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Roles for lysine acetyltransferases during mammalian hibernation
Author’s Accepted Manuscript Roles for lysine acetyltransferases mammalian hibernation
during
Andrew N. Rouble, Liam J. Hawkins, Kenneth B. Storey www.elsevier.com/locate/jtherbio
PII: DOI: Reference:
S0306-4565(18)30049-4 https://doi.org/10.1016/j.jtherbio.2018.03.013 TB2078
To appear in: Journal of Thermal Biology Received date: 9 February 2018 Revised date: 13 March 2018 Accepted date: 13 March 2018 Cite this article as: Andrew N. Rouble, Liam J. Hawkins and Kenneth B. Storey, Roles for lysine acetyltransferases during mammalian hibernation, Journal of Thermal Biology, https://doi.org/10.1016/j.jtherbio.2018.03.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Roles for lysine acetyltransferases during mammalian hibernation Rouble, Andrew N.1, Hawkins, Liam J.1, and Storey, Kenneth B.1,*
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Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive,
Ottawa, Ontario K1S 5B6, Canada * Corresponding Author: Dr. Kenneth B. Storey Phone: +1 (613) 520-3678; Email: [email protected] Abbreviations: KAT, lysine acetyltransferase; HAT, histone acetyltransferase; H3K9ac, histone H3 lysine 9 acetylation; BAT, brown adipose tissue; WAT, white adipose tissue; Tb, body temperature; HDAC, histone deacetylase; IA, interbout arousal; EC, euthermic in cold room; EN, entrance into torpor; ET, early torpor; LT, late torpor; EA, early arousal; Conflicts of Interest: The authors report no conflicts of interest.
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Abstract The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is a well-known model for studying hibernation. While in a torpid state, these animals globally suppress energy expensive processes, while supporting specialized pathways necessary for survival. Lysine acetyltransferases (KATs) play a crucial role in modulating the expression and activity of a wide-variety of cellular pathways and processes, and therefore, may play a role during hibernation when the cell is shifting to an energy conservative, cytoprotective state. Here we measured protein levels of four KATs (CBP, PCAF, GCN5L2, HAT1), total histone acetyltransferase (HAT) activity, and the levels of acetylation of histone H3 lysine 9 (H3K9ac), in multiple tissues across the torpor-arousal cycle. Our results show a tissue-specific response of KATs, particularly in the adipose tissues where specific KATs (PCAF and GCN5L2), HAT activity, and H3K9ac increased in the metabolically active BAT while HAT1, HAT activity and H3K9ac decreased in WAT. Liver showed significant increases in the KAT PCAF whereas skeletal muscle had decreased CBP and GCN5L2. Both liver and skeletal muscle showed no change in HAT activity and H3K9me3 increased in muscle during torpor. Together, these results suggest KATs may play specialized roles in the different tissues of the ground squirrel to contribute to the hibernator phenotype.
Keywords: Hibernation; Acetyltransferases; Skeletal muscle; Liver; Adipose tissue
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Introduction Some mammals have adapted to survive the winter months, when food resources become limited and temperatures drop below 0°C, by entering hibernation. During hibernation, these animals lower their metabolic rate for weeks to months at a time, with interspersed but brief periods of arousal where their metabolism temporarily returns to euthermic levels (French, 1985). While there is considerable variety in hibernation parameters (e.g. duration, feeding vs. non-feeding, range of heterothermicity) depending on taxa and climatic region (Giroud et al., 2008; Nespolo et al., 2010; Weitten et al., 2016), during torpor, animals such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) display drastic reductions in body temperature (Tb), heart rate, breathing rate, and organ perfusion rates occur (Bullard and Funkhouser, 1962; Carey et al., 2003; Frerichs et al., 1994). Congruent with these physiological changes are alterations to energy metabolism pathways that result in a shift away from carbohydrate metabolism towards lipid catabolism, particularly in the liver, and fatty acid oxidation of lipid reserves in white adipose tissue (WAT) accumulated during the summer months (Carey et al., 2003; Dark, 2005). These lipid deposits are essential in placental hibernators during periods of arousal, when increases in oxygen consumption and fatty acid oxidation fuel thermogenesis from uncoupled respiration in the specialized mitochondria of brown adipose tissue (BAT). Remarkably, these animals endure little to no damage to their tissues, or atrophy of their muscles (Andres-Mateos et al., 2013), despite prolonged periods of inactivity, rapid tissue reperfusion, and exposure to the production of high levels of reactive oxygen species due to heightened oxygen consumption during arousal (Storey, 2010). Interestingly, it is generally accepted that these extraordinary metabolic and protective adaptations are the result of the regulation of gene expression, rather than novel hibernation-specific genes (Carey et al., 2003). Therefore, the mechanisms of genetic regulation employed by hibernators have made these animals particularly interesting subjects in the fields of
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gerontology, obesity, diabetes, and neurodegenerative diseases (Carey et al., 2003; Härtig et al., 2007; Martin, 2008; Storey, 2010; Wu and Storey, 2016). The molecular regulation of hibernation is in large part dependent on the reversible posttranslational modification of proteins (Bell and Storey, 2017; Rouble and Storey, 2015; Storey, 2010; Tessier et al., 2017). We have previously investigated the role of reversible protein acetylation in this context, with specific focus on the expression and activity of NAD+-dependent SIRT deacetylases (Rouble and Storey, 2015) and histone deacetylases (HDACs) (Morin and Storey, 2006). The counter-parts to these deacetylase enzymes are lysine acetyltransferases (KATs), which function to add acetyl-moieties to lysine residues. The addition of acetyl-groups to target proteins can alter important molecular parameters, such as DNA-binding affinities, enzymatic activities, protein stability, target specificity, and complex formation (Carrozza et al., 2003; Chen et al., 2001; Spange et al., 2009). KATs also enable the histone-acetylation-dependent epigenetic remodeling of chromatin, thereby controlling the activation of transcription at specific loci (Carrozza et al., 2003; Lee and Workman, 2007; Verdone et al., 2006). Four of the best-characterized KATs are CREB Binding Protein (CBP), P300/CBP-associated factor (PCAF), General Control Of Amino Acid Synthesis Protein 5-Like 2 (GCN5L2), and Histone Acetyltransferase 1 (HAT1). The integral functions of these proteins in processes such as cell death, DNA repair, proliferation, metabolism, and general transcriptional activation (Chen et al., 2001; Spange et al., 2009; Xiong and Guan, 2012). Because of these important roles, these KATs are of great interest to researchers seeking novel approaches to the treatment of various diseases, and similarly, as potential targets for the regulation processes that allow hibernating mammals to undergo metabolic rate depression during torpor. In this study, we explore KATs in the context of a mammalian hibernator, the thirteen-lined ground squirrel. Protein levels of CBP, PCAF, GCN5L2, and HAT1 were measured over the five standardized time points of the torpor-arousal cycle (Carey et al., 2003; Storey, 2010) with a euthermic control in four tissues: liver, skeletal muscle, BAT, and WAT. These tissues were selected due
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to their previously mentioned physiology, metabolism, and resilience during hibernation. Additionally, total histone acetyltransferase (HAT) activities were measured, comparing euthermic animals to those in deep-torpor. Finally, the levels of acetylated histone H3 lysine 9 (H3K9ac), a KAT-targeted histone residue, were measured across the torpor-arousal cycle. The results reinforce previous studies on the role for reversible protein acetylation in the context of mammalian hibernation (Rouble and Storey, 2015).
