FIG. 1.

Chicken HSF1 has little potential to activate heat shock genes in response to heat shock. (A) The phylogenetic tree generated in Clustal W (43) for members of the HSF family. The molecular tree was constructed by the neighbor-joining method. Gaps were excluded from all phylogenetic analyses. The numerals show bootstrap values (1,000 bootstrap replicates were performed). The unrooted tree was drawn with the program TreeView (31). The bar represents 0.1 substitutions per site. The amino acid sequences used in the tree construction are human HSF1 (hHSF1, SP accession no. Q00613), mouse HSF1 (mHSF1, SP accession no. P38532), chicken HSF1 (cHSF1, SP accession no. P38529), zebrafish HSF1 (Zebra HSF1, DDBJ accession no. AB062117), and Xenopus HSF1 (Xenopus HSF1, SP accession no. P41154), human HSF2 (hHSF2, SP accession no. Q03933), mouse HSF2 (mHSF2, SP accession no. P38533), and chicken HSF2 (cHSF2, SP accession no. P38530), chicken HSF3 (cHSF3, SP accession no. P38531), human HSF4b (hHSF4, SP accession no. Q9ULV5) and mouse HSF4b (mHSF4, SP accession no. Q9R0L1), and HSF from Saccharomyces cerevisiae (ScHSF, SP accession no. P10961), D. melanogaster (DmHSF, SP accession no. P22813), and Caenorhabditis elegans (CeHSF, PIR accession no. T27125). (B) HSF3-null cells were transfected with an expression vector for cHSF1, hHSF1, D. melanogaster HSF, or cHSF3. Clones grown in the presence of zeocine were selected for Western blot analysis with HSF1-specific antiserum αHSF1β and antiserum for HA or HSF3-specific antiserum αHSF3γ (26). Representative clones are shown. Arrows in the upper and lower columns indicate endogenous cHSF1 and cHSF3, respectively. An asterisk shows an unmodified form of HSF3 (42). Arrowheads indicate ectopically expressed HSFs. (C) Heat shock response in HSF3-null cells expressing the indicated HSFs. RNAs were extracted from cells untreated or treated with heat shock at 45°C for 60 min, and Northern blot analysis was performed with cDNA for Hsp70 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Schematic representation of wild-type and mutant HSF1 loci together with the targeting vector, mHSF1-neo. Exons are represented by solid boxes, and the DNA-binding domain is encoded by exons 2 and 3. The 3′ probe for Southern blot analysis (solid bar) and primer sites (arrowheads) are indicated. Restriction enzyme sites are: Bg, BglII; Hp, HpaI; S, SalI. (E) Southern blot analysis of DNA prepared from wild-type, HSF1^+/−, and HSF1^−/− ES cells. DNA was digested with BglII and hybridized with the ³²P-labeled 3′ probe or neo probe. (F) HSF1^−/− ES cells were transfected with expression vector pcHSF1-Hyg or phHSF1-Hyg, and clones which expressed a high level of cHSF1 or hHSF1 were selected. HSF1 protein levels in each representative clone were shown by Western blot analysis with αHSF1β antiserum. The lower column represents a longer exposure. (G) Examination of heat shock response by Northern blot analysis. HSF1^−/− ES cells expressing cHSF1 or hHSF1 as well as wild-type and HSF1^−/− ES cells were heat shocked at 43°C for 30 min. RNAs were extracted from these cells, and Northern blot analysis was performed with ³²P-labeled cDNAs for mouse Hsp70-1 and human β-actin.