Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis
© Pelzel et al; licensee BioMed Central Ltd. 2010
Received: 16 November 2009
Accepted: 26 May 2010
Published: 26 May 2010
Silencing of normal gene expression occurs early in the apoptosis of neurons, well before the cell is committed to the death pathway, and has been extensively characterized in injured retinal ganglion cells. The causative mechanism of this widespread change in gene expression is unknown. We investigated whether an epigenetic change in active chromatin, specifically histone H4 deacetylation, was an underlying mechanism of gene silencing in apoptotic retinal ganglion cells (RGCs) following an acute injury to the optic nerve.
Histone deacetylase 3 (HDAC3) translocates to the nuclei of dying cells shortly after lesion of the optic nerve and is associated with an increase in nuclear HDAC activity and widespread histone deacetylation. H4 in promoters of representative genes was rapidly and indiscriminately deacetylated, regardless of the gene examined. As apoptosis progressed, H4 of silenced genes remained deacetylated, while H4 of newly activated genes regained, or even increased, its acetylated state. Inhibition of retinal HDAC activity with trichostatin A (TSA) was able to both preserve the expression of a representative RGC-specific gene and attenuate cell loss in response to optic nerve damage.
These data indicate that histone deacetylation plays a central role in transcriptional dysregulation in dying RGCs. The data also suggests that HDAC3, in particular, may feature heavily in apoptotic gene silencing.
Intrinsic apoptosis in neurons culminates in BAX activation and translocation to the mitochondria, the release of cytochrome c, and the activation of the caspase cascade. BAX translocation marks the committed step of the cell death process . Therefore, investigation of the apoptotic pathway prior to BAX involvement is an important element of developing strategies to intervene in neuronal cell death.
An early event in apoptosis is silencing of normal gene expression. In addition to this, new transcription, required for apoptosis, is activated. This change in transcriptional profile occurs in several models of neurodegeneration, including Huntington's Disease, Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, spinocerebellar ataxia type 3, and the optic neuropathy glaucoma [2–10]. In glaucoma, retinal ganglion cells (RGCs) execute a typical intrinsic apoptotic program. Changes in transcription of several genes in injured RGCs have been shown in experimental glaucoma and after acute injury to the optic nerve. Genes that decrease in expression in RGCs include several that are specifically expressed in these cells, such as Thy1, Brn3b, Nrn1, Fem1c, and Sncg, [3–5, 11–16], as well as several non-cell type specific genes, including BclX l , TrkB, and members of the neurofilament gene family [3, 13, 14, 17]. Of the genes with increased expression, the majority are proapoptotic or stress response genes, such as Bim, cJun, and several Hsp s and caspases [4, 5, 18–20]. This change in the pattern of gene expression in RGCs occurs before detectable cell loss [4, 11, 21] and can also be induced by optic nerve crush (ONC) of Bax knock-out RGCs, indicating that this event occurs early in the apoptotic pathway. Little investigation has been conducted to understand the mechanism underlying the down-regulation of normal gene expression. The global nature of gene silencing in RGCs, however, suggests that epigenetic changes of the chromatin of actively transcribed genes may be an early step in apoptosis.
Post-translational modifications of histones are well known epigenetic changes that regulate chromatin folding, organization, and gene activity . Histone modifications include phosphorylation, methylation, ubiquitination, and/or acetylation of lysine residues principally in the N-terminal tails . While all of these modifications have an effect on the transcriptional activity, acetylation has the most direct effect . Acetylated histones are typically found in transcriptionally active euchromatic chromatin, whereas transcriptionally inactive heterochromatic chromatin is rich in deacetylated histones. Theoretically, deacetylation is thought to lead to a more compact chromatin structure, which limits access of transcription factors . Alternatively, acetyl groups may facilitate the interactions of chromatin with specific transcription factors containing bromodomains, which recognize and bind acetylated amino acids of other proteins, including histone tails . The acetylation and deacetylation of histones is controlled by opposing protein families called histone acetyltransferases (HATs) and histone deacetylases (HDACs).
Here we show that several RGC specific genes, which decrease in expression after ONC, exhibit a decrease in promoter histone acetylation. This deacetylation is accompanied by an increase in both HDAC2 and HDAC3 expression and the translocation of HDAC3 to the nuclei of dying RGCs. Additionally, inhibition of HDAC activity is able to prevent the ONC-mediated silencing of at least one RGC-specific gene and attenuate the level of RGC death. These results represent one of the first documentations of epigenetic changes associated with neuronal cell death and may provide insight into some of the earliest changes occurring in dying RGCs.
Nuclear HDAC activity is increased after ONC
Dose-dependent inhibition of nuclear HDAC activity was also performed with different inhibitors to help evaluate which HDACs were active both before and after ONC. TSA-mediated inhibition of control and crush nuclear extracts from day 5 retinas showed a dose-dependent decrease in HDAC activity with an IC50 ranging from 15 to 22 nM (Figure 1B). A similar loss of activity was observed in extracts treated with the selective class I inhibitor, valproic acid (data not shown). To further refine which class I HDACs may be contributing to the retinal nuclear activity, we repeated this experiment using apicidin, which is selective for HDACs 2 and 3. HDAC activity was also nearly completely inhibited with apicidin, yielding an IC50 ranging from 1.88 to 2.55 nM (Figure 1C). These values agreed with the reported IC50 value for HDAC3 (2.5 nM) using this inhibitor  and suggest that HDACs 2 and 3 contribute the majority of nuclear HDAC activity in the retina.