Results Analysis of KAT protein levels over the torpor-arousal cycle Relative protein levels of select KATs (CBP, PCAF, GCN5L2, and HAT1) were measured in the liver (Fig. 1), skeletal muscle (Fig. 2), BAT (Fig. 3), and WAT (Fig. 4) of the ground squirrel over the six timepoints of the torpor arousal cycle. In liver, protein levels of CBP were significantly reduced to 0.4 ± 0.08 during interbout arousal (IA) as compared to values for animals that were euthermic in the cold room (EC), while levels of PCAF were significantly elevated over controls during four stages: entrance into torpor (EN), early torpor (ET), late torpor (LT) and early arousal (EA) (by 4.7 ± 0.2, 2.2 ± 0.09, 3.0 ± 0.2, and 4.6 ± 0.3-fold, respectively). Protein levels of GCN5L2 were also significantly decreased to 0.6 ± 0.03 during LT when compared to EC, and subsequently increased during EA by 1.4 ± 0.2-fold over controls. No significant fluctuations were observed for protein levels of HAT1 in liver over the torpor-arousal cycle. In skeletal muscle, CBP protein levels were significantly reduced during LT to 0.3 ± 0.06 of the corresponding EC values, while levels of GCN5L2 were also significantly lowered during EN, LT, EA and IA (to 0.7 ± 0.03, 0.6 ± 0.03, 0.7 ± 0.1, and 0.7 ± 0.06 of EC, respectively), but not during ET. Protein levels of PCAF and HAT1 did not change significantly over torpor-arousal.
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In BAT, protein levels of PCAF were significantly elevated during EN by 1.9 ± 0.1-fold over EC, while levels of GCN5L2 were also elevated during LT by 1.4 ± 0.04-fold before significantly decreasing during IA to 0.7 ± 0.01 of EC. Protein levels of HAT1 were also significantly reduced during ET and IA to 0.6 ± 0.02 and 0.4 ± 0.05 of EC values, respectively. In contrast, levels of CBP did not fluctuate significantly over the course of the torpor-arousal cycle. In WAT, protein levels of HAT1 decreased significantly during LT to 0.5 ± 0.04 of the EC value, whereas levels of CBP, PCAF and GCN5L2 did not change significantly at any of the sampled time points.
Analysis of total HAT activity Total relative HAT activity was measured in the total soluble protein fraction of liver, skeletal muscle, BAT, and WAT, comparing EC and LT stages (Fig. 5). In liver and skeletal muscle, relative HAT activity did not change significantly during LT as compared to EC control (Liver EC – 2869 ± 743 ng/h/mg, LT – 2377 ± 297 ng/h/mg; Muscle EC – 749 ± 39 ng/h/mg, LT – 808 ± 105 ng/h/mg). In contrast, relative HAT activity in BAT was significantly elevated during LT by 2.0 ± 0.2-fold as compared to EC (EC – 2407 ± 581 ng/h/mg, LT – 4754 ± 497 ng/h/mg), whereas total KAT activity in WAT was significantly reduced during LT to 0.5 ± 0.07 of the EC value (EC – 96 ± 14 ng/h/mg, LT – 46 ± 7 ng/h/mg).
Analysis of acetylation status of histone H3 lysine 9 during hibernation Relative protein levels of histone H3 acetylated at lysine 9 (H3K9ac) (Fig. 6) were measured in ground squirrel liver, muscle, BAT and WAT, over the six sampling-points of the torpor-arousal cycle. Relative amounts of H3K9ac protein increased significantly in BAT during ET and LT (by 2.4 ± 0.2 and 2.2 ± 0.2-fold, respectively) and in muscle during ET (by 1.8 ± 0.1-fold) as compared to the respective EC controls. In WAT, H3K9 levels decreased significantly during ET to 0.4 ± 0.02 before increasing by 2.2 ± 0.2-fold during IA, as compared to EC. In liver, no significant fluctuations in H3K9 levels were observed.
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Discussion KATs have been implicated in the regulation of a wide variety of cellular processes through their modification of histones and direct action in transcriptional complexes (Chan and La Thangue, 2001; Nagy and Tora, 2007; Roth et al., 2001). Since transcription is tightly controlled in hibernating mammals, it may be the case that KATs such as CBP, PCAF, GNC5L2, and HAT1 are also regulated or involved in the regulation of this energy intensive process. Our previous studies have identified possible roles for HDACs and SIRT-deacetylases in metabolic rate depression and cellular protective pathways during hibernation (Morin and Storey, 2006; Rouble and Storey, 2015). However, given that KATs have never previously been characterized in hibernators, their regulation in the context of this form of metabolic suppression has been unknown, representing a major gap in knowledge within the field of hibernation research. With the goal of filling this gap, this study has attempted to provide an initial characterization of KAT involvement in hibernation, and succeeds in providing evidence to suggest a role for these enzymes within this context.
Hibernation and KAT protein levels Our results show that, of the four KATs considered, these enzymes are differentially expressed in a tissue-specific manner at various points during hibernation, but do not exhibit an overall pattern of universal protein suppression or activation (Fig. 1-4). This observation would suggest that, in these animals, each of the studied factors likely serves some distinct role(s) at different times, and in different tissues, throughout hibernation. For example, PCAF protein expression was strongly enhanced in liver from EN through to EA (returning to euthermic levels during the interbout period), while this same pattern was not observed in any other tissue. This suggests that the increased expression of PCAF serves a specific role in liver during torpor that does not appear to be required in other tissues. Interestingly,
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under conditions that inhibit glycolysis, PCAF is known to acetylate and reduce the activity of pyruvate kinase (Lv et al., 2011), a major glycolytic enzyme that is also suppressed by RPP during hibernation (Storey and Storey, 2010). The increased expression of PCAF in liver throughout hibernation may therefore contribute to the inhibition of certain metabolic processes such as glycolysis, in a manner analogous to the increased activities of specific kinases/phosphatases that also occur during this time in the liver. Protein levels of CBP also demonstrated a tissue-specific response, most notably being suppressed during late torpor only in muscle tissue. Given CBP’s role as a diverse transcriptional coactivator (Chan and La Thangue, 2001; Kalkhoven, 2004), this fluctuation may serve an important purpose in the regulation of the metabolic suppression in hibernator muscle. This is because its presence and acetyltransferase activity in certain transcriptional complexes is responsible and necessary for the regulation of many different genes; therefore, a decrease in CBP expression could contribute to the widespread transcriptional suppression that is characteristic of the torpid state. In fact, this proposed function would also complement current evidence that suggests an epigenetic contribution to global transcriptional arrest. In hibernator muscle, the acetylation of histone H3 at lysine residue 23 (H3K23) is known to be significantly reduced during torpor, which is consistent with the notion of suppressed transcriptional activity at loci associated with this histone modification (Morin and Storey, 2006). This change is also correlated with an increase in the expression levels of several HDACs which target this residue, in addition to levels of total HDAC activity (Morin and Storey, 2006). CBP also targets this residue as a substrate for its acetyltransferase activity (Henry et al., 2013). Thus, the observed decrease in CBP expression during late torpor may compliment the increase in HDAC activity to promote the deacetylation of certain histone residues, and thereby promote transcriptional suppression. This may also be true for GCN5L2, the protein levels of which were also significantly reduced during late torpor in muscle. GCN5L2 targets H3K23 for acetylation (Grant et al., 1999), so downregulation of GCN5L2 may contribute to the reduced acetylation of histone H3. Like CBP, GCN5L2 is also a major
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transcriptional co-activator in a non-epigenetic sense (Nagy and Tora, 2007), so its decreased expression in muscle and liver may also generally reflect the global suppression of transcription during torpor. In contrast, the enhanced expression of GCN5L2 at the same time in BAT could suggest an increase in the transcriptional activation of GCN5L2 target-genes, which could be functional in this tissue given its potential role in regulating thermogenesis when Tb drops below acceptable limits during torpor(Boyer and Barnes, 1999), or to initiate arousal (Nizielski et al., 1989). While the actual functions of these and other observed changes in KAT protein expression cannot be conclusively determined by the current study, the fact that such changes do occur is evidence to suggest that these enzymes likely serve roles in the regulation of the various processes implicated during hibernation.
