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Endocrinology Vol. 148, No. 10 4592-4600
Copyright © 2007 by The Endocrine Society

Acetylation of Histone H3 and Adrenergic-Regulated Gene Transcription in Rat Pinealocytes

A. K. Ho, D. M. Price, W. G. Dukewich, N. Steinberg, T. G. Arnason and C. L. Chik

Departments of Physiology (A.K.H., D.M.P., W.G.D., N.S.) and Medicine (T.G.A., C.L.C.), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta Canada T6G 2H7

Address all correspondence and requests for reprints to: A. K. Ho, Department of Physiology, 7-26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: anho{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study we investigated the effect of histone acetylation on the transcription of adrenergic-induced genes in rat pinealocytes. We found that treatment of pinealocytes with trichostatin A (TSA), a histone deacetylase inhibitor, caused hyperacetylation of histone H3 (H3) Lys14 at nanomolar concentrations. Hyperacetylation of H3 was also observed after treatment with scriptaid, a structurally unrelated histone deacetylase inhibitor. The effects of TSA and scriptaid were inhibitory on the adrenergic induction of arylalkylamine-N-acetyltransferase (aa-nat) mRNA, protein, and enzyme activity, and on melatonin production. TSA at higher concentrations also inhibited the adrenergic induction of mapk phosphatase-1 (mkp-1) and inducible cAMP early repressor mRNAs. In contrast, the effect of TSA on the norepinephrine induction of the c-fos mRNA was stimulatory. Moreover, the effect of TSA on adrenergic-induced gene transcription was dependent on the time of its addition; its effect was only observed during the active phase of transcription. Chromatin immunoprecipitation with antibodies against acetylated Lys14 of H3 showed an increase in DNA recovery of the promoter regions of aa-nat, mkp-1, and c-fos after treatment with TSA. Together, our results demonstrate that histone acetylation differentially influences the transcription of adrenergic-induced genes, an enhancing effect for c-fos but inhibitory for aa-nat, mkp-1, and inducible cAMP early repressor. Moreover, both inhibitory and enhancing effects appear to be mediated through specific modification of promoter-bound histones during gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MAMMALIAN pineal gland, the nightly release of norepinephrine (NE) from the sympathetic neurons stimulates both {alpha}1 and ß-adrenergic receptors, resulting in a 100-fold increase in intracellular cAMP levels (1, 2, 3). In turn, the increase in cAMP activates cAMP-dependent protein kinase A (PKA), which translocates to the nucleus and phosphorylates cAMP response element binding (CREB) protein, a transcription factor (4). Phosphorylated CREB binds to cAMP response elements (CREs) in the promoter region of cAMP-regulated genes and causes activation of their transcription (5, 6, 7). This activation of transcription results in a 150-fold increase in the mRNA of arylalkylamine-N-acetyltransferase (aa-nat), which encodes the rhythm-generating enzyme in the production of melatonin (8, 9). In addition to induction of AA-NAT activity, NE stimulation, primarily through PKA activation, also increases the expression of other proteins that include enzymes such as MAPK phosphatase-1 (MKP-1) (10, 11), tryptophan hydroxylase (12) and phosphodiesterase 4B (13), transcription factors such as c-Fos (14), inducible cAMP early repressor (ICER) (15), and Fra-2 (16), as well as transporter proteins such as pep-T (17).

Histones control gene expression by modulating the structure of chromatin, and the accessibility of regulatory DNA sequences to transcriptional activators and repressors (18, 19). Various posttranslational modifications, including acetylation, phosphorylation, and methylation, can occur on histone tails (20). These posttranslational modifications are thought to form a "code," specifying patterns of cellular gene expression (21). The acetylation and deacetylation of lysines in the tails of the core histones are among the most extensively studied aspects of chromatin structure (22). In general, acetylation of histones by histone acetyltransferases (HATs) stimulates gene expression by relaxing chromatin structure, allowing access of transcription factors to DNA; deacetylation of histones by histone deacetylases (HDACs) promotes chromatin condensation and transcriptional repression. However, in PC12 cells, HDAC activity is required for cAMP activation of a subset of CREB target genes (23). Thus, it appears that the expression of certain genes in some cell type may not conform to the general model of HATs mediating activation and HDACs mediating repression of gene transcription.

