Fig 2: The eGFP-tagged Blimp1 knock-in allele efficiently rescues plasma cell differentiation.(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) [25] or Blimp1-IRES-GFP (Blimp1-GFP/+) [26] reporter strains. Prdm1?Ex1A splenocytes lacking the ability to generate plasma cells were included as a negative control [27]. (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.

Fig 3: BLIMP1 and IRF1 are co-expressed in fetal human intestine.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.

Fig 4: The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition.(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 IFN?-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.

Fig 5: ChIP-seq analysis of genome-wide Blimp1 binding sites in E18.5 small intestine.(A) Distribution of all Blimp1 peak locations in comparison to whole genome at defined genomic regions. * P = 2.5 x 10-45 in comparison to whole genome for the same region. (B) The distance of each Blimp1 ChIP-seq peak from the nearest TSS binned at 5 kb intervals. (C) De novo motif analysis (MEME) identifies significant enrichment of a consensus Blimp1 binding motif underlying Blimp1 peaks (n = 2689, MEME E value = 4.7 x 10-712) that closely resembles previously reported mouse (STAMP E value = 3.4 x 10-7) and human BLIMP1 motifs (TOMTOM P value = 2.8 x 10-9). (D) Functional annotation analysis using GREAT reveals Blimp1 preferentially binds to promoter regions upstream of genes associated with metabolism, transcription, developmental and immune processes. (E) A subset of overlapping Blimp1 peaks shared with previously reported mouse and human ChIP-seq datasets. (F) Up-regulated genes in conditional mutants are significantly (P = 1.1 x 10-22) enriched for Blimp1 binding (+/- 5 kb of TSS) relative to all genes on the array. (G) UCSC track view of ChIP (red) and input (blue) wiggle plot overlays showing enrichment of GFP ChIP-seq density in Prdm1 BEG/BEG embryonic small intestine proximal to the promoters of Cyp4v3, Slc16a5, Myo18b, Prdm1, Myc and Il33.