Histone acetyltransferase activity through the torpor-arousal cycle To further characterize the possible function of KATs in the context of the hibernator, total HAT activity (HAT enzymatic activity is carried out by KATs in the four tissues was compared between euthermic (EC) and late torpor (LT) stages (Fig. 5). While no change in HAT activity occurred in liver or muscle between these stages, the adipose tissues demonstrated significant fluctuations in HAT activity. In BAT, HAT activity levels doubled during torpor as compared to control, thereby further supporting a role for increased HAT function in this tissue during hypometabolism. As mentioned, increased HAT activity in BAT might reflect the need for this tissue to maintain the expression of certain pathways involved in the thermoregulatory response, perhaps via acetylation-induced increases in the transcriptional activation of specific genes. In contrast, HAT activity in WAT was reduced by half during torpor, suggesting that a reduction in the function of these enzymes is required in this tissue during metabolic depression. Interestingly, the activity fluctuations in both tissues seem to correlate with some of the observed protein data – in BAT, GCN5L2 levels increase with total HAT activity during torpor, while levels of HAT1 decrease with total activity in WAT, possibly supporting the notion that these
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increases/decreases in protein levels make an actual contribution to the total measurable acetyltransferase activity within the tissues. However, given that these measurements only accounted for total HAT activity – and not the activity of specific KATs – no conclusions can be made regarding the relationship between relative changes in the protein expression of one or two enzymes, and total HAT activity. This concept also applies to the lack of change in activity observed in muscle and liver – although protein fluctuations occurred in both tissues, observable changes in total activity will not necessarily follow, even if the activities of specific KATs do change. Regardless of the specificity of the measurements, however, the current data support a role for overall enhanced HAT activity in BAT and decreased HAT activity in WAT during torpor, thereby providing further evidence to implicate these enzymes in the regulation of hibernation.
Hibernation responsive acetyl-histone levels Transcriptional control through KAT-mediated acetylation of histone residues has been shown to be integral to the activation of countless genes and the regulation of crucial cellular processes (Carrozza et al., 2003; Jenuwein and Allis, 2001; Verdone et al., 2006). Thus, to more fully characterize KATs in the context of hibernation, we measured the acetylation levels of KAT-targeted histone H3 lysine 9 (H3K9ac) across the torpor-arousal cycle (Fig. 6). Acetylation of H3K9 is generally associated with active transcription, and multiple KATs measured in this study (PCAF, GCN5L2 and CBP) target this residue (Karmodiya et al., 2012). Similar to the measured KAT protein levels and HAT activities, protein levels of H3K9ac showed tissue-specific changes, suggesting that transcriptional activation/deactivation of certain processes by differential histone acetylation may occur at various points throughout the torpor-arousal cycle. Notably, H3K9ac was significantly elevated in BAT during torpor, which is consistent with the transcriptional activation of gene programs regulated by this residue being a part of this tissue’s response to torpor. In fact, Evidence exists to suggest that the regulation of uncoupling
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protein-1 (UCP1, the main protein responsible for uncoupled respiration and non-shivering thermogenesis in the mitochondria of BAT) is controlled by histone H3 acetylation, whereby decreased levels of the modification are associated with reductions in ucp1 gene expression (Kiskinis et al., 2007). Given that UCP1 expression is absolutely integral to the thermoregulatory function of BAT (Golozoubova et al., 2001), and is therefore indispensable for the survival of the hibernating mammal, the transcriptional activation of the ucp1 gene by H3K9 acetylation in BAT during torpor would be unsurprising. Similarly, the significant fluctuation in H3K9 acetylation between early torpor and interbout arousal in WAT may reflect general changes in transcriptional activity that occur over this period (i.e. suppression during the initial metabolic decline, followed by strong reactivation during arousal). Interestingly, the results in BAT correlate with the observed increases in HAT activity and GCN5L2 protein levels that also occur in BAT at this time, possibly reflecting the expected change in downstream substrate acetylation that should occur with fluctuations in HAT activity/expression. While the exact functions of the observed changes in H3K9 acetylation remain unknown, the current data support the idea that epigenetic mechanisms likely contribute to the adaptation involved in the torporarousal cycle.
Conclusion This study serves as the first known investigation to provide significant evidence to suggest that the differential expression of KATs may be characteristic of the hibernation phenotype. Indeed, the results discussed herein identify fluctuations in the protein levels of four of the best-studied KATs in the literature, changes in HAT activity, and differential acetylation of a downstream KAT histone target, at various points throughout the torpor-arousal cycle and in a tissue-specific manner. Some of these changes also appear to correlate. For example, during torpor in BAT, the increased expression of the major transcriptional co-activator GCN5L2 occurs concurrently with increases in HAT activity and H3K9
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acetylation, all of which are changes that point towards enhanced transcriptional activation via KATmediated regulation. While specific impacts on the hibernation phenotype by these proteins will need to be explored further, these data likely represent further examples of the widespread function of reversible protein acetylation in the regulation of diverse cellular processes, and suggests its importance to hibernator biology.
Materials and Methods Animal care and treatment Animal experiments were performed as previously described (McMullen and Hallenbeck, 2010; Rouble et al., 2013) by Dr. J.M. Hallenbeck and were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke (NIH; animal protocol no. ASP 1223– 05). Ictidomys tridecemlineatus were used in this study and are small mammalian winter hibernators that enter bouts of torpor lasting days to weeks with periods of rewarming and arousal that can last ~24 hours. Animals were sacrificed throughout the torpor-arousal cycle, and liver, skeletal muscle, BAT, and WAT were quickly excised and frozen in liquid nitrogen. Animals were sampled at the following time points: (1) euthermic in the cold room (EC) maintained at 4°C, as previously described (McMullen and Hallenbeck, 2010) – these animals had not entered torpor for at least 72 h and had Tb of 36-37°C. (2) Entrance into torpor (EN) – these animals have shown a decline in Tb (18-31°C) and have begun to enter torpor. (3) Early torpor (ET) – animals that are in torpor for 24 h with a constant Tb of 5-8°C. (4) Late torpor (LT) – these animals had been in torpor for at least 5 days with a constant Tb of 5-8°C. (5) Early arousal (EA) – these animals show a rising Tb and are sampled when Tb was 9-12°C. (6) Interbout arousal (IA) – these animals have arisen from torpor and their Tb has return to euthermic levels (~37°C) for ~18 h. Tissues were transported on dry ice to Carleton University and stored at -80°C until use.
