Influence of Regulatory Elements on HSF1 Activation

Heat shock factor 1 (HSF1) is the key regulator of the heat shock response (HSR), a conserved cellular mechanism that maintains proteostasis under cellular stress. Central to HSF1 regulation is its multi-domain structure, including a regulatory domain (RD) previously thought to be essential for activation via phosphorylation. To elucidate the RDs function, we utilized a luciferase reporter assay in HSF1 knockout HEK293 cells reconstituted with either full-length HSF1 (HSF1 fl), a phosphorylation- deficient mutant (HSF1 11A), or a variant lacking the RD (HSF1 ls). Our findings reveal that while all constructs induce robust HSR activation upon heat stress, their responses diverge under chemical stressors such as CdSO₄ and Geldanamycin. The reduced activity of the phosphorylation-deficient mutant compared to the RD-deleted variant suggests that RD phosphorylation modulates HSF1 activity in a stressor-specific manner, and that removal of the RD may relieve inhibitory constraints. These results demonstrate that the RD acts as a stressor-specific regulatory element, offering new insight into HSF1’s activation dynamics and potential therapeutic targeting in stress-related diseases.