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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination.

ABSTRACT: Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner consistent with activation. Most striking, however, was loss of the Pol II peak near the 3' ends of mRNA genes, coupled to a gain in polymerase occupancy extending tens of kilobases downstream of 3' ends. Typical patterns of 3' end occupancy were largely restored 60 min after cells returned to normal growth temperatures. These changes in polymerase occupancy revealed a heat shock-induced loss of normal termination, which was potent, global, and reversible. The occupancy of the termination factor CPSF73 at the 3' ends of representative genes was reduced after heat shock, suggesting a mechanism for impaired termination. The data support a model in which heat shock induces widespread repression of transcriptional initiation and loss of transcription termination, which reverses as cells return to homeostasis.

SUBMITTER: Cardiello JF

PROVIDER: S-EPMC6113597 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination.

Cardiello Joseph F JF Goodrich James A JA Kugel Jennifer F JF

Molecular and cellular biology 20180828 18

Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner ...[more]

PMID: 29967245

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Project description:ObjectiveCortisol clearance is reduced in sepsis and may contribute to the development of impaired adrenocortical function that is thought to contribute to the pathophysiology of critical illness-related corticosteroid insufficiency. We sought to assess adrenocortical function using computer-assisted numerical modeling methodology to characterize and compare maximal cortisol secretion rate and free cortisol half-life in septic shock, sepsis, and healthy control subjects.DesignPost hoc analysis of previously published total cortisol, free cortisol, corticosteroid-binding globulin, and albumin concentration data.SettingSingle academic medical center.PatientsSubjects included septic shock (n = 45), sepsis (n = 25), and healthy controls (n = 10).InterventionsI.v. cosyntropin (250 μg).Measurements and main resultsSolutions for maximal cortisol secretion rate and free cortisol half-life were obtained by least squares solution of simultaneous, nonlinear differential equations that account for free cortisol appearance and elimination as well as reversible binding to corticosteroid-binding globulin and albumin. Maximal cortisol secretion rate was significantly greater in septic shock (0.83 nM/s [0.44, 1.58 nM/s] reported as median [lower quartile, upper quartile]) compared with sepsis (0.51 nM/s [0.36, 0.62 nM/s]; p = 0.007) and controls (0.49 nM/s [0.42, 0.62 nM/s]; p = 0.04). The variance of maximal cortisol secretion rate in septic shock was also greater than that of sepsis or control groups (F test, p < 0.001). Free cortisol half-life was significantly increased in septic shock (4.6 min [2.2, 6.3 min]) and sepsis (3.0 min [2.3, 4.8 min] when compared with controls (2.0 min [1.2, 2.6 min]) (both p < 0.004).ConclusionsResults obtained by numerical modeling are consistent with comparable measures obtained by the gold standard stable isotope dilution method. Septic shock is associated with generally not only higher levels but also greater variance of maximal cortisol secretion rate when compared with control and sepsis groups. Additional studies would be needed to determine whether assessment of cortisol kinetic parameters such as maximal cortisol secretion rate and free cortisol half-life is useful in the diagnosis or management of critical illness-related corticosteroid insufficiency.

Project description:Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combination-treated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GA-treated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination.

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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination.

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