Induction of PKA-regulated gene transcription involves binding of PKA-phosphorylated CREB to CREs in the promoters of cAMP-regulated genes and the recruitment of the coactivator, CREB binding protein (p300) (24). Because CREB binding protein is a protein with HAT activity (25), and trichostatin A (TSA), an HDAC inhibitor, enhances the activation of CRE reporter genes by cAMP (23), modulation of the acetylation status of histone could have a significant effect on the transcription of NE -> cAMP/PKA-induced aa-nat and other genes in the rat pineal gland (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Our recent finding that histone phosphorylation is one of the early events occurring after NE stimulation also supports a role for histone modification in adrenergic-induced gene transcription in this tissue (26). However, the effect on adrenergic-induced gene transcription of other histone modifications such as acetylation has not been determined. Therefore, in this study we investigated the role of acetylation of histone H3 Lys14 (Ac-H3) in the adrenergic induction of aa-nat and other adrenergic-regulated genes in rat pinealocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
NE was obtained from Sigma Chemical Co. (St. Louis, MO). TSA, scriptaid, and nullscript were obtained from Calbiochem Corp. (San Diego, CA). [3H]Acetyl-coenzyme A (specific activity, 1 mCi/mmol) was from Amersham Biosciences (Piscataway, NJ). [3H]Melatonin was obtained from NEN Life Science Products (Boston, MA). Antibodies against histone H3 (H3) (07-690) and Lys-14-acetylated H3 (07-353) were obtained from Upstate Biochemicals (Lake Placid, NY). Monoclonal antibody against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was obtained from Ambion Inc. (Austin, TX). Polyclonal antibodies for the RIA of melatonin were obtained from CIDTech Co (Mississauga, Ontario, Canada). Polyclonal antibodies against AA-NAT25–200 (3314) were a gift from Dr. D. C. Klein (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD). All other chemicals were of the purest grades available commercially.

Preparation of cultured pinealocytes and drug treatment
This study was reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta (Edmonton, Alberta). Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. Animals were housed in a 12-h light, 12-h dark lighting cycle. Pinealocytes were prepared by papain dissociation of freshly dissected rat pineal glands using a system from Worthington Biochemical Corp. (Lakewood, NJ) according to the manufacturer’s instructions. Cell yield was approximately 7 x 105 cells per gland. Cells were suspended in DMEM containing 10% fetal calf serum, and maintained before the experiment at 37 C for at least 18 h in a mixture of 95% air and 5% CO2. Aliquots of pinealocytes (1 x 105 cells/0.3 ml) were treated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide for the duration indicated. Treated cells were collected by centrifugation (1 min, 8000 g). Cell pellets for Western blot analysis were lysed in 1x sample buffer [20 mM Tris-HCl (pH 6.8) containing 2 mM EDTA, 5 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 5% 2-mercaptoethanol, 10% glycerol, 2% SDS, and 0.002% bromphenol blue] and boiled for 5 min. Cell lysates were stored at –20 C until electrophoresis. Samples for RNA isolation were immediately homogenized in Trizol (Invitrogen Corp., Carlsbad, CA).

Western blot
SDS-PAGE was performed as described previously (27) using 10% acrylamide in the presence of 1 mg/ml sodium dodecyl sulfate (Mini-Protein II gel system; Bio-Rad Laboratories, Hercules, CA). Twenty-five micrograms of protein were loaded per lane. After electrophoresis, gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1 h, 100 V), which were then incubated with a blocking solution [5% dried skim milk in 100 mM Tris (pH 7.5) with 140 mM NaCl and 0.01% Tween 20] for a minimum of 1 h. Blots were then incubated overnight at 4 C with diluted specific antisera as indicated. The specificities of the antibodies were confirmed by preincubation with the respective blocking peptides. After washing three times with the blocking solution, blots were incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad Laboratories) for 1 h at room temperature. Blots were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).

RT-PCR
Total RNA was isolated from cultured pineal cells using Trizol. First-strand cDNA was synthesized using an Omniscript reverse-transcriptase kit (QIAGEN, Inc., Valencia, CA) with an oligo-dT primer. PCR reactions performed and primers used were as previously reported (10, 11).

