Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA-binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8-mer palindromic core 5¢-TTCTAGAA-3¢. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes.Keywords: heat shock transcription factor-2; protein purification; cooperativity; SELEX; consensus binding sequence.Heat shock factor 2 (HSF2) belongs to the vertebrate heat shock factor family that also includes HSF1, HSF3 and HSF4 [1][2][3][4][5]. The members of the HSF family are defined by their ability to specifically bind the regulatory sequence heat shock element (HSE) [6]. Located in the regulatory regions of heat shock genes, HSE consists of the inverted repeat of a basal element nGAAm [7]. Two inverted repeats are sufficient for Drosophila HSF binding, but optimal binding is obtained with three repeats [8]. In agreement with this observation, the activated form of HSFs has been demonstrated to be a trimer in yeast [9], in Drosophila [10], in human [11,12] or in mouse [13]. The HSE-binding activity of heat shock factors is not constitutive, but induced by various stresses, by differentiation or developmental processes. HSF1 and HSF3 are activated by stresses that elicit the so-called Ôheat shock responseÕ and induce the transcription of heat shock genes. HSF1 corresponds to the paradigm member of the family and is the functional homolog, for its function in the heat shock response, of the unique HSF found in yeast and Drosophila. Avian HSF3 is activated by more severe stresses than HSF1, but is also required for an optimal response to stress [14,15]. Indeed, avian cells expressing HSF1, but in which the HSF3 gene has been disrupted, exhibit a diminished response to stress, even at mild heat shock temperatures [14]. Athough heterotrimers were never detected, HSFs may interact with each other in a more complex way.HSF4 is an exception and constitutively binds DNA as a trimer in the absence of stress. Its expression is regulated in a tissue-specific manner [5,16]. The Hsf4 gene generates both an activator or a repressor of heat shock genes by alternative splicing; the tissue-specificity of the two forms may create a modulation of expression of hsps in the different tissues.In contrast to HSF1 and HSF3, HSF2 is no...