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Protein extraction and immunoblotting Total protein extraction and immunoblotting was performed as previously described (Rouble et al., 2013). Membranes were blocked with milk (2.5-5%, 20-30 min) or polyvinyl alcohol (1mg/mL, 30-70 kDa PVA, 45-60 sec) in tris-buffered saline with Tween-20 (TBST). Targets were probed with antibodies for HAT1 (Genetex, GTX110643), GCN5L2 (Cell Signaling, #3305), PCAF (Cell Signaling, #3378), CBP (Cell Signaling, #7389), or H3K9ac (Cell Signaling, #9649) (1:1000-12000 v/v in TBST) overnight at 4°C. Membranes were then probed with HRP-conjugated anti-rabbit secondary antibodies (1:1500-8000 v/v in TBST, 30-60 min) at room temperature and visualized with chemiluminescence using the ChemiGenius Bio Imaging System (Syngene, Frederick, MD, USA). Membranes were then stained with Coomassie blue (0.25% w/v Coomassie brilliant blue, 7.5% v/v acetic acid, 50% methanol) for loading standardization.
Lysine acetyltransferase activity assay Total histone acetyltransferase (HAT) activity during EC and LT was assayed in liver, skeletal muscle, BAT, and WAT total protein extracts using the EpiQuick HAT Activity/Inhibition Assay Kit (Epigentek, P-4003) as per the manufacturer’s instructions. Samples were prepared as above with the exception that extracts were not mixed with 2X SDS loading buffer. Briefly, in each assay well, 50 µL of 1:50 HAT substrate (supplied by the manufacturer) was incubated at room temperature for 45 min, which was then aspirated and each well was washed with 150 µL of wash buffer (supplied by the manufacturer) three times. Then, added to each well was 2 µL of protein extracts, 26 µL of HAT assay buffer (supplied by the manufacturer), and 2 µL of acetyl CoA (1:20 v/v from 30 mM stock in HAT assay buffer), which were then incubated for 60 min at 37°C. The wells were then washed three times as above and 50 µL of capture antibody (supplied by the manufacturer) was added and incubated for 60 min at room temperature on an orbital shaker. The wells were wash four times, and 50 µL of detection antibody (1:1000, supplied by the manufacturer) was added and incubated for 30 min at room 13
temperature on an orbital shaker. Each well was then washed five times and 100 µL of developer solution was added to each well and incubated for 10 min at room temperature on an orbital shaker in the dark. 50 µL of stop solution was then added to each well and the absorbance of each well was read at 450 nm using a Powerwave HT spectrophotometer (BioTek). Three wells with additional assay buffer instead of protein extracts were run during the assay to act as negative controls as per the manufacturer’s instructions.
Quantification and statistics Band densities on chemiluminescent immunoblots were quantified using GeneTools (Syngene, Frederick, MD). Band densities were standardized against the summed intensity of Coomassie stained protein bands in the same lane and then normalized to their respective EC condition. Data are expressed as mean ± SEM, n = 4. Statistical analysis of the data was performed by a one-way ANOVA with a Dunnett’s post-hoc test (p < 0.05) to correct for multiple comparisons using SigmaPlot 12 statistical package software (Systat Software Inc., San Jose, CA, USA). HAT activity assays were corrected using negative control wells, data are expressed as mean ± SEM, n = 4 and normalized to the EC samples. Statistical analysis of HAT activity assay results was performed by Student’s t-tests (p < 0.05).
Acknowledgments The authors thank Dr. J.M. Hallenbeck and Dr. D.C. McMullen (NINDS, NIH, Bethesda) for providing the tissue samples for this study and Jan Storey for assistance in the editing of the manuscript.
Funding
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This work was supported by a Discovery grant (Grant # 6793) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. ANR held a NSERC CGSM Scholarship, and KBS holds the Canada Research Chair in Molecular Physiology.
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Nespolo, R.F., Verdugo, C., Cortés, P.A., Bacigalupe, L.D., 2010. Bioenergetics of torpor in the microbiotherid marsupial, monito del monte (Dromiciops gliroides): the role of temperature and food availability. J. Comp. Physiol. B. 180, 767–73. https://doi.org/10.1007/s00360-010-0449-y Nizielski, S.E., Billington, C.J., Levine, a S., 1989. Brown fat GDP binding and circulating metabolites during hibernation and arousal. Am. J. Physiol. 257, R536-41. https://doi.org/10.1152/ajpregu.1989.257.3.R536 Roth, S.Y., Denu, J.M., Allis, C.D., 2001. Histone acetyltransferases. Annu. Rev. Biochem. 70, 81–120. https://doi.org/10.1146/annurev.biochem.70.1.81 Rouble, A.N., Hefler, J., Mamady, H., Storey, K.B., Tessier, S.N., 2013. Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation. PeerJ 1, e29. https://doi.org/10.7717/peerj.29 Rouble, A.N., Storey, K.B., 2015. Characterization of the SIRT family of NAD+-dependent protein deacetylases in the context of a mammalian model of hibernation, the thirteen-lined ground squirrel. Cryobiology 71, 334–43. https://doi.org/10.1016/j.cryobiol.2015.08.009 Spange, S., Wagner, T., Heinzel, T., Krämer, O.H., 2009. Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int. J. Biochem. Cell Biol. 41, 185–98. https://doi.org/10.1016/j.biocel.2008.08.027 Storey, K.B., 2010. Out cold: biochemical regulation of mammalian hibernation - a mini-review. Gerontology 56, 220–30. https://doi.org/10.1159/000228829 Storey, K.B., Storey, J.M., 2010. Metabolic rate depression: the biochemistry of mammalian hibernation. Adv. Clin. Chem. 52, 77–108. https://doi.org/10.1016/S0065-2423(10)52003-1 Tessier, S.N., Luu, B.E., Smith, J.C., Storey, K.B., 2017. The role of global histone post-translational modifications during mammalian hibernation. Cryobiology 75, 28–36. https://doi.org/10.1016/j.cryobiol.2017.02.008 Verdone, L., Agricola, E., Caserta, M., Di Mauro, E., 2006. Histone acetylation in gene regulation. Brief. Funct. Genomic. Proteomic. 5, 209–21. https://doi.org/10.1093/bfgp/ell028 Weitten, M., Oudart, H., Habold, C., 2016. Maintenance of a fully functional digestive system during hibernation in the European hamster, a food-storing hibernator. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 193, 45–51. https://doi.org/10.1016/j.cbpa.2016.01.006 Wu, C.-W., Storey, K.B., 2016. Life in the cold: links between mammalian hibernation and longevity. Biomol. Concepts 7, 41–52. https://doi.org/10.1515/bmc-2015-0032 Xiong, Y., Guan, K.-L., 2012. Mechanistic insights into the regulation of metabolic enzymes by acetylation. J. Cell Biol. 198, 155–64. https://doi.org/10.1083/jcb.201202056
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Figure Legends Figure 1. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in liver of I. tridecemlineatus over the torpor-arousal cycle. Representative protein bands are shown for selected sampling points (labelled to the left and right of the blots). Histogram shows mean standardized band densities (±SEM, n=4). Protein bands were standardized against the summed intensity of a group of Coomassie-stained protein bands from the same sample lane. Data were analyzed using a one-way ANOVA with a post hoc Dunnett’s test. The symbol * indicates significant difference from the respective EC control, p < 0.05. Figure 2. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in skeletal muscle of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 3. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in brown adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 4. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in white adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 5. Total relative HAT activity in liver, skeletal muscle, brown adipose tissue and white adipose tissue of I. tridecemlineatus comparing euthermic control (EC) and late torpor (LT) points of the torporarousal cycle. Histograms show means ± SEM, n = 4. Data were analyzed using the Student’s t-test. The symbol * indicates significant difference from the respective EC control, p < 0.05. Figure 6. Relative protein expression of histone H3 acetylated at lysine 9 (H3K9ac) in liver, skeletal muscle, brown adipose tissue and white adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1.
