Fig. 8: HSF5 possesses an unique DNA binding specificity.
a The purified HSF5 N-terminal (aa1-209) protein was used for EMSA assay (left). Shown on the right is the corresponding HSF5 N-terminal part is shown (red) on the ribbon model that was predicted from AlphaFold2. b DNA binding ability of HSF5 DNA binding domain was examined by EMSA assay. Shown on the top are the target (T) and mutant (M) sequences of the DNA probes. Target (T) sequence was designed according to the enriched motif that was predicted by Chip-seq as shown in Fig. S8e. Increasing amount of the purified protein was mixed with 0.04 pmol of 32P-labeled DNA probes (T or M) at the protein/DNA molar ratio of 15.6 − 250. Arrowhead: unbound DNA. The protein-DNA complexes are shown by * with a bracket. c HSF5 N-terminal protein (1 μM) was mixed with 0.04 pmol of the 32P-labeled target (T) DNA probe. DNA binding specificity of HSF5- DNA complex was assessed by adding the unlabeled target or mutant competitor DNA (1 – 125 fold excess to the 32P-labeled DNA probes). A single experiment was performed in (a–c). d Amino acid sequences of the DNA-binding domain in the mouse HSFs are aligned. HSF1, HSF2, and HSF4 possess a DNA recognition helix (red) containing a conserved Ser-Phe-Val-Arg-Gln amino acid sequence, which is known to insert into the major groove of the HSE. HSF5 possesses a Ser-Phe-Ile-Arg-Gln amino acid sequence at the corresponding position. HSF5 possesses insertion of amino acid sequence between the helix 2 and DNA recognition helix. e Ribbon models of HSF5-DBD (1-167 a.a.) are shown that were predicted from AlphaFold2. Helixes are colored as shown in (a).