Chromatin immunoprecipitation (ChIP)
Primers for ChIP assay were selected from genomic DNA sequences upstream of the translation start site for aa-nat, c-fos, icer, and mkp-1. Each amplicon is approximately 300 bp in length and extends from approximately 200–500 bp upstream of the translation start site for each gene. The primer sequences (5'-3') were as follows: aa-nat forward ChIP primer, GGA GAC CTT CCT GTT CTC CTG; aa-nat reverse ChIP primer, TAG GGA GAG CAT GGG CTA AG; mkp-1 forward ChIP primer, CCG ATG ACG TCT TTG CTT TT; mkp-1 reverse ChIP primer, GTC GAT CTT GTG CGG TTT TT; icer forward ChIP primer, CCA CAC TCA GCA TTT CCT CA; icer reverse ChIP primer, CAA AGA AGG AAG CGT TTG GA; c-fos forward ChIP primer, GGG GCG TAG AGT TGA TGA CA; and c-fos reverse ChIP primer, GAA ACC CGA GAA CAT CAT GG. Samples for ChIP were prepared using 2 x 106 pineal cells for each treatment; cells in suspension were treated with NE (3 µM) for 2 h before immunoprecipitation assay. ChIP assays were performed using the EZ-ChIP kit from Upstate Biochemicals according to the manufacturer’s protocol. Appropriate modifications were incorporated for use with suspended rather than adherent cells. PCR on purified DNA brought down by immunoprecipitation was performed using 35 cycles of a program consisting of denaturing at 94 C for 40 sec, annealing at 63 C for 30 sec, and extension at 72 C for 30 sec. Initial denaturing and final extension were 5 min in duration. PCR products were run out on ethidium bromide-stained 1.5% agarose gels following standard procedures.

AA-NAT assay
AA-NAT activity was determined as described previously (28). Briefly, treated pinealocytes were stored frozen in dry ice until homogenization in a reaction mixture of 0.1 M phosphate buffer (pH 6.8), containing 30 nmol [3H]acetyl coenzyme A (specific activity 1 mCi/mmol) and 1 µmol tryptamine hydrochloride in a final volume of 60 µl. The reaction mixture was incubated at 37 C for 1 h. At the end of the incubation period, the reaction was stopped by the addition of 1 ml methylene chloride. After vortexing, the aqueous phase was removed, and the organic phase was washed three times with 0.1 M phosphate buffer (pH 6.8). The organic phase was transferred to a scintillation vial, evaporated to dryness, and the radioactive acetylated product determined by scintillation counting. AA-NAT activity was expressed as nmol/h·105 cells.

Melatonin assay
Briefly, medium melatonin was extracted from 300 µl medium by vortexing with 1 ml methylene chloride. After centrifugation, the organic phase was collected and evaporated to dryness. The residue was reconstituted in 500 µl assay buffer [0.01 M phosphate buffer (pH 6.5) containing 0.1% gelatin]. The recovery of medium melatonin was more than 98%. The extracted melatonin was assayed by a RIA as described previously (28).

Results and statistical analysis
For quantitation of RT-PCR analyses, gel images were acquired with Kodak 1D software on a Kodak 2000R imaging station (Kodak Co., Rochester, NY) (29). For analyses of Western blots, exposed films were scanned, and band densitometry of acquired images was performed with Kodak 1D software. Densitometric values were initially expressed relative to the values of GAPDH or H3 to account for the variability of loading. Using the values relative to GAPDH or H3, a paired t test was used to determine differences between groups, and ANOVA with the Newman-Keuls test was used for comparisons within multiple groups. Because of the variability of the densitometric values between experiments, in some studies the results were expressed as percentage of control or NE response based on the mean ± SEM from at least three independent experiments. For RIAs or radioenzymatic assays, data were presented as the mean ± SEM from three independent experiments. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment with HDAC inhibitors causes hyperacetylation of H3
Treatment of pinealocytes with TSA (100 nM), an HDAC inhibitor, for 2 h caused hyperacetylation of Lys 14 on the 17-kDa H3 protein (Fig. 1AGo). A similar hyperacetylation of H3 was observed when pinealocytes were treated with scriptaid (1 µM), a structurally unrelated HDAC inhibitor. In contrast, the inactive analog nullscript (1 µM) had no effect on the acetylation of H3. Changes in the level of acetylated H3 occurred without changes in the level of total (acetylated and unacetylated) H3. The hyperacetylation of H3 caused by TSA was concentration dependent (0.1–100 nM; Fig. 1BGo) with a significant effect observable at 1 nM. Treatment of pinealocytes with NE (3 µM) for 2 h did not affect the basal level of H3 acetylation or the TSA-mediated hyperacetylation of H3 (Fig. 1CGo).


Figure 1
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  		FIG. 1. Effect of HDAC inhibitors on H3 acetylation. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated with: TSA (10 nM), scriptaid (1 µM), or nullscript (1 µM) for 2 h (A); TSA (0.1–100 nM) for 2 h (B); or TSA (10 nM) in the presence of absence of NE (3 µM) for 2 h (C). Cells were collected by centrifugation, dissolved in 1x sample buffer, and analyzed by Western blotting using an antibody against Ac-H3 and reprobed with an antibody against H3, as described in Materials and Methods. Representative immunoblots from three independent experiments (left) and corresponding densitometric measurements of Ac-H3 presented as fold increase vs. control (Con), n = 3 (right). *, P < 0.05, significantly different from control.

