Crosstalk between HSF1 and HSF2 during the heat shock response in mouse testes

Abstract

Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellular stress, while HSF2 becomes activated during development and differentiation, including spermatogenesis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heat shock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mechanisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heat shock response. For this purpose we used chromatin immunoprecipitation and the proximity ligation approaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32 °C) or heat shocked (at 38 °C or 43 °C) spermatocytes, which are cells the most sensitive to hyperthermia. At the physiological temperature or after mild hyperthermia at 38 °C, the sharing of binding sites for both HSFs was observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermia at 43 °C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of promoter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence of HSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resulted in a decreased number of such complexes and they were barely detected after strong hyperthermia at 43 °C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermatogenic cells. However, temperature elevation causes remodeling of chromatin binding and interactions between HSFs are disrupted. This potentially affects the regulation of stress response and contributes to the heat sensitivity of these cells.

Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells

“…Data on HSF1 binding to Pmaip1 assessed in genomewide ChIP-Seq analysis were extracted from the dataset available on Gene Expression Omnibus, accession no. GSE56735 [34]. The ChIP assay was carried out according to the protocol of a ChIP kit from Upstate Biotechnology (Lake Placid, NY, USA) using protein A-sepharose beads (GE Healthcare Europe GmbH, Freiburg, Germany) or according to the Dynabeads™ Protein A from Life Technologies protocol (Thermo Fisher Scientific, Waltham, MA, USA), or according to the protocol from the iDeal ChIP-Seq Kit for Transcription Factors (Diagenode, Denville, NJ, USA).…”

“…Using a genome-wide ChIP-Seq approach we found that HSF1 became localized in the second intron of the Pmaip1 gene in the chromatin of heat-shocked mouse spermatocytes [34]. This HSF1 binding occurred at a perfect HSE motif.…”

Identification of Novel Response and Predictive Biomarkers to Hsp90 Inhibitors Through Proteomic Profiling of Patient-derived Prostate Tumor Explants

“…Heat-shock cognate 70 protein, HSPA8, significantly increased upon treatment with both 17-AAG and AUY922 compared with control. HSPA8 regulates the heat shock response (HSR) and maintains homeostasis (64). The induction of the heat shock response is established as a bona fide component of the molecular signature of Hsp90 inhibition observed in numerous cancer cell line studies (18,(65)(66)(67) and is used to indicate target modulation in clinical trials (68,69).…”

SPEN protein expression and interactions with chromatin in mouse testicular cells

“…For heat shock, equal volumes of CO 2 saturated, pre-heated media (to 53°C or 60°C) were added to the testicular cells suspensions, which immediately raised their temperature from 32°C (physiological temperature) to 38°C or 43°C respectively. Tubes were submerged in a water bath at the appropriate temperature for an additional 15-30 min (time estimated previously as optimal for activation of the heat shock response mediated by HSF1) (Kus-Liśkiewicz et al 2013, Korfanty et al 2014 with recovery at 32°C in a cell culture incubator. All animal experiments were carried out according to Polish legislation and were approved by the Local Committee of Ethics and Animal Experimentation at the Medical University of Silesia in Katowice, Poland (Decision No 10/2012) and by the Institutional Animal Care Policy of the Maria Sklodowska-Curie Institute -Oncology Center (Gliwice, Poland).…”