Unraveling Heat Acclimation Memory: From Epigenetic Mechanisms to the Expressed Phenotype Thesis submitted for the degree of ―Doctor of Philosophy‖ By Anna Tetievsky Submitted to the Senate of the Hebrew University of Jerusalem June 2012 This work was carried out under the supervision of: Proffesor Michal Horowitz Abstract Heat acclimation (AC) is a reversible ‘within lifetime‗ phenotypic adaptation to long-term elevations in environmental temperature that evolves via a continuum of temporally varying processes. Successful AC is characterized by enhanced thermal tolerance manifesting as improved endurance and resistance to temperature extremes, collectively delaying the onset of heat injury. Heat acclimation can reinforce or interfere with the ability to combat novel acute stressors. In this way, AC was found to confer protection to a variety of stressors (namely, cross-tolerance) with impaired oxygen supply/oxygen demand ratios. Among these, cross-tolerance between ischemia-reperfusion insult in the heart and hyperoxia in the brain have been extensively studied. The available evidence substantiates that a reprogramming of gene expression and translational processes are essential events in the pathway to heat acclimation. The important hallmarks of AC and AC-mediated cross-tolerance identified to date include enhanced reserves of heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), anti-apoptotic and anti-oxidative pathways, accelerated (vs. non- acclimated) stress-mediated transcriptional activation of these genes and in turn, the heat shock response (HSR). Although the benefits of acclimation are considered transitory, the few published investigations on the time course of AC loss indicate that reacquisition of the acclimated phenotype [re-acclimation-(ReAC)] is more rapid upon return to the hot environment than the time required to acclimate initially. Our previous findings show that the cardiac acclimated phenotype has a molecular memory and regains both acclimatory enhanced performance and cardioprotection after loss of acclimation within only 2 days of exposure to the acclimating conditions. Interestingly, the protein levels of hsp70 and anti-apoptotic bcl-xL were upregulated even after 30 days of deacclimation (DeAC) conditions. Given that the evolvement of AC depends on a reprogramming of gene expression, the dichotomy between the molecular and physiological phenotypes led us to conclude that AC induces a long-lasting, transcriptional program that enables individuals who have undergone an initial AC session to achieve faster ReAC. We hypothesized that AC memory involves upstream epigenetic information that predisposes to rapid reacclimation and cytoprotective memory. In a broad sense, the transfer of epigenetic information is associated with chromatin remodeling via molecular and biochemical processes that maintain the chromatin- DNA package in active or silent states. Post-translational modifications (e.g. lysine acetylation, serine phosphorylation) of the N-terminal tails of histone proteins H3 and H4 that protrude from the nucleosome are the most common forms of chromatin remodeling. Such modifications can be controlled by intracellular signaling and are likely to be important in selective epigenetic tagging when environmental stressors are involved. My goal in this study was twofold: To test our hypothesis that “acclimation memory” stems from epigenetic adaptations and to unravel the mechanisms involved. To exploit the DeAC/ReAC model and discover “core gene clusters” and their master regulators that are possibly involved in the generation of “acclimation memory”. To achieve these goals my specific aims are: 1) To study the transcriptional kinetics of genes of interest: hsp70, hsp90 and hsf1 to validate the hypothesis that transcriptional dynamics is a part of the acclimatory memory repertory. 2) To focus on the molecular and biochemical processes that maintain the chromatin-DNA package in an active state, allowing accessibility and binding of transcription factors to DNA recognition sites. 3) To take a genome-wide approach using a whole rat genome array, to screen for alterations in the expression of genes involved with chromatin remodeling and transcriptional regulation following AC, DeAC, and ReAC. All experiments were conducted on male rats, Rattus norvegicus, divided into five experimental groups: 1. Control rats (C) maintained at normothermic conditions (24 ºC). 2. Heat acclimated for 2 days (AC2d) at 34ºC. 3. Heat acclimated for 30 days (AC) at 34ºC. 4. Deacclimated (DeAC) at normothermic conditions for 30 days (after AC for 30 days). 5. Reacclimated for 2 days (ReAC) after DeAC for 30 days. Because faster transcriptional activation in response to stress is a hallmark of successful acclimation, we studied the kinetics of hsp70 and hsp90 transcription following acute heat stress (HS) at 41º C in all experimental groups. Our results here show that both hsp70 and hsp90, similar to the AC group, exhibit faster (than Control) transcriptional dynamics in the DeAC and ReAC groups, with an mRNA peak of hsp70 at 40 min post-HS and mRNA peak of hsp90 immediately after the heat shock (HS) treatment (0 min post-HS). This is the first evidence that the altered ―acclimated‖ HS response is retained after 30 days at normothermic conditions (DeAC), and also exists at ReAC conditions. This finding supports our hypothesis that faster transcriptional dynamics is part of the memory repertory. Interestingly, the DeAC phenotype is characterized by a mismatch between the greater HSP70 basal mRNA/protein levels and the loss of cardioprotection. One explanation for the dichotomy between the phenotypic-physiological and the genotypic response at DeAC group could stem from the decrease in HSP90 levels in this group. In contrast to the elevated protein HSP70 levels, the DeAC hearts did not display high HSP90 protein or mRNA levels. Given that HSP90 is an essential component in the HS response and the duration of its upregulation is critical to cellular integrity we hypothesized that an inverse relationship between HSP70 and HSP90 in DeAC causes the loss of cardioprotection in DeAC group but does not interfere with predisposition to fast restoration of cardioprotection upon ReAC. As HSPs are essential in the AC process and the HS response, we used these genes as a prototype model for proving the concept that epigenetic mechanisms contribute to cytoprotective memory. Histone acetylation, referred to as euchromatin modification, is associated with active transcription. Hence, to substantiate our hypothesis that ReAC involves the activation of the epigenetic machinery, we first measured the levels of acetylated H4 and phosphorylated H3 (Serine10) in the promoter regions of all the experimental treatments. The rationale for measuring H4 acetylation was based on the report of Thomson et al., (2004), which demonstrated the involvement of H4 acetylation in transcription of HSP70 during heat stress in a mammalian species. We screened H3 acetylation/phosphorylation because of the vast body of evidence supporting its involvement in the HS response in non-mammalian species and its role in stress memory. To confirm the binding of transcription factors to the euchromatin, as an indication of the initiation of transcriptional events, we measured the binding of HSF1 to the heat shock element (HSE) on the HSP70 and HSP90 genes (an essential step in HSP transcription). Our results revealed a profile of chromatin remodeling at the HSE of the promoter site of hsp70 and hsp90 and HSF1 binding, which provides a conceptual model of the cytoprotective memory: (i) At the onset of AC, in an ambient-temperature dependent manner, histone H3 phosphorylation by MSK1 kinase switches on HSF1 binding at the HSE of hsp70 and hsp90, with subsequent histone H4 acetylation by a specific acetyl transferase TIP60 at the HSE of both genes, (ii) The acetylation persists throughout DeAC and ReAC, resulting in constitutive HSF1 binding to the hsp70 promoter, irrespective of the transitions in ambient temperatures (from acclimating temperature (34ºC) to 24ºC during DeAC and again returning to 34ºC during ReAC). In contrast, HSF1 binding to hsp90 is temperature dependent. No HSF1 binding occurs in the DeAC state, despite the maintenance of histone H4 acetylation in the HSE in the promoter area of this gene. HSF1-hsp90 binding requires elevated ambient temperatures. The maintenance of elevated histone H4 acetylation in the hsp90 promoter and constitutively elevated HSP70 reserve during DeAC may facilitate the rapid resumption of HSF1 binding to hsp90 HSE, hsp90 transcript translation and the reformation of a cytoprotective milieu upon ReAC. This investigation delineates, for the first time, the whole-genomic response in a mammalian species during the AC process and also at DeAC and ReAC regimes. From our analyses, we 1) outline the dynamics of the genomic response of different sets of genes in all the experimental groups, thus allowing some perception of the global acclimatory molecular strategy underlying heat acclimation and, 2) discuss the likely pathways leading to the ―molecular memory‖ formation conferred by AC. Our data demonstrate that from a total of 27,342 distinct probes, 651 genes showed a significantly changed transcriptional behavior (either upregulated or downregulated > 1.5 - fold) at least in one of the experimental groups. The clustering by a bioinformatics tool revealed five gene clusters characterized by a significantly identical transcriptional