 
Treatment with HDAC inhibitors suppresses the NE-stimulated AA-NAT protein and enzyme activity
Treatment of pinealocytes with TSA sufficient to cause hyperacetylation of H3 Lys14 was effective in inhibiting the NE induction of AA-NAT protein, with an estimated IC50 value of 10 nM (Fig. 2Go, A and B). TSA also inhibited NE-stimulated AA-NAT activity (Fig. 2CGo) and melatonin production (Fig. 2DGo) with a similar potency. Whereas another HDAC inhibitor, scriptaid (1 µM), also significantly reduced NE induction of AA-NAT protein (Fig. 3Go, A and B), AA-NAT activity, and melatonin production (Fig. 3Go, C and D), the inactive analog nullscript (1 µM) had no effect. These results indicate that the histone hyperacetylation mediated by HDAC inhibitors suppresses adrenergic-stimulated AA-NAT expression and melatonin synthesis.


Figure 2
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  		FIG. 2. Concentration response to TSA on NE induction of AA-NAT protein level, AA-NAT activity, and melatonin accumulation. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated for 3 h with NE (3 µM) in the absence or presence of varying concentrations of TSA. Cells were collected by centrifugation, dissolved in 1x sample buffer, and analyzed by Western blotting using an antibody against Ac-H3 or AA-NAT; GAPDH was included to control for loading. A, Representative immunoblots from three independent experiments. B, Corresponding densitometric measurements of AA-NAT protein levels presented as fold increase vs. NE (n = 3). C, Pinealocytes (0.2 x 105 cells/0.3 ml) were treated for 4 h with NE (3 µM) in the absence or presence of varying concentrations of TSA. Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity. D, Media were collected for melatonin production as described in Materials and Methods. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 

Figure 3
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  		FIG. 3. Effect of HDAC inhibitors on NE-induced AA-NAT protein levels, AA-NAT activity, and melatonin accumulation. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated for 3 h with NE (3 µM) in the absence or presence of TSA (10 nM), scriptaid (1 µM), or nullscript (1 µM). Cells were collected by centrifugation, dissolved in 1x sample buffer, and analyzed by Western blotting using an antibody against Ac-H3 or AA-NAT; GAPDH was included to control for loading. A, Representative immunoblots from three independent experiments. B, Corresponding densitometric measurements of AA-NAT presented as fold increase vs. NE (n = 3). C, Pinealocytes (0.2 x 105 cells/0.3 ml) were treated for 4 h with NE (3 µM) in the absence or presence of TSA (10 nM), scriptaid (1 µM), or nullscript (1 µM). Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity. D, Media were collected for melatonin production as described in Materials and Methods. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
HDAC inhibitors reduce NE-stimulated aa-nat expression by suppressing transcription
The aforementioned single time point experiment suggested that hyperacetylation of H3 Lys14 had a negative impact on the adrenergic stimulated aa-natexpression. This could occur through either a delay in the induction of aa-nat or suppression of aa-nat transcription. These alternatives were examined by determining the time profile effect of TSA treatment on NE-stimulated aa-nat mRNA, enzyme activity, and melatonin production. Treatment of pinealocytes with NE (3 µM) caused a time-dependent increase in the level of aa-nat mRNA, which peaked between 3 and 4 h after NE treatment and declined afterwards (Fig. 4Go, A and B). Cotreatment with TSA (10 nM) significantly reduced NE-stimulated aa-nat mRNA levels between 2 and 5 h after NE stimulation (Fig. 4BGo). In parallel with the inhibitory effect of TSA on NE induction of aa-nat mRNA expression, TSA also reduced the NE induction of AA-NAT activity and melatonin production (Fig. 4Go, C and D). These results indicate that the effect of the HDAC inhibitor occurs at a site upstream of aa-nat mRNA synthesis and that the reduction of AA-NAT expression observed is not due to a change in the time profile of the response.