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Graphical abstract
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Highlights
There is tissue specific expression of lysine acetyltransferases during torpor Increased levels of PCAF seen in liver throughout torpor cycle Histone acetylation increases in metabolically active brown adipose tissue Lysine acetylation plays tissue specific role in conveying hibernator phenotype
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Andrew N. Rouble, Liam J. Hawkins, Kenneth B. Storey www.elsevier.com/locate/jtherbio
PII: DOI: Reference:
S0306-4565(18)30049-4 https://doi.org/10.1016/j.jtherbio.2018.03.013 TB2078
To appear in: Journal of Thermal Biology Received date: 9 February 2018 Revised date: 13 March 2018 Accepted date: 13 March 2018 Cite this article as: Andrew N. Rouble, Liam J. Hawkins and Kenneth B. Storey, Roles for lysine acetyltransferases during mammalian hibernation, Journal of Thermal Biology, https://doi.org/10.1016/j.jtherbio.2018.03.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Roles for lysine acetyltransferases during mammalian hibernation Rouble, Andrew N.1, Hawkins, Liam J.1, and Storey, Kenneth B.1,*
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Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive,
Ottawa, Ontario K1S 5B6, Canada * Corresponding Author: Dr. Kenneth B. Storey Phone: +1 (613) 520-3678; Email: [email protected] Abbreviations: KAT, lysine acetyltransferase; HAT, histone acetyltransferase; H3K9ac, histone H3 lysine 9 acetylation; BAT, brown adipose tissue; WAT, white adipose tissue; Tb, body temperature; HDAC, histone deacetylase; IA, interbout arousal; EC, euthermic in cold room; EN, entrance into torpor; ET, early torpor; LT, late torpor; EA, early arousal; Conflicts of Interest: The authors report no conflicts of interest.
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Abstract The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is a well-known model for studying hibernation. While in a torpid state, these animals globally suppress energy expensive processes, while supporting specialized pathways necessary for survival. Lysine acetyltransferases (KATs) play a crucial role in modulating the expression and activity of a wide-variety of cellular pathways and processes, and therefore, may play a role during hibernation when the cell is shifting to an energy conservative, cytoprotective state. Here we measured protein levels of four KATs (CBP, PCAF, GCN5L2, HAT1), total histone acetyltransferase (HAT) activity, and the levels of acetylation of histone H3 lysine 9 (H3K9ac), in multiple tissues across the torpor-arousal cycle. Our results show a tissue-specific response of KATs, particularly in the adipose tissues where specific KATs (PCAF and GCN5L2), HAT activity, and H3K9ac increased in the metabolically active BAT while HAT1, HAT activity and H3K9ac decreased in WAT. Liver showed significant increases in the KAT PCAF whereas skeletal muscle had decreased CBP and GCN5L2. Both liver and skeletal muscle showed no change in HAT activity and H3K9me3 increased in muscle during torpor. Together, these results suggest KATs may play specialized roles in the different tissues of the ground squirrel to contribute to the hibernator phenotype.
Keywords: Hibernation; Acetyltransferases; Skeletal muscle; Liver; Adipose tissue
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Introduction Some mammals have adapted to survive the winter months, when food resources become limited and temperatures drop below 0°C, by entering hibernation. During hibernation, these animals lower their metabolic rate for weeks to months at a time, with interspersed but brief periods of arousal where their metabolism temporarily returns to euthermic levels (French, 1985). While there is considerable variety in hibernation parameters (e.g. duration, feeding vs. non-feeding, range of heterothermicity) depending on taxa and climatic region (Giroud et al., 2008; Nespolo et al., 2010; Weitten et al., 2016), during torpor, animals such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) display drastic reductions in body temperature (Tb), heart rate, breathing rate, and organ perfusion rates occur (Bullard and Funkhouser, 1962; Carey et al., 2003; Frerichs et al., 1994). Congruent with these physiological changes are alterations to energy metabolism pathways that result in a shift away from carbohydrate metabolism towards lipid catabolism, particularly in the liver, and fatty acid oxidation of lipid reserves in white adipose tissue (WAT) accumulated during the summer months (Carey et al., 2003; Dark, 2005). These lipid deposits are essential in placental hibernators during periods of arousal, when increases in oxygen consumption and fatty acid oxidation fuel thermogenesis from uncoupled respiration in the specialized mitochondria of brown adipose tissue (BAT). Remarkably, these animals endure little to no damage to their tissues, or atrophy of their muscles (Andres-Mateos et al., 2013), despite prolonged periods of inactivity, rapid tissue reperfusion, and exposure to the production of high levels of reactive oxygen species due to heightened oxygen consumption during arousal (Storey, 2010). Interestingly, it is generally accepted that these extraordinary metabolic and protective adaptations are the result of the regulation of gene expression, rather than novel hibernation-specific genes (Carey et al., 2003). Therefore, the mechanisms of genetic regulation employed by hibernators have made these animals particularly interesting subjects in the fields of
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gerontology, obesity, diabetes, and neurodegenerative diseases (Carey et al., 2003; Härtig et al., 2007; Martin, 2008; Storey, 2010; Wu and Storey, 2016). The molecular regulation of hibernation is in large part dependent on the reversible posttranslational modification of proteins (Bell and Storey, 2017; Rouble and Storey, 2015; Storey, 2010; Tessier et al., 2017). We have previously investigated the role of reversible protein acetylation in this context, with specific focus on the expression and activity of NAD+-dependent SIRT deacetylases (Rouble and Storey, 2015) and histone deacetylases (HDACs) (Morin and Storey, 2006). The counter-parts to these deacetylase enzymes are lysine acetyltransferases (KATs), which function to add acetyl-moieties to lysine residues. The addition of acetyl-groups to target proteins can alter important molecular parameters, such as DNA-binding affinities, enzymatic activities, protein stability, target specificity, and complex formation (Carrozza et al., 2003; Chen et al., 2001; Spange et al., 2009). KATs also enable the histone-acetylation-dependent epigenetic remodeling of chromatin, thereby controlling the activation of transcription at specific loci (Carrozza et al., 2003; Lee and Workman, 2007; Verdone et al., 2006). Four of the best-characterized KATs are CREB Binding Protein (CBP), P300/CBP-associated factor (PCAF), General Control Of Amino Acid Synthesis Protein 5-Like 2 (GCN5L2), and Histone Acetyltransferase 1 (HAT1). The integral functions of these proteins in processes such as cell death, DNA repair, proliferation, metabolism, and general transcriptional activation (Chen et al., 2001; Spange et al., 2009; Xiong and Guan, 2012). Because of these important roles, these KATs are of great interest to researchers seeking novel approaches to the treatment of various diseases, and similarly, as potential targets for the regulation processes that allow hibernating mammals to undergo metabolic rate depression during torpor. In this study, we explore KATs in the context of a mammalian hibernator, the thirteen-lined ground squirrel. Protein levels of CBP, PCAF, GCN5L2, and HAT1 were measured over the five standardized time points of the torpor-arousal cycle (Carey et al., 2003; Storey, 2010) with a euthermic control in four tissues: liver, skeletal muscle, BAT, and WAT. These tissues were selected due
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to their previously mentioned physiology, metabolism, and resilience during hibernation. Additionally, total histone acetyltransferase (HAT) activities were measured, comparing euthermic animals to those in deep-torpor. Finally, the levels of acetylated histone H3 lysine 9 (H3K9ac), a KAT-targeted histone residue, were measured across the torpor-arousal cycle. The results reinforce previous studies on the role for reversible protein acetylation in the context of mammalian hibernation (Rouble and Storey, 2015).
