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. 2016 Jun 28;5:e14470. doi: 10.7554/eLife.14470

Figure 8. Streptavidin ChIP-Seq of biotinylated PROP1 in GHFT1 cells.

(A) Schematic diagram of biotin tagging system. The birA recognition motif tag (red) is shown fused to the amino terminus of PROP1. The biotin acceptor lysine (K) is indicated with *. (B) Western blot of protein extracts from GHFT1 cell clones expressing either BirA alone, or BirA and the recombinant Prop1Tag. Upper panel, probed with anti-alpha-V5 antibody for BirA detection; Middle panel, probed with streptavidin horse radish peroxidase (SA-HRP) and lower panel, probed with anti-Histone 3 antibody for protein loading control. (C) Quantitative ChIP assay at a known PROP1 occupancy site in the Pou1f1 promoter and a negative control site (Hoxd10 promoter) using streptavidin-based ChIP. (D) Streptavidin ChIP-Seq enrichment profiles for PROP1 at the Pou1f1 gene. The known peak at −7.8 kb is indicated with *. A novel peak at -6 kb is indicated with ** and (E) validated N = 3 OWA and * indicates p<0.05 relative to controls. Data are mean + SEM. Each ChIP experiment was repeated at least twice with similar results. (F) Table containing coordinates of the peaks at the Pou1f1 promoter and the motifs found in the peaks.

DOI: http://dx.doi.org/10.7554/eLife.14470.012

Figure 8—source data 1. RNA-Seq performed on GHFT1 BirA and GHFT1 BirA Prop1Tag cells (N = 3).
The table shows fkpm values for each sample. The authentication of this cell line was based on the expression of Gapdh as an internal control, Pou1f1, Cga, Prl, Gh and Tsh.
DOI: http://dx.doi.org/10.7554/eLife.14470.013
elife-14470-fig8-data1.xlsx (8.8KB, xlsx)
DOI: 10.7554/eLife.14470.013

Figure 8.

Figure 8—figure supplement 1. Characterization of Prop1-biotag transgenic mice.

Figure 8—figure supplement 1.

(A) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). (B) Body weight at P21. Prop1-/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p<0.05. (C) Dysmorphology characteristic of the Prop1 mutant pituitary gland is rescued by the biotin-tagged Prop1 transgene. Upper panel: Hematoxylin and eosin staining of e16.5 embryos. Lower panel: Immunostaining with POU1F1-1 antibody at e16.5 paraffin sections. Scale bars, 100 µm.
DOI: http://dx.doi.org/10.7554/eLife.14470.014