A transcription factor screen identifies Tead1 as a direct regulator of apelin transcription
(A) Overview of the yeast one-hybrid (Y1H) assay to screen 745 transcription factors for their ability to interact with the Apelin (Apln) promoter. Binding of a transcription factor is readout via expression of the HIS3 reporter which enables yeast growth on a selective 3AT-containing medium plate.
(B) Y1H screen results using the −200/-1 bp fragment of the mouse Apln promoter as bait and 745 mouse TFs as prey (n = 4 replicates per tested TF, hence the formation of a quadrant in case of positive interaction).
(C) Z-score-normalized Y1H spot intensities for all 745 TFs. Six TFs with Z-scores above the background threshold (red line) are noted here and in (B).
(D) Relative mRNA expression of the six TF candidates in bulk mRNA profiling of human and mouse tissues. Microarray data for humans (left) from the GeneAtlas UI33A and mouse (right) from the GeneAtlas MOE430 of the bioGPS gene annotation portal. n = 2 replicates per tissue. Expression data are normalized and presented in a log2-scaled heatmap by species.
(E) mRNA expression of Apln and the six TFs in C2C12 myoblasts measured by RT-qPCR relative to Hprt. n.d., not detected. Mean ± SE of mean (SEM) of n = 16 replicates.
(F) mRNA expression of Tead1 in C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3 d n = 16 replicates.
(G) ChIP-qPCR assay of Tead1 testing for binding to known target promoters (Ctgf, Ankrd1), Apln promoter (−177/-77 bp), or a random negative control in C2C12 myoblasts treated with either scrambled control or Tead1-targeted siRNA for 3 days. ChIP was performed with Tead1 or IgG control antibodies and qPCR was normalized to IP input. n = 1 biological replicate.
(H) Luciferase activity of five Apln promoter fragments transfected into C2C12 myoblasts at D3 with scrambled or Tead1 siRNA. Vector control contains no Apln promoter. Mean ± SEM of n = 8 biological replicates. p values are reported from two-tailed, unpaired t-tests between siRNA conditions.