Under circumstances of heat stress, heat shock factor 1 (HSF1) plays important roles in heat shock protein expression. In the normal state, HSF1 exists in the cytoplasm in an inert monomeric state. However, when cells are stressed, HSF1 forms a homotrimeric complex and is translocated to the nucleus, where it specifically binds heat shock element (HSE), a conserved regulatory DNA sequence comprised of at least three contiguous inverted repeats of 5'-nGAAn-3' upstream of heat shock genes In this graduation thesis, we focus on two key processes (trimerization and DNA-binding) during human HSF1 heat-induced activation.In Chapter 2, an increasing concentration of dithiothreitol (DTT) was found to either enhance or inhibit the heat-induced trimerization of HSF1, suggesting the involvement of dual redox-dependent HSF1 activation mechanisms. Our in vitro experiments show that the heatinduced bonding between the cysteine C36 and C103 residues of HSF1 forms an intermolecular disulfide covalent bond (SS-I bond) and that it directly causes HSF1 to trimerize and bond to DNA. Gel filtration assays show that HSF1 can form intermolecular hydrophobic interaction-mediated (iHI-m) noncovalent oligomers. Moreover in Chapter 3, we found that the lack of a trimerization domain prevents HSF1 activation, which suggests that iHI-m noncovalent trimerization is a precondition of SS-I bond formation. On the other hand, intramolecular SS-II bond (in which the C153, C373, and C378 residues of HSF1 participate) formation inhibits this iHI-m trimerization, thereby preventing SS-I bond formation and DNA binding. Thus, HSF1 activation is regulated positively by intermolecular SS-I bond formation and negatively by intramolecular SS-II bond formation. Importantly, these two SS bonds confer different DTT sensitivities (the SS-II bond is more sensitive). Therefore, a low concentration of DTT cleaves the SS-II bond but not the SS-I bond and thus improves DNA binding of HSF1, whereas a high concentration DTT cuts both SS bonds and inhibits HSF1 activation.In Chapter 4, we demonstrate that three conserved aromatic amino acids (Trp37, Tyr60, and Phe104) are essential for HSF1 trimerization. Point mutation and fluorescence spectroscopy experiments show that an intramolecular interaction between Tyr60 and alpha-helix 1 in the DNA-binding domain stabilizes the HSF1 structure upon heat stress. Furthermore, intermolecular aromatic-aromatic interaction between the Trp37 and Phe104 supports the approach with the Cys36 and Cys103. Thus, the existence of two differential interactions facilitates the formation of intermolecular disulfide bonds, leading to the heat-induced HSF1 trimerization.In Chapter 5, we further found the role of alpha-helix1 (H1) in its DNA-binding domain (DBD) during HSF1 activation. HSF1 lacking H1 loses its heat-induced activity, suggesting the importance of the latter. Furthermore, the CD spectra and AMBER prediction show that this H1 deficiency does not change the structure of HSF1 monomer, but does impact its heat-induced trimerization. Point mutation showed that Phe18 in H1 interacts with Tyr60, and that Trp23 interacts with Phe104 by an aromatic?Caromatic interaction. Thus, the presence of H1 stabilizes the DBD structure, which facilitates the heat-induced trimerization and DNA-binding of HSF1.