Results Analysis of KAT protein levels over the torpor-arousal cycle Relative protein levels of select KATs (CBP, PCAF, GCN5L2, and HAT1) were measured in the liver (Fig. 1), skeletal muscle (Fig. 2), BAT (Fig. 3), and WAT (Fig. 4) of the ground squirrel over the six timepoints of the torpor arousal cycle. In liver, protein levels of CBP were significantly reduced to 0.4 ± 0.08 during interbout arousal (IA) as compared to values for animals that were euthermic in the cold room (EC), while levels of PCAF were significantly elevated over controls during four stages: entrance into torpor (EN), early torpor (ET), late torpor (LT) and early arousal (EA) (by 4.7 ± 0.2, 2.2 ± 0.09, 3.0 ± 0.2, and 4.6 ± 0.3-fold, respectively). Protein levels of GCN5L2 were also significantly decreased to 0.6 ± 0.03 during LT when compared to EC, and subsequently increased during EA by 1.4 ± 0.2-fold over controls. No significant fluctuations were observed for protein levels of HAT1 in liver over the torpor-arousal cycle. In skeletal muscle, CBP protein levels were significantly reduced during LT to 0.3 ± 0.06 of the corresponding EC values, while levels of GCN5L2 were also significantly lowered during EN, LT, EA and IA (to 0.7 ± 0.03, 0.6 ± 0.03, 0.7 ± 0.1, and 0.7 ± 0.06 of EC, respectively), but not during ET. Protein levels of PCAF and HAT1 did not change significantly over torpor-arousal.
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In BAT, protein levels of PCAF were significantly elevated during EN by 1.9 ± 0.1-fold over EC, while levels of GCN5L2 were also elevated during LT by 1.4 ± 0.04-fold before significantly decreasing during IA to 0.7 ± 0.01 of EC. Protein levels of HAT1 were also significantly reduced during ET and IA to 0.6 ± 0.02 and 0.4 ± 0.05 of EC values, respectively. In contrast, levels of CBP did not fluctuate significantly over the course of the torpor-arousal cycle. In WAT, protein levels of HAT1 decreased significantly during LT to 0.5 ± 0.04 of the EC value, whereas levels of CBP, PCAF and GCN5L2 did not change significantly at any of the sampled time points.
Analysis of total HAT activity Total relative HAT activity was measured in the total soluble protein fraction of liver, skeletal muscle, BAT, and WAT, comparing EC and LT stages (Fig. 5). In liver and skeletal muscle, relative HAT activity did not change significantly during LT as compared to EC control (Liver EC – 2869 ± 743 ng/h/mg, LT – 2377 ± 297 ng/h/mg; Muscle EC – 749 ± 39 ng/h/mg, LT – 808 ± 105 ng/h/mg). In contrast, relative HAT activity in BAT was significantly elevated during LT by 2.0 ± 0.2-fold as compared to EC (EC – 2407 ± 581 ng/h/mg, LT – 4754 ± 497 ng/h/mg), whereas total KAT activity in WAT was significantly reduced during LT to 0.5 ± 0.07 of the EC value (EC – 96 ± 14 ng/h/mg, LT – 46 ± 7 ng/h/mg).
Analysis of acetylation status of histone H3 lysine 9 during hibernation Relative protein levels of histone H3 acetylated at lysine 9 (H3K9ac) (Fig. 6) were measured in ground squirrel liver, muscle, BAT and WAT, over the six sampling-points of the torpor-arousal cycle. Relative amounts of H3K9ac protein increased significantly in BAT during ET and LT (by 2.4 ± 0.2 and 2.2 ± 0.2-fold, respectively) and in muscle during ET (by 1.8 ± 0.1-fold) as compared to the respective EC controls. In WAT, H3K9 levels decreased significantly during ET to 0.4 ± 0.02 before increasing by 2.2 ± 0.2-fold during IA, as compared to EC. In liver, no significant fluctuations in H3K9 levels were observed.
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Discussion KATs have been implicated in the regulation of a wide variety of cellular processes through their modification of histones and direct action in transcriptional complexes (Chan and La Thangue, 2001; Nagy and Tora, 2007; Roth et al., 2001). Since transcription is tightly controlled in hibernating mammals, it may be the case that KATs such as CBP, PCAF, GNC5L2, and HAT1 are also regulated or involved in the regulation of this energy intensive process. Our previous studies have identified possible roles for HDACs and SIRT-deacetylases in metabolic rate depression and cellular protective pathways during hibernation (Morin and Storey, 2006; Rouble and Storey, 2015). However, given that KATs have never previously been characterized in hibernators, their regulation in the context of this form of metabolic suppression has been unknown, representing a major gap in knowledge within the field of hibernation research. With the goal of filling this gap, this study has attempted to provide an initial characterization of KAT involvement in hibernation, and succeeds in providing evidence to suggest a role for these enzymes within this context.
