Fig 2: 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.

Fig 3: ChIP-seq analysis of genome-wide Irf1 binding sites in E18.5 small intestine.(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) De novo motif analysis identifies significant enrichment of the consensus IRF1 binding motif underlying Irf1 peaks (n = 1996, MEME E value 1.9 x 10−802) that closely resembles the motif identified by Garber et al. [42] (STAMP E 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 Psmb8, Psmb9, Tap1, Tap2, Tapbp and Erap1. 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.

Fig 4: The eGFP-tagged Blimp1-fusion protein is efficiently expressed in the developing placenta and embryonic small intestine, but fails to rescue germ cell defects.(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.

Fig 5: PGCs down-regulate p-Smad159 to become refractory to the high-Bmp signalling environment. a p-Smad159 staining of BV+ cell populations. Arrowhead confirms e6.5 Smad2ΔVE embryos lack p-Smad159 in the VE, while arrows indicates weak activity in BV-expressing epiblast cells. b p-Smad159 staining in control and Smad2ΔVE BV+ embryos at e7.5. Arrows indicate loss of p-Smad159 staining in BV high-expressing cells. c p-Smad159 staining in BV+ wild-type d6 PGCLC aggregates. Arrows indicate BV+ cell populations lacking p-Smad159 reactivity. d p-Smad159 staining in control and Blimp1−/− BV+ embryos at e7.5. Arrows indicate reduction of p-Smad159 reactivity in BV high-expressing cells in expanded panels. Arrowheads indicate retained p-Smad159 activity in BV+ cells in the Blimp1−/− mutant embryo. All IF images are counter stained with DAPI. Scale bars = 100 μm. e Genome browser track view of GFP-ChIP wiggle plots from GFP-Blimp1 expressing PGCLC cultures at day (d)2 (blue) and d6 (red) showing enrichment of GFP-Blimp1 density at a Smad1 intronic region. The sequence below the GFP-Blimp1 ChIP site contains a Blimp1 binding motif indicated in red. The consensus Blimp1 binding motif previously identified by genome-wide ChIP-seq data sets is also indicated65