Figure 4
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  		FIG. 4. Time course response to TSA on NE induction of the mRNA levels of aa-nat, AA-NAT activity, and melatonin accumulation. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated with NE (3 µM) in the absence or presence of TSA (10 nM) for the duration indicated. Cells were collected by centrifugation and prepared for RT-PCR or AA-NAT enzymatic assay, and medium was collected for melatonin assay as described in Materials and Methods. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat mRNA levels; the gapdh signal is included to demonstrate the consistency of sample preparation. B, Densitometric measurements of aa-nat mRNA levels presented as fold changes vs. maximal OD value. C, AA-NAT enzymatic activity from the cell pellets. D, Cumulative melatonin production in the media. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
Relative sensitivities of adrenergic-induced genes to treatment with HDAC inhibitors
To determine whether the suppressive effect of HDAC inhibitors on aa-nat transcription reflects a general inhibition of adrenergic-stimulated transcription, we compared the responses to HDAC inhibitor treatment of three additional adrenergic-induced genes, mkp-1, icer, and c-fos, with that of aa-nat. Pinealocytes were cotreated with NE (3 µM) and TSA (10 nM), scriptaid (1 µM), or nullscript (1 µM) for 3 h, and the adrenergic induction of mRNA levels of aa-nat, mkp-1, icer, and c-fos were determined. As shown in Fig. 5Go, TSA and scriptaid inhibited the adrenergic induction of aa-nat expression but had little effect on the mRNA levels of mkp-1or icer. In contrast, these treatments enhanced the adrenergic induction of c-fos mRNA.


Figure 5
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  		FIG. 5. Effect of HDAC inhibitors on the expression of four adrenergic-induced genes. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated for 2 h with NE (3 µM) in the absence or presence of TSA (10 nM), scriptaid (1 µM), or nullscript (1 µM). Cells were collected by centrifugation and prepared for RT-PCR as described in Materials and Methods. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat, mkp-1, icer, and c-fos mRNA levels; the gapdh signal is included to demonstrate the consistency of sample preparation. B, Corresponding histograms of densitometric measurements of mRNA levels presented as fold changes vs. NE. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
The relative sensitivities of these adrenergic-induced genes toward HDAC inhibition were characterized using varying concentrations of TSA. As shown in Fig. 6Go, A and B, whereas the NE-stimulated aa-nat response was significantly reduced by TSA at a concentration of 10 nM or higher, transcription of mkp-1 and icer was only reduced by TSA at 1 µM. In the case of c-fos, treatment with TSA enhanced the NE-stimulated response in a concentration-dependent manner, and the enhancement was significant at 10 nM. These results suggest that the nature of the response and the sensitivity toward histone acetylation are gene specific in the rat pineal gland.


Figure 6
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  		FIG. 6. Concentration response to TSA on the expression of four adrenergic-induced genes. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated for 2 h with NE (3 µM) in the absence or presence of varying concentrations of TSA as indicated. Cells were collected by centrifugation and prepared for RT-PCR as described in Materials and Methods. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat, mkp-1, icer, and c-fos mRNA levels; the gapdh signal is included to demonstrate the consistency of sample preparation. B, Corresponding densitometric measurements of mRNA levels presented as fold changes vs. NE. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
The effects of TSA on adrenergic-induced aa-nat mRNA and enzymatic activity are dependent on the time of exposure to the inhibitor
The previous experiment based on a single time point suggested that inhibition of transcription was probably involved in the suppressive effects of HDAC inhibitors on NE-induced aa-nat expression. To characterize further this suppression, the importance of time of exposure to TSA, relative to NE stimulation, on the suppressive effect of TSA on aa-nat expression was determined. Measurements of the mRNA levels of mkp-1, icer, and c-fos were included for comparison. Pinealocytes were treated with NE (3 µM) for 2 and 4 h with TSA (30 nM) present during the first 2 h (range 0–2) or the last 2 h (range 2–4) of a 2 or 4-h NE treatment, thus maintaining a constant duration of TSA exposure (2 h) for all treatment groups. As shown in Fig. 7Go, A and B, the presence of TSA during the first 2-h NE stimulation significantly reduced the NE induction of aa-nat mRNA. The inhibitory effect of TSA was reversible because washout of TSA allowed the mRNA of aa-nat to recover within 2 h to a level similar to that stimulated by NE alone for 2 or 4 h. Conversely, addition of TSA to pinealocytes previously stimulated with NE for 2 h enhanced the level of aa-nat transcription stimulated by NE (Fig. 7BGo).