Hibernation and KAT protein levels Our results show that, of the four KATs considered, these enzymes are differentially expressed in a tissue-specific manner at various points during hibernation, but do not exhibit an overall pattern of universal protein suppression or activation (Fig. 1-4). This observation would suggest that, in these animals, each of the studied factors likely serves some distinct role(s) at different times, and in different tissues, throughout hibernation. For example, PCAF protein expression was strongly enhanced in liver from EN through to EA (returning to euthermic levels during the interbout period), while this same pattern was not observed in any other tissue. This suggests that the increased expression of PCAF serves a specific role in liver during torpor that does not appear to be required in other tissues. Interestingly,
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under conditions that inhibit glycolysis, PCAF is known to acetylate and reduce the activity of pyruvate kinase (Lv et al., 2011), a major glycolytic enzyme that is also suppressed by RPP during hibernation (Storey and Storey, 2010). The increased expression of PCAF in liver throughout hibernation may therefore contribute to the inhibition of certain metabolic processes such as glycolysis, in a manner analogous to the increased activities of specific kinases/phosphatases that also occur during this time in the liver. Protein levels of CBP also demonstrated a tissue-specific response, most notably being suppressed during late torpor only in muscle tissue. Given CBP’s role as a diverse transcriptional coactivator (Chan and La Thangue, 2001; Kalkhoven, 2004), this fluctuation may serve an important purpose in the regulation of the metabolic suppression in hibernator muscle. This is because its presence and acetyltransferase activity in certain transcriptional complexes is responsible and necessary for the regulation of many different genes; therefore, a decrease in CBP expression could contribute to the widespread transcriptional suppression that is characteristic of the torpid state. In fact, this proposed function would also complement current evidence that suggests an epigenetic contribution to global transcriptional arrest. In hibernator muscle, the acetylation of histone H3 at lysine residue 23 (H3K23) is known to be significantly reduced during torpor, which is consistent with the notion of suppressed transcriptional activity at loci associated with this histone modification (Morin and Storey, 2006). This change is also correlated with an increase in the expression levels of several HDACs which target this residue, in addition to levels of total HDAC activity (Morin and Storey, 2006). CBP also targets this residue as a substrate for its acetyltransferase activity (Henry et al., 2013). Thus, the observed decrease in CBP expression during late torpor may compliment the increase in HDAC activity to promote the deacetylation of certain histone residues, and thereby promote transcriptional suppression. This may also be true for GCN5L2, the protein levels of which were also significantly reduced during late torpor in muscle. GCN5L2 targets H3K23 for acetylation (Grant et al., 1999), so downregulation of GCN5L2 may contribute to the reduced acetylation of histone H3. Like CBP, GCN5L2 is also a major
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transcriptional co-activator in a non-epigenetic sense (Nagy and Tora, 2007), so its decreased expression in muscle and liver may also generally reflect the global suppression of transcription during torpor. In contrast, the enhanced expression of GCN5L2 at the same time in BAT could suggest an increase in the transcriptional activation of GCN5L2 target-genes, which could be functional in this tissue given its potential role in regulating thermogenesis when Tb drops below acceptable limits during torpor(Boyer and Barnes, 1999), or to initiate arousal (Nizielski et al., 1989). While the actual functions of these and other observed changes in KAT protein expression cannot be conclusively determined by the current study, the fact that such changes do occur is evidence to suggest that these enzymes likely serve roles in the regulation of the various processes implicated during hibernation.
Histone acetyltransferase activity through the torpor-arousal cycle To further characterize the possible function of KATs in the context of the hibernator, total HAT activity (HAT enzymatic activity is carried out by KATs in the four tissues was compared between euthermic (EC) and late torpor (LT) stages (Fig. 5). While no change in HAT activity occurred in liver or muscle between these stages, the adipose tissues demonstrated significant fluctuations in HAT activity. In BAT, HAT activity levels doubled during torpor as compared to control, thereby further supporting a role for increased HAT function in this tissue during hypometabolism. As mentioned, increased HAT activity in BAT might reflect the need for this tissue to maintain the expression of certain pathways involved in the thermoregulatory response, perhaps via acetylation-induced increases in the transcriptional activation of specific genes. In contrast, HAT activity in WAT was reduced by half during torpor, suggesting that a reduction in the function of these enzymes is required in this tissue during metabolic depression. Interestingly, the activity fluctuations in both tissues seem to correlate with some of the observed protein data – in BAT, GCN5L2 levels increase with total HAT activity during torpor, while levels of HAT1 decrease with total activity in WAT, possibly supporting the notion that these
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increases/decreases in protein levels make an actual contribution to the total measurable acetyltransferase activity within the tissues. However, given that these measurements only accounted for total HAT activity – and not the activity of specific KATs – no conclusions can be made regarding the relationship between relative changes in the protein expression of one or two enzymes, and total HAT activity. This concept also applies to the lack of change in activity observed in muscle and liver – although protein fluctuations occurred in both tissues, observable changes in total activity will not necessarily follow, even if the activities of specific KATs do change. Regardless of the specificity of the measurements, however, the current data support a role for overall enhanced HAT activity in BAT and decreased HAT activity in WAT during torpor, thereby providing further evidence to implicate these enzymes in the regulation of hibernation.
Hibernation responsive acetyl-histone levels Transcriptional control through KAT-mediated acetylation of histone residues has been shown to be integral to the activation of countless genes and the regulation of crucial cellular processes (Carrozza et al., 2003; Jenuwein and Allis, 2001; Verdone et al., 2006). Thus, to more fully characterize KATs in the context of hibernation, we measured the acetylation levels of KAT-targeted histone H3 lysine 9 (H3K9ac) across the torpor-arousal cycle (Fig. 6). Acetylation of H3K9 is generally associated with active transcription, and multiple KATs measured in this study (PCAF, GCN5L2 and CBP) target this residue (Karmodiya et al., 2012). Similar to the measured KAT protein levels and HAT activities, protein levels of H3K9ac showed tissue-specific changes, suggesting that transcriptional activation/deactivation of certain processes by differential histone acetylation may occur at various points throughout the torpor-arousal cycle. Notably, H3K9ac was significantly elevated in BAT during torpor, which is consistent with the transcriptional activation of gene programs regulated by this residue being a part of this tissue’s response to torpor. In fact, Evidence exists to suggest that the regulation of uncoupling
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protein-1 (UCP1, the main protein responsible for uncoupled respiration and non-shivering thermogenesis in the mitochondria of BAT) is controlled by histone H3 acetylation, whereby decreased levels of the modification are associated with reductions in ucp1 gene expression (Kiskinis et al., 2007). Given that UCP1 expression is absolutely integral to the thermoregulatory function of BAT (Golozoubova et al., 2001), and is therefore indispensable for the survival of the hibernating mammal, the transcriptional activation of the ucp1 gene by H3K9 acetylation in BAT during torpor would be unsurprising. Similarly, the significant fluctuation in H3K9 acetylation between early torpor and interbout arousal in WAT may reflect general changes in transcriptional activity that occur over this period (i.e. suppression during the initial metabolic decline, followed by strong reactivation during arousal). Interestingly, the results in BAT correlate with the observed increases in HAT activity and GCN5L2 protein levels that also occur in BAT at this time, possibly reflecting the expected change in downstream substrate acetylation that should occur with fluctuations in HAT activity/expression. While the exact functions of the observed changes in H3K9 acetylation remain unknown, the current data support the idea that epigenetic mechanisms likely contribute to the adaptation involved in the torporarousal cycle.
