Fig. 7

Evaluation of Tshb regulatory elements in transfection and transgenic mice. a Tracks illustrating the results of RNA-seq, CUT&RUN for POU1F1, H3K27Ac, and H3K4Me1, ATAC-seq, and CUT&RUN for H3K27Me3 are shown for the Tshb locus in TαT1 cells (red) and GHF-T1 cells (blue). Elements that were tested functionally are highlighted. The significance is indicated with asterisks: p value < 0.05 = *; p value < 0.01 = **. b TαT1 cells were transfected with a 438 bp Tshb promoter-proximal element fused to luciferase and putative regulatory elements in the forward (filled circles) or reverse (x) orientation. Luciferase activity for each construct is normalized to the promoter-only luciferase activity and significant differences from promoter alone are indicated with asterisks (p < 0.05 = * and p < 0.01 = **). c Heterologous CV1 cells were transfected with the Tshb promoter-only construct or the promoter plus element 4 construct, along with expression vectors for POU1F1 and/or GATA2 as indicated by + or −. Significant differences from the reporter gene alone are indicated with asterisks (p < 0.05 = * and p < 0.01 = **). The values obtained for both constructs with POU1F1 or GATA2 expression vectors were significantly different, p < 0.01. d Genome browser track illustrating element 4 (1.4 kb located 6.6–7.7 kb upstream of the Tshb transcription start site) with experimentally determined sites for POU1F1 binding and ATAC-seq, and predicted binding sites for POU1F1, GATA2, and PITX1 that reach a confidence level of at least 0.8 in JASPAR. An extended list of binding motifs predicted in Element 4 is presented is in Supplemental Table 5. e A section of the pituitary gland from transgenic founder 399 was co-immunostaining for YFP (red) and TSHB (green), revealing overlap in expression (yellow). Nuclei are stained with DAPI (blue). f Same as e, in founder mouse 423. For transfections, N = 6/reporter gene