Figure 7
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  		FIG. 7. Importance of the time of addition of TSA on the NE-stimulated mRNA level and enzyme activity of AA-NAT. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated with NE (3 µM) for 2 or 4 h in the presence or absence of TSA (10 nM) during the first 2-h (range 0–2) or last 2-h (range 2–4) NE treatment. Cells were collected by centrifugation and prepared for RT-PCR as described in Materials and Methods. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat mRNA levels; the gapdh signal is included to demonstrate the consistency of sample preparation. B, Corresponding densitometric measurements of mRNA levels presented as fold changes vs. NE. Each value represents the mean ± SEM (n = 3). C, Pinealocytes (0.2 x 105 cells/0.3 ml) were cultured for 24 h and treated with NE (3 µM) for 3 or 6 h in the presence or absence of TSA (10 nM) during the first 3-h (range 0–3) or last 3-h (range 3–6) NE treatment. Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
To confirm a lack of inhibitory effect on the expression of aa-nat with delayed addition of TSA, the activity of the enzyme was also measured. To optimize the measurement of AA-NAT activity, TSA (30 nM) was present either during the first 3 h (range 0–3) or the last 3 h (range 3–6) of a 6-h treatment with NE (3 µM). As shown in Fig. 7CGo, cotreatment with TSA during the first 3 h suppressed the NE-stimulated AA-NAT activity when measured 3 h posttreatment. However, once TSA was washed out, continuous stimulation with NE allowed the AA-NAT activity to recover to a level similar to that stimulated by NE in the absence of TSA during the first 3 h. In contrast, addition of TSA during the last 3 h of the 6-h stimulation with NE had no effect on the NE-stimulated AA-NAT activity. These results indicate that the timing of exposure to TSA determines the specific effect of HDAC inhibition on NE-stimulated aa-nat transcription. However, the elevated aa-nat mRNA level that follows delayed addition of TSA does not appear to lead to increased AA-NAT enzyme activity.

The effect of TSA on adrenergic-induced mkp-1, icer, and c-fos mRNAs is also dependent on the time of exposure to the inhibitor
With regards to other adrenergic-stimulated genes, whereas cotreatment with TSA during the initial NE stimulation (0–2 h) reduced the mRNA levels of mkp-1 and icer and enhanced the mRNA level of c-fos, addition of TSA during the last 2 h of the 4-h treatment with NE had no effect on the mRNA levels of these three genes (Fig. 8Go). These results indicate that TSA needs to be present during the initial induction by NE to exert its effect, regardless of inhibition or enhancement, on these three adrenergic-induced genes.


Figure 8
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  		FIG. 8. Importance of the time of addition of TSA on the adrenergic-induced expression of mkp-1, icer, and c-fos. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and treated with NE (3 µM) for 2 or 4 h in the presence or absence of TSA (10 nM) during the first 2-h (range 0–2) or last 2-h (range 2–4) NE treatment. Cells were collected by centrifugation and prepared for RT-PCR as described in Materials and Methods. A, Representative ethidium bromide-stained agarose gels from three experiments showing mkp-1, icer, and c-fos mRNA levels; the gapdh signal is included to demonstrate the consistency of sample preparation. B–D, Corresponding densitometric measurements of mRNA levels presented as fold changes vs. NE. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE. Con, Control.

 
Time-dependent effects of NE on the induction of aa-nat and c-fos expression
A previous study has indicated that activation kinetics is a critical factor that determines the nature of the role that HDACs play in activation of specific CREB target genes (23). In view of the opposite effects of HDAC inhibitors on the transcription of aa-nat and c-fos, the activation kinetics of these two genes were compared. As shown in Fig. 9Go, treatment of pinealocytes with NE (3 µM) caused a rapid increase in the mRNA level of c-fos, with the maximal level observed around 1 h after treatment, and was followed by a gradual decline over the next 4 h. In comparison, aa-nat mRNA showed a gradual increase after NE stimulation, with the maximal level observed around 3 h posttreatment. Interestingly, whereas the aa-nat mRNA level was not detectable in the control cells, the mRNA level of c-fos mRNA was detectable in the control cells.


Figure 9
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  		FIG. 9. Time-dependent effects of NE on the induction of aa-nat and c-fos expression. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and stimulated with NE (3 µM) for the duration indicated. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat and c-fos mRNA levels in pinealocytes treated as indicated; the gapdh signal is included to demonstrate the consistency of sample preparation. B, Relative mRNA levels of aa-nat and c-fos after NE stimulation as determined from densitometric measurements. Maximal OD value was assigned a value of one. Each value represents the mean ± SEM (n = 3). Con, Control.

