Systematic interrogation of the effect of homotypic TF binding site numbers on expression. (A) Illustration of different expression functions when adding homotypic binding sites for different TFs. (B) The design of 1024 synthetic oligos to systematically investigate the effect of site numbers on expression. Four different TF binding sites were planted in all possible combinations of one to seven sites in seven predefined positions within two different background sequences. (C) Shown is the number of homotypic clusters for TF binding sites (HCTs) of different TFs in the human genomes. Data were taken from Gotea et al. (2010). Each gray bar represents a single TF. Denoted are the four TFs chosen for the design of the synthetic oligos representing different numbers of HCTs. (D–G) Expression measurements of oligos with increasing number of sites for SP1 (D), ETS1 (E), YY1 (F), and CREB (G) in the ACTB background. Each dot represents a single oligo in the library. A logistic function was fitted (Methods), and the correlations between the expression measurements and the fitted values are shown for each TF. Missing data points in panel D are oligos with NaN value (less than 100 reads; see Methods). (H) A summary plot of the four expression curves that were computed in D through G for direct comparison between TFs.
