Page 126 - 2021_06-Haematologica-web

R. Famà et al.
Identification of key Ets-responsive elements in the F8 proximal promoter region
In order to decipher the importance of each Ets BS in pF8 activity, we generated several pF8 mutants and tested their impact. We adopted two different strategies. The
first aimed at eliminating progressively the Ets BS by per- forming deletions of pF8 (Figure 3A and B). Using this approach in ECV-304 cells, we observed that the basal activity of the full-length promoter (pF8-1,175) was main- tained in pF8-600 and pF8-446, where one Ets-2 BS was
A
B
CD
EF
Figure 3. Crucial E26 transformation-specific-responsive elements are localized in the first 342 bases of F8 promoter region. (A) Table summarizing the Ets-1 and Ets-2 binding sites distributed across the F8 promotor (pF8) and scheme of mutagenesis strategies used to investigate the role of each E26 transformation-specific binding site (Ets-BS). Ets-BS destroyed by mutagenesis are highlighted in black and are indicated with E followed by a number. Only Ets-BS with a dissimilarity score <3 were disrupted by mutagenesis. (B) Schematic representation of mutagenesis strategies used to perform single or multiple deletions of Ets-BS on pF8. Colored boxes highlight the Ets-BS which underwent to a single deletion. Colored scissors indicate the points on pF8 where it was cut to generate the shortened forms with multiple ETS-BS deletions. (C and D) Histograms representing fold change in luciferase activity of shortened promoters at (C) basal level or after overexpression of (D) Ets-1, Ets-2 or both. (E and F) Histograms showing changing in luciferase activity of Ets-BS single deleted promoters at (E) basal level or after overexpression of (F) Ets-1, Ets-2 or both. All experiments were performed in ECV-304 cells. Results are expressed as mean ± standard deviation from three independent experiments performed in triplicate. *P<0.05.
1628
haematologica | 2021; 106(6)