 
Sensitivities of adrenergic-stimulated genes toward TSA treatment correlate with their increased association with acetylated H3
Given the global changes in H3 acetylation that we observed in pinealocytes after TSA treatment and the selective effects of TSA on different adrenergic-induced genes, it was essential to determine if the TSA-induced histone hyperacetylation was occurring in the proximity of the respective genes. To assay for local changes in H3 acetylation, we designed PCR primers that amplified genomic regions upstream of the aa-nat, mkp-1, c-fos, and icer loci, and used these primers to PCR amplify DNA fragments that were brought down by immunoprecipitation with an antibody specific for Lys14 acetylated H3. The ChIP assay showed a marked increase in DNA recovery from the promoter regions of aa-nat and c-fos, and to a lesser degree mkp-1, in immunoprecipitates prepared from cells treated with TSA (100 nM) compared with untreated controls (Fig. 10Go). The increase in DNA recovery from the promoter regions of icer in immunoprecipitates prepared from cells treated with TSA (100 nM) was not significant. These results demonstrate parallel changes between genes that are sensitive to TSA treatment (both inhibitory or stimulatory) and genes that are associated with histone acetylation. This further supports the interpretation that histone acetylation selectively affects adrenergic-induced genes with different sensitivities.


Figure 10
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  		FIG. 10. Effect of TSA on local increases in histone acetylation in rat pinealocytes. ChIP with an antibody specific for Lys14 acetyl-H3 was performed on pinealocytes (2 x 106 cells per treatment) treated with or without TSA (100 nM) for 2 h, as described in Materials and Methods. Immunoprecipitated genomic DNA was subjected to PCR amplification with primer sets that amplified regions upstream of the aa-nat, mkp-1, icer, and c-fos genes. A, PCR signal generated from Lys14 acetyl-H3 immunoprecipitated DNA (IP) amplified with the respective primer sets for aa-nat, mkp-1, icer, or c-fos. Water was included as template for the negative control (Blank); total rat genomic DNA was included as template for the positive control (genomic DNA). Images are representative of three separate experiments. B, Densitometric measurements of aa-nat, mkp-1, icer, and c-fos signals presented as fold increase over control (Con) in OD value. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Histone modifications are regarded as integral and dynamic components of the machinery responsible for regulating gene expression (19). In particular, histone acetylation can impact transcriptional activity of CREB/CRE-driven genes in other cell types (30). In the rat pineal gland, numerous nocturnally activated and adrenergic-regulated genes are primarily under the regulation of a ß-adrenergic receptor -> PKA mechanism (8, 11, 12, 13, 14, 15, 16, 17). Of the various adrenergic-regulated genes, the best characterized in terms of function and regulation is aa-nat (2, 9). Moreover, the involvement of CREB phosphorylation in the adrenergic induction of aa-nat transcription is also well established (4). Therefore, in this study we determined the effect of Ac-H3 on the transcription of aa-nat and compared it with three other established adrenergic -> CRE-driven genes, namely mkp-1, icer, and c-fos.

Findings from the present study show that, in cultured pineal cells, adrenergic-mediated aa-nat induction is highly sensitive to HDAC inhibition. At nanomolar concentrations, TSA is effective in reducing the NE-stimulated induction of aa-nat mRNA, the enzymatic activity, and protein levels of AA-NAT as well as melatonin production. These effects of TSA are likely related to inhibition of HDAC activity because the potency of this inhibition parallels the hyperacetylation of H3 caused by TSA. Furthermore, scriptaid, a structurally unrelated HDAC inhibitor, is also effective in inhibiting NE-stimulated aa-nat gene expression and melatonin synthesis, whereas nullscript, the inactive analog of scriptaid, has no effect. These findings indicate that HDAC activity is probably required for the adrenergic induction of aa-nat gene expression.

Comparison of the sensitivities among the four adrenergic-induced genes, aa-nat, mkp-1, icer, and c-fos, toward HDAC inhibition reveals a differential response as well as a difference in sensitivity. Whereas the adrenergic-induced transcription of aa-nat is inhibited by TSA at nanomolar concentrations, the transcription of mkp-1 and icer requires µM concentration of TSA before significant inhibition is observed. In contrast, the NE-stimulated transcription of c-fos is enhanced by treatment with TSA. A similar pattern of response is also observed with scriptaid. Together, these results indicate that inhibition of HDAC activity can have distinct effects on the activation of different adrenergic- regulated genes in the rat pinealocyte. Furthermore, HDAC, typically associated with repression (22), appears to contribute to activation of a subset of adrenergic-stimulated genes in the rat pineal gland.

