PMC

(a). Venn diagrams showing the total number of target sites overlapping by one base-pair or more between BLIMP1 on one hand and the indicated transcription factors on the other. (b) Venn diagrams showing the total number of genes overlapping between BLIMP1 on one hand and indicated transcription factors on the other hand. In (a) and (b), the circles for the “self-renewal” cluster genes are indicated in green and the “polycomb” cluster genes in blue (c) Track-view of BLIMP1 binding on example genomic loci including the views for EZH2, RING1B, NELFA as well as SIN3A where appropriate.

(a). Bright-field and fluorescent microscopy images of the Oct4 reporter ESC line (GOF) in culture. The images show ESCs, EpiLCs (stage preceding PGCLC), and PGCLCs as indicated. The left panels show the induction of PGCLCs using cytokines, and the right panels show the induction of PGCLCs without cytokines using doxycycline dependent induction of BLIMP1, AP2γ and PRDM14 from GOF cells harbouring stable doxycycline-responsive constructs. The numbers on the figure panels indicate the ratio of fluorescent cells induced at each time-point. (b). RT-qPCR analysis of sorted fluorescent PGCLCs on Day2 and 4 of either cytokine or doxycycline induction, as well as EpiLCs. Panels (a and b) show a representative of two identical experiments.

(a). Cluster analysis of single-cell RNA-seq expression profiles of PGCs and somatic neighbours. The bar indicates the mean Euclidian distance between the samples. Note that E7.5 BLIMP1 mutant (KO) cells cluster next to E7.5 somatic cells, while the PGCs form distinct clusters following specification (b). Expression levels of selected gene transcripts during PGC specification (RPM=read numbers pr. million reads). The y-axis represents read numbers pr. million reads sequenced. The region shaded in blue represents BLIMP1 mutant (KO) cells. The dotted line represents the onset of PGC specification. (c). Expression of Dnmt3b, T-Brachyury, Tcfap2c and Cbx7 during PGC specification, which are all bound by BLIMP1 during PGC specification. (d). Track views of BLIMP1 binding to the genomic loci of Dnmt3b,T-Brachyury, and Tcfap2c (encoding AP2γ) and Cbx7.

(a) Design and overview of the experimental approaches towards transcriptional network for PGC specification.The arrow pointing to PGCs refers to EGFP reporter labelled PGCs in mouse embryos. (b) RT-qPCR analysis of differentially regulated genes downstream of BLIMP1 in P19ECs, showing induction of the PGC genes Nanos3, Rhox9, Dppa3, Tcfap2c, Dnd1, Prdm14, Dazl and Ddx4. (c) RT-qPCR analysis showing the individual and combinatorial effect of BLIMP1, AP2γ and PRDM14 in P19ECs on the mesendodermal transcription factor genes, Eomes and T-Brachyury, the DNA methyltransferase Dnmt3b on Myc and the PGC genes Nanos3, Dnd1, Prdm1, Ddx4/Vasa and Dppa3. The error bars in panels (b) and (c) represent standard deviations for 3 independent cell cultures.

(a). Relative enrichment of BLIMP1, AP2γ and PRDM14 targets on differentially expressed genes between E7.5 PGCs and soma, and the combinatorial association of the peak regions to the differentially expressed genes. (b).The plots depict the hypergeometric p-values for the corresponding enrichment of BLIMP1, AP2γ and Prdm14 targets on differentially expressed genes shown in Fig 6a. (c). Venn diagrams showing the total number of genomic target sites overlapping by one base-pair or more between BLIMP1, AP2γ and PRDM14. The p-value for the enrichment of overlap between the factors of p < 0.0001 was calculated using a permutation test. See details in . (d) Venn diagrams showing the total number of genes overlapping between BLIMP1, AP2γ and PRDM14. The p-value of p < 1×10⁻²⁹⁹ was calculated using a ch-square test. See details in (e). Track-view of BLIMP1, PRDM14 and AP2γ binding to selected repressed and induced PGC targets. (f). A transcriptional network model depicting the role of PRDM14, BLIMP1 and AP2γ during PGC specification.

(a). De novo motif analysis revealed high enrichment for the AP2γ consensus binding site with a p-value of 1.1×10⁻²⁴¹. (b). Distribution of AP2γ binding relative to promoters revealing a mean distance of +53 bp from the TSS. (c). Track-view of AP2γ binding on the Pou5f1 distal enhancer, to the PGC genes Dppa3 and Nanos3, and the somatic differentiation regulators HoxA10, HoxA11, Hes7 and T-Brachyury. (d). A ChIP-qPCR of AP2γ binding to the promoter of Nanos3, Dppa3/Stella as well as the distal enhancer of Oct4 (Oct4-DE). (e). Over represented gene categories sorted by cell-type specific expression at different Theiler Stages (TS) during embryonic development, TS8 corresponds roughly to E6.0, TS17 to E10.5, TS20 to E17 and TS21 to E21/P0. (f). Over represented functional categories of genes bound by AP2γ showing the p-value for the enrichment of biological process GO-terms.

(a). Relative enrichment of BLIMP1 binding regions and the scores associated with genes differentially expressed between E7.5 PGCs and BLIMP1 mutant (KO) cells, and between E7.5 PGCs and E7.5 somatic cells, respectively. The x-axes indicate the log₂ (fold change) and the y-axes indicate the log₂ of the BLIMP1 target enrichment at each fold change-interval of differentially expressed genes over the average target frequency of the whole expression data set. (Peaks: the enrichment of peaks associated with genes in each interval at E7.5. Scores; the enrichment of binding scores calculated for genes in each interval at E7.5). (b). Binding frequency of BLIMP1 to genes associated with features on the whole microarray as well as differentially regulated genes (c). Heat map depicting the genes repressed by BLIMP1 in both P19ECs and during PGC specification. The first 4 (blue) columns refer to normalized gene expression levels in PGCs. The next 3 columns (red) reflect values of differential expression between the samples indicated. The asterisks in the final column indicate a high confidence BLIMP1 binding region associated with a gene. BLIMP1 binding was detected in 34/59 repressed genes in both data sets. (RPM: reads per million. FC: fold change).

(a) Track-view of BLIMP1 ChIP-Seq density profile displayed using the UCSC genome browser centred on the Id3 and Myc genes. (b). BLIMP1 peaks distributed roughly evenly between inter- and intragenic positions, with 1248 of 2480 intergenic peaks falling within 1kb 5′ of TSS, and 527 peaks were more than 10Kb away from promoters. (TSS: transcriptional start site; TES: transcriptional end site). (c) Distribution of BLIMP1 binding relative to promoters revealing a median distance of +171.5 bp from the TSS. (d) De novo motif analysis revealed high enrichment for the BLIMP1 consensus (p-value of 1.2×10⁻³⁸⁸). (e). BLIMP1 binding profiles on Hox gene clusters are indicated in the views with their respective numbers. For example, Hoxa1 is indicated by a1, Hoxa2 is a2 and so forth. (f). Functional categories of genes bound by BLIMP1 showing the p-value for the molecular function as well as biological process GO-terms. (g). Validation of novel BLIMP1 binding regions by ChIP-qPCR in P19 EC cells. (h). A validation of novel BLIMP1 binding regions in PGCLCs by ChIP followed by whole genome amplification of precipitated and input DNA assayed by qPCR. The y-axis represents the % of signal from amplified input material at the same starting quantity of the immunoprecipitated DNA to ensure proportional amplification.