When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins. Display omitted •RNAi identifies heat stress response modulators in different cellular locations•Chromatin modifiers and nuclear proteasome are critical stress response regulators•EP300 acetylates HSF1 and regulates its stability and cellular level•The proteasome attenuates the stress response by degrading activated HSF1 EP300-mediated acetylation stabilizes HSF1, a key regulator of the cellular response to proteotoxic stress, but further acetylation during the stress response attenuates HSF1 activity and leads to proteasomal degradation, underscoring the complexity of the cellular stress response.