HDACs 2 and 3 in injured retinas
Because subcellular localization of HDACs is a common mechanism of controlling HDAC activity , we also examined the distribution of HDAC2 and HDAC3 after ONC. In sections from retinas 3 days post ONC (Figure 3B, lower panels), HDAC2 remained localized to nuclei of the GCL. HDAC3 localization in the GCL, however, appeared to change after ONC, with cells showing both cytoplasmic and nuclear localization, as determined by colabeling with DAPI staining (Figure 3B and additional file 1: Localization of HDACs 2 and 3 before and after optic nerve crush). To confirm these findings, western blot analysis was conducted on nuclear and cytoplasmic fractions isolated from control and crush retinas. As shown in Figure 3C, a band at 59 kD, corresponding to HDAC2, was present in the nuclear fractions of both control and ONC retinas. A 49 kD band, corresponding to HDAC3, was present in the cytoplasmic fraction from control retinas, but was both cytoplasmic and nuclear in the experimental crush retinas consistent with the nuclear translocation of this protein after ONC. As a control for the fractionation, the blots were also probed for acetylated histone H4 and GAPDH as nuclear and cytoplasmic controls, respectively.
Histone H4 acetylation in the GCL decreases following ONC
Characterization of HDAC3 translocation and deacetylation of histone H4 in dying cells
Promoter deacetylation is associated with a downregulation of gene expression in injured RGCs
Inhibition of HDAC activity blocks ONC-induced silencing of the Fem1c R3 reporter gene
Although promoter histone deacetylation is associated with silenced genes in RGCs, these experiments do not conclusively demonstrate that this epigenetic change is the controlling mechanism for transcriptional downregulation. To address this, we examined if inhibitors of HDAC activity could block the ONC-mediated downregulation of RGC-specific gene expression. For these experiments, we used Fem1cRosa3 (R3) mice, which contain the βGeo promoter trap reporter in the first intron of the Fem1c gene. Previously, we showed that mice express βGEO in an RGC-specific manner . Additionally, we have observed a 75% decrease in βGEO total protein and enzyme activity , and a 50% decrease in Fem1c transcript levels, by 5 days post ONC (Figure 8A, B). Thus, the R3 reporter allows for the rapid detection and quantification of changes in RGC gene expression.
Inhibition of HDAC activity attenuates cell loss following ONC
Previous studies by our group and others have shown that silencing of normal gene expression is an early event in the apoptotic pathway of neurons, including RGCs [5, 11, 12, 15]. Although microarray studies have carefully documented early gene expression changes in dying neurons, little attention has been given to understanding the causative mechanism leading to these widespread changes. Here we propose that epigenetic changes in active chromatin, specifically histone deacetylation, are part of the underlying mechanisms of apoptotic gene silencing.
We were able to detect an increase in whole retinal nuclear HDAC activity at the earliest time point examined (1 day post ONC), however, it was not significantly higher until day 5. This lag in HDAC activity may reflect that the increase was mainly occurring in the RGCs, which only comprise 1-2% of the cell population in the retina. Therefore, day 5 post ONC may represent a point when a maximum number of RGCs were exhibiting an increase in HDAC activity. Conversely, because this experiment was performed on whole retina extracts and not on RGC-enriched samples, the increase in activity could possibly be due to changes in other cell types within the retina. The immunofluorescent studies examining changes in nuclear histone H4 acetylation, however, suggest that the changes in HDAC activity are likely limited to dying cells in the GCL.
The question that remains, however, is why does there appear to be a progressive increase in the level of nuclear HDAC activity and a similar progressive decrease in histone deacetylation after this initial silencing event? Part of the answer to this may lie in the relative sensitivity of the different assays used to detect changes in transcript abundance, promoter acetylation, HDAC activity, and H4 acetylation levels, but more likely the consequences of deacetylation are two-fold. The early onset of deacetylation may selectively target actively expressed genes, leading to gene silencing. Later, progressive and global deacetylation may be required as the cell continues through the apoptotic pathway, in order to facilitate the condensation of the nuclear chromatin into a heterochromatic state. Chromatin condensation is one of the morphological hallmarks of apoptosis, and has been clearly documented in apoptotic RGCs [34, 35]. Histone tails in condensed heterochromatin are generally hypermethylated and hypoacetylated . HDAC3, in particular, may play a key role in chromatin condensation. Previous studies have shown that deacetylation of histone H3 by HDAC3 initiates chromatin condensation during mitosis by creating a preferred binding site for Aurora B kinase, which then phosphorylates H3 . This modification of the histone code is the first of several events that eventually lead to chromatin condensation.
Although our experiments show that deacetylation of histones may be a central mechanism of transcriptional silencing, consistent with other reports [37, 38], they do not exclude involvement of other chromatin modifications such as the methylation and demethylation of histones. Unlike the association between acetylation status of histones and transcriptional activity, the role of methylation in regulating transcription is more complicated. Trimethylation of lysine 4 in histone H3 (H3K4me3), for example, is associated with transcriptional activity, while trimethylation of H4K20 is associated with silent chromatin [24, 39]. Additionally, HDAC activity is often closely linked to the activity of demethylases, since these enzymes are part of larger protein complexes. In both humans and mice, HDAC2 is often part of the REST complex, which also includes the histone demethylases RBP2 and AOF2 . Similarly, HDAC3 is a component of the SMRT/N-CoR complex, which is able to associate with the histone demethylase JMJD2A . Preliminary RT-PCR results from our laboratory have shown that transcripts for both Ncor1 and Ncor2 (the mouse homologs to SMRT and N-COR), as well as Rcor2 (COREST) and Aof