbs as indicated, followed by FITC-conjugated avidin. The shifts were detectable with conformationally dependent mAbs directed against distinct epitopes contributed by both chains and thus reflect decreased expression rather than a serological change. Representative data from one of three identical experiments with similar outcomes are shown.</p