Figure 3. Ratio between HSF2α and HSF2β controls HSF1β transcriptional activity.
(A) Representative Western-blot showing the expression of HSF2 isoforms after transfection. Hsf1.2−/− iMEFs were co-transfected with pCR3.1-HSF1β and pCR3.1-HSF2α/β to obtain the expression of equivalent amounts of HSF1 and HSF2, and increasing concentration of HSF2β relatively to HSF2α. Cells were treated with 2.5 µM MG132 for 8 h. Protein extracts were loaded on 12% polyacrylamide gel and submitted to a long migration to separate efficiently HSF2 isoforms. HSF2 was revealed by immunobloting. (B) Transcriptional activity induced, with fixed concentrations of HSF1 and total HSF2, but varying combinations of HSF2β/HSF2α. Hsf1.2−/− iMEFs were co-transfected with 12.5 ng of pCR3.1-HSF1β and with the same quantity of pCR3.1-HSF2α/β, as previously described. Diamonds correspond to the experimental data obtained in independent triplicates, and expressed as percentage of maximal activity. Solid line drawn to equation 2 with random multimerization and considering that HSF1 is active only in absence of HSF2β in the trimer. (C) HSE-driven transcriptional activities expected from Eq. 2, with a constant and identical amounts of HSF1 and total HSF2, and when increasing the ratio HSF2β/HSF2α. The transcriptional strength of a trimer is assumed to be proportional to the number of HSF1 monomers present, and the unit of transcriptional strength (k = 1) corresponds to that of an HSF1 monomer. The strength of trimerization is considered as either (i) identical between hetero- and homodimers (plain line), (ii) 10 fold higher for homodimers (dashed line), or (iii) 10 fold higher for heterodimers (dotted line). (D) HSE-driven transcriptional activities drawn to Eq. 2, with constant and equivalent amounts of HSF1 and HSF2, capable to either randomly homo- or heterotrimerize. Plain line: as for panel C, the transcriptional strength of a trimer is proportional to the number of HSF1 monomers included in the trimer and the strength of a HSF1 monomer is set to 1 (k 3 = 3 k 1 and k 2 = 2 k 1 and k 1 = 1). Dashed line: Transcriptional strength independent on whether the trimer contains 1, 2 or 3 HSF1 monomers (k 1 = k 2 = k 3 = 1). Dotted line: same rule with k 1 = k 2 = k 3 = 3.