Changes in mRNA levels can be the result of changes in the rate of transcription or alteration of mRNA stability. In the case of the effect of HDAC inhibitors on NE-stimulated mRNA levels, our results show that, regardless of the response, enhancing or inhibiting, TSA treatment is most effective when it is present during the first 2 h of NE stimulation when transcription is most active. In contrast, addition of TSA after 2-h NE stimulation has no effect on the mRNA levels of the respective genes. This suggests that once the mRNA has been synthesized, HDAC inhibition is not effective in causing any changes, indicating that HDAC inhibitors are probably acting at the level of transcription rather than on the stability of the mRNA to produce their effects on the adrenergic-induced genes.

The precise mechanism through which HDAC influences gene transcription in the rat pineal gland is not clear. Possible mechanisms include interference with the process of transcriptional activation such as formation of a preinitiation complex, or attenuating the rate or efficiency of transcription itself. Another possibility is that the target of HDAC is a transcription factor, and acetylation of this factor may alter the transcription rate. However, our results from the ChIP assay using antibodies against acetyl-H3 show an increase in recovery of DNA of the promoter regions of aa-nat, c-fos, and, to a lesser degree, mkp-1 after TSA treatment. This indicates that interaction between acetyl-histone and the promoter regions of the adrenergic genes appears to be an essential step involved in this mechanism. Furthermore, the relative increases in the association of the adrenergic-stimulated genes with acetyl-histone correlate reasonably well with their sensitivities toward treatment with TSA. This lends support to the idea that acetylation of histone is an important step through which adrenergic gene expression is modified by HDAC inhibition. Also noteworthy is the observation that the increase in promoter association with acetyl-histone applies to the four adrenergic-stimulated genes, regardless of the effects of TSA on their transcription, inhibitory for aa-nat, icer, and mkp-1, and enhancement for c-fos. This suggests that association with acetyl-histone alone is not sufficient to determine if this leads to an enhancement or inhibition of transcription.

Although all four genes investigated in the present study are NE-stimulated and CREB target genes in the pineal, they showed differential responses toward HDAC inhibition. The specific mechanism that determines the suppressive effect of HDAC inhibition on the expression of aa-nat, icer, and mkp-1, and an enhancing effect on the expression of c-fos in the pinealocyte is not clear. A previous study with PC12 cells has indicated that activation kinetics and preinitiation complex assembly are two important determinants that govern the role that HDACs play in activation of specific CREB target genes (23). It was suggested that CREB target genes with slower activation kinetics may require HDAC for preinitiation complex formation, whereas genes with faster kinetics, as a result of having the preinitiation complex already assembled under basal condition, may not require HDAC activity for activation. Consistent with this suggestion, we found in the time course study that, in rat pinealocytes, the NE-stimulated c-fos mRNA accumulation, which is enhanced by TSA, has a faster kinetics than that of aa-nat, which is inhibited by TSA. In addition, the level of c-fos mRNA is detectable in the control cells, suggesting that the preinitiation complex for c-fos transcription is probably assembled to a certain degree to allow for the basal transcription of c-fos. In the case of aa-nat, the finding that addition of TSA during the first 2 h of NE stimulation inhibits whereas delayed addition of TSA enhances aa-nat mRNA synthesis is supportive of the requirement of HDAC for the formation of preinitiation complex for this gene upon NE stimulation. However, once the complex has been assembled, as observed 2-h post-NE stimulation, subsequent treatment with TSA has an enhancing effect on aa-nat gene expression, as in the case of c-fos.

In summary, our results indicate that histone acetylation plays a significant role in the transcriptional regulation of adrenergic-regulated genes in the rat pineal gland. The effect of increased Ac-H3 is gene selective, stimulatory for c-fos, but inhibitory for aa-nat, icer, and mkp-1. Because only global changes in acetyl-histone are measured in this study, one could not exclude the contributions of dynamic changes in histone turnover (22) or interactions with other covalent histone modifications to the effects observed in our study. Recently, we have demonstrated that phosphorylation of H3 in the rat pineal gland is under adrenergic control (26). Findings from the present study further add to the importance of the role of histone modifications in regulating gene transcription in this tissue.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 12, 2007

Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; Ac-H3, acetylation of histone H3 Lys14; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, cAMP response element binding; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; H3, histone H3; HAT, histone acetyltransferase; HDAC, histone deacetylase; ICER, inducible cAMP early repressor; MKP-1, MAPK phosphatase-1; NE, norepinephrine; PKA, protein kinase A; TSA, trichostatin A.

Received May 1, 2007.

Accepted for publication June 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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