Conclusion This study serves as the first known investigation to provide significant evidence to suggest that the differential expression of KATs may be characteristic of the hibernation phenotype. Indeed, the results discussed herein identify fluctuations in the protein levels of four of the best-studied KATs in the literature, changes in HAT activity, and differential acetylation of a downstream KAT histone target, at various points throughout the torpor-arousal cycle and in a tissue-specific manner. Some of these changes also appear to correlate. For example, during torpor in BAT, the increased expression of the major transcriptional co-activator GCN5L2 occurs concurrently with increases in HAT activity and H3K9
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acetylation, all of which are changes that point towards enhanced transcriptional activation via KATmediated regulation. While specific impacts on the hibernation phenotype by these proteins will need to be explored further, these data likely represent further examples of the widespread function of reversible protein acetylation in the regulation of diverse cellular processes, and suggests its importance to hibernator biology.
Materials and Methods Animal care and treatment Animal experiments were performed as previously described (McMullen and Hallenbeck, 2010; Rouble et al., 2013) by Dr. J.M. Hallenbeck and were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke (NIH; animal protocol no. ASP 1223– 05). Ictidomys tridecemlineatus were used in this study and are small mammalian winter hibernators that enter bouts of torpor lasting days to weeks with periods of rewarming and arousal that can last ~24 hours. Animals were sacrificed throughout the torpor-arousal cycle, and liver, skeletal muscle, BAT, and WAT were quickly excised and frozen in liquid nitrogen. Animals were sampled at the following time points: (1) euthermic in the cold room (EC) maintained at 4°C, as previously described (McMullen and Hallenbeck, 2010) – these animals had not entered torpor for at least 72 h and had Tb of 36-37°C. (2) Entrance into torpor (EN) – these animals have shown a decline in Tb (18-31°C) and have begun to enter torpor. (3) Early torpor (ET) – animals that are in torpor for 24 h with a constant Tb of 5-8°C. (4) Late torpor (LT) – these animals had been in torpor for at least 5 days with a constant Tb of 5-8°C. (5) Early arousal (EA) – these animals show a rising Tb and are sampled when Tb was 9-12°C. (6) Interbout arousal (IA) – these animals have arisen from torpor and their Tb has return to euthermic levels (~37°C) for ~18 h. Tissues were transported on dry ice to Carleton University and stored at -80°C until use.
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Protein extraction and immunoblotting Total protein extraction and immunoblotting was performed as previously described (Rouble et al., 2013). Membranes were blocked with milk (2.5-5%, 20-30 min) or polyvinyl alcohol (1mg/mL, 30-70 kDa PVA, 45-60 sec) in tris-buffered saline with Tween-20 (TBST). Targets were probed with antibodies for HAT1 (Genetex, GTX110643), GCN5L2 (Cell Signaling, #3305), PCAF (Cell Signaling, #3378), CBP (Cell Signaling, #7389), or H3K9ac (Cell Signaling, #9649) (1:1000-12000 v/v in TBST) overnight at 4°C. Membranes were then probed with HRP-conjugated anti-rabbit secondary antibodies (1:1500-8000 v/v in TBST, 30-60 min) at room temperature and visualized with chemiluminescence using the ChemiGenius Bio Imaging System (Syngene, Frederick, MD, USA). Membranes were then stained with Coomassie blue (0.25% w/v Coomassie brilliant blue, 7.5% v/v acetic acid, 50% methanol) for loading standardization.
Lysine acetyltransferase activity assay Total histone acetyltransferase (HAT) activity during EC and LT was assayed in liver, skeletal muscle, BAT, and WAT total protein extracts using the EpiQuick HAT Activity/Inhibition Assay Kit (Epigentek, P-4003) as per the manufacturer’s instructions. Samples were prepared as above with the exception that extracts were not mixed with 2X SDS loading buffer. Briefly, in each assay well, 50 µL of 1:50 HAT substrate (supplied by the manufacturer) was incubated at room temperature for 45 min, which was then aspirated and each well was washed with 150 µL of wash buffer (supplied by the manufacturer) three times. Then, added to each well was 2 µL of protein extracts, 26 µL of HAT assay buffer (supplied by the manufacturer), and 2 µL of acetyl CoA (1:20 v/v from 30 mM stock in HAT assay buffer), which were then incubated for 60 min at 37°C. The wells were then washed three times as above and 50 µL of capture antibody (supplied by the manufacturer) was added and incubated for 60 min at room temperature on an orbital shaker. The wells were wash four times, and 50 µL of detection antibody (1:1000, supplied by the manufacturer) was added and incubated for 30 min at room 13
temperature on an orbital shaker. Each well was then washed five times and 100 µL of developer solution was added to each well and incubated for 10 min at room temperature on an orbital shaker in the dark. 50 µL of stop solution was then added to each well and the absorbance of each well was read at 450 nm using a Powerwave HT spectrophotometer (BioTek). Three wells with additional assay buffer instead of protein extracts were run during the assay to act as negative controls as per the manufacturer’s instructions.
Quantification and statistics Band densities on chemiluminescent immunoblots were quantified using GeneTools (Syngene, Frederick, MD). Band densities were standardized against the summed intensity of Coomassie stained protein bands in the same lane and then normalized to their respective EC condition. Data are expressed as mean ± SEM, n = 4. Statistical analysis of the data was performed by a one-way ANOVA with a Dunnett’s post-hoc test (p < 0.05) to correct for multiple comparisons using SigmaPlot 12 statistical package software (Systat Software Inc., San Jose, CA, USA). HAT activity assays were corrected using negative control wells, data are expressed as mean ± SEM, n = 4 and normalized to the EC samples. Statistical analysis of HAT activity assay results was performed by Student’s t-tests (p < 0.05).
Acknowledgments The authors thank Dr. J.M. Hallenbeck and Dr. D.C. McMullen (NINDS, NIH, Bethesda) for providing the tissue samples for this study and Jan Storey for assistance in the editing of the manuscript.
Funding
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This work was supported by a Discovery grant (Grant # 6793) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. ANR held a NSERC CGSM Scholarship, and KBS holds the Canada Research Chair in Molecular Physiology.
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Figure Legends Figure 1. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in liver of I. tridecemlineatus over the torpor-arousal cycle. Representative protein bands are shown for selected sampling points (labelled to the left and right of the blots). Histogram shows mean standardized band densities (±SEM, n=4). Protein bands were standardized against the summed intensity of a group of Coomassie-stained protein bands from the same sample lane. Data were analyzed using a one-way ANOVA with a post hoc Dunnett’s test. The symbol * indicates significant difference from the respective EC control, p < 0.05. Figure 2. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in skeletal muscle of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 3. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in brown adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 4. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in white adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. Figure 5. Total relative HAT activity in liver, skeletal muscle, brown adipose tissue and white adipose tissue of I. tridecemlineatus comparing euthermic control (EC) and late torpor (LT) points of the torporarousal cycle. Histograms show means ± SEM, n = 4. Data were analyzed using the Student’s t-test. The symbol * indicates significant difference from the respective EC control, p < 0.05. Figure 6. Relative protein expression of histone H3 acetylated at lysine 9 (H3K9ac) in liver, skeletal muscle, brown adipose tissue and white adipose tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1.
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Graphical abstract
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Highlights
There is tissue specific expression of lysine acetyltransferases during torpor Increased levels of PCAF seen in liver throughout torpor cycle Histone acetylation increases in metabolically active brown adipose tissue Lysine acetylation plays tissue specific role in conveying hibernator phenotype
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