We used the conditional chemical genetics approach known as “anchor away” (AA) to rapidly inactivate the essential yeast transcription factor Hsf1. We coupled Hsf1-AA to RNA-seq and NET-seq to define the genes whose expression depends on Hsf1 and performed Hsf1-3xFLAG-V5 ChIP-seq to validate direct targets. We also carried out a number of other perturbations to yeast stress pathways to show that most of the gene expression changes during heat shock are Hsf1-independent but depend on PKA signaling and the Msn2/4 general stress transcription factors. Finally, we generated RNA-seq in mouse ES cells and MEFs in wild type and hsf1-/- cells to define HSF1 targets in murine cells. Overall design: yeast RNA-seq samples, 4 yeast NET-seq samples, 8 murine RNA seq samples and 1 yeast ChIP-seq sample