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. 2021 Jun 1;49(11):6249–6266. doi: 10.1093/nar/gkab443

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — TF residence time and concentration stimulate transcription activation. (A) Example images of the smFISH methodology: Lectin-FITC-stained cell membrane (blue; contrast 0 3000), smFISH of specific mRNA transcripts and nascent sites of transcription (arrow) (red; z-projection; contrast 2 40), TMR-stained HaloTag-TALE-TF (green; contrast 0 500) and composite image. Dashed line indicates the cell outline. Dotted line indicates the nucleus. The bright aggregate visualized by membrane stain and seen in the TALE-TF channel due to bleed-through probably corresponds to endoplasmic reticulum or Golgi apparatus. (B) mRNA distribution in cells expressing no TALE-TF (empty) and in cells expressing the different TALE-TF with < 300 nM. Cell numbers: N = 752 (empty); N = 888 (T19R); N = 690 (T9R); N = 664 (T13R); N = 790 (T15R). Mean (line), 25th/75th percentile (box) and 10th/90th percentile (whiskers) define features of box plot. Significance was determined with Wilcoxon–Mann–Whitney two-sample rank test. (C) Mean mRNA numbers from B) of cells expressing TALE-TF subtracted with the mean mRNA number of cells expressing no TALE-TF plotted versus TALE-TF residence time. Error bars denote s.e.m. for mean mRNA numbers and resampling error estimation for residence times. (D) Mean mRNA numbers of cells expressing TALE-TF at the indicated concentration subtracted with the mean mRNA number of cells expressing no TALE-TF. Cell numbers (<200 nM, 200 to <400 nM, 400 to <600 nM): T19R N = 655, 375, 113; T9R N = 509, 282, 102; T13R N = 478, 256, 33; T15R N = 580, 365, 155). Error bars denote s.e.m. (E) mRNA distributions of TALE-TFs together with the distribution inferred by BIRD for the case where TF residence time modifies k_on,eff of the gene. (F) Comparison of mRNA distributions inferred by BIRD with the measured mRNA distributions of TALE-TFs by means of the lowest sum of squared residues in cases were TF residence time modifies k_on,eff, k_off,eff or k_tx.