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Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor PPARα

ABSTRACT: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through activation by HS factor-1 (HSF1). We hypothesized that there are interactions on a genetic level between PPARα and the HS response mediated by HSF1. Wild-type and PPARα-null mice were exposed to HS, the PPARα agonist WY-14,643 (WY), or both; gene and protein expression was examined in the livers of the mice 4 or 24 hrs after HS. Gene expression profiling identified a number of Hsp family members that were altered similarly in both mouse strains. However, most of the targets of HS did not overlap between strains. A subset of genes was shown by microarray and RT-PCR to be regulated by HS in a PPARα-dependent manner. HS also down-regulated a large set of mitochondrial genes specifically in PPARα-null mice that are known targets of PPARg co-activator 1 (PGC-1) family members. Pretreatment of PPARα-null mice with WY increased expression of PGC-1b and target genes and prevented the down-regulation of the mitochondrial genes by HS. A comparison of HS genes regulated in our dataset with those identified in wild-type and HSF1-null mouse embryonic fibroblasts indicated that although many HS genes are regulated independently of both PPARα and HSF1, a number require both factors for HS responsiveness. These findings demonstrate that the PPARα genotype has a dramatic effect on the transcriptional targets of HS and support an expanded role for PPARα in the regulation of proteome maintenance genes after exposure to diverse forms of environmental stress including HS. Keywords: Gene Expresssion-Heat shock

ORGANISM(S): Mus musculus

PROVIDER: GSE14869 | GEO | 2009/05/01

SECONDARY ACCESSION(S): PRJNA111933

REPOSITORIES: GEO

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Project description:Perfluorooctane sulfonate (PFOS) is a perfluoroalkyl acid (PFAA) and a persistent environmental contaminant found in the tissues of humans and wildlife. Although blood levels of PFOS have begun to decline, health concerns remain because of the long half-life of PFOS in humans. Like other PFAAs, such as perfluorooctanoic acid (PFOA), PFOS is an activator of peroxisome proliferator-activated receptor-alpha (PPARα) and exhibits hepatocarcinogenic potential in rodents. PFOS is also a developmental toxicant in rodents where, unlike PFOA, it’s mode of action is independent of PPARα. Wild-type (WT) and PPARα-null (Null) mice were dosed with 0, 3, or 10 mg/kg/day PFOS for 7 days. Animals were euthanized, livers weighed, and liver samples collected for histology and preparation of total RNA. Gene profiling was conducted using Affymetrix 430_2 microarrays. In WT mice, PFOS induced changes that were characteristic of PPARα transactivation including regulation of genes associated with lipid metabolism, peroxisome biogenesis, proteasome activation, and inflammation. PPARα-independent changes were indicated in both WT and Null mice by altered expression of genes related to lipid metabolism, inflammation, and xenobiotic metabolism. Such results are similar to prior studies done with PFOA and are consistent with modest activation of the constitutive androstane receptor (CAR) and possibly PPARγ and/or PPARβ/δ. Unique treatment-related effects were also found in Null mice including altered expression of genes associated with ribosome biogenesis, oxidative phosphorylation and cholesterol biosynthesis. Of interest was up-regulation of Cyp7a1, a gene which is under the control of various transcription regulators. Hence, in addition to its ability to modestly activate PPARα, PFOS induces a variety of “off-target” effects as well. PPARalpha-null and wild-type male mice at 6-9 months of age were dosed by gavage for 7 consecutive days with either 0, 3, or 10 mg/kg PFOS (potassium salt) in 0.5% Tween 20. Five biological replicates consisting of individual animals were included in each dosage group. Data were compared to results previously published by our group for PFOA and Wy-14,643, a commonly used agonist of PPARalpha (Rosen et al., Toxicol Pathol. 36:592-607, 2008; GSE9796)

Project description:HSF1 binds DNA via the DBD domain, causing gene upregulation during HS. We assessed the effect of RD-mediated phase separation on chromatin targeting of HSF1 using Cut&Tag followed by high-throughput sequencing to map genome-wide binding of LLPS-competent versus LLPS-incompetent HSF1 mutants under both HS and NHS conditions. Comparing with WT HSF1 under NHS, both WT under HS and M1 under NHS showed increased and broad binding to enhancers and distal intergenic region, with binding most enriched in expected motifs of HSF-related transcription factors. To further assess the role for IDR-induced LLPS in chromatin targeting of HSF1, we used several additional strategies. First, the treatment of 1,6-hexanediol markedly decreased chromatin occupancy both of WT under HS and M1 under NHS conditions. Second, we interrogate chromatin binding of LLPS-deficient mutant M3. Cut&Tag analysis revealed that M3 showed decreased chromatin binding compared to WT under HS and M1 under NHS. The decreased chromatin binding of M3 to chromatin was not due to the loss of its DNA binding ability, as the EMSA assay revealed that M3 was still capable of binding HSE. Instead, the decreased chromatin binding reflects the loss of inter-molecular interaction between HSF1 that holds LLPS. Furthermore, M3 in heat shocked cells shows similar reduced genomic targeting and shallow binding pattern as NHS cells . Third, the enrichment of transcriptional apparatus RNA Pol II, CYCT1, BRD4 to HSF1 target genes also depend on whether HSF1 can phase separate at these sites. Lastly, we conducted live cell single molecule imaging to evaluate chromatin binding kinetics of LLPS-deficient mutant M3 relative to WT HSF1. Measurements of single molecule displacement and diffusion coefficient showed M3 to be significantly more mobile than WT under HS, which suggests M3 was less confined within phase-separated puncta compared with LLPS-competent HSF1. Consistent with this result, super resolution imaging of M3 also showed decreased cluster formation at HSP gene foci but maintained nSBs formation. Altogether, LLPS-forming capability of HSF1 is essential for the efficient recruitment of HSF1 and transcriptional apparatus to HSP gene loci.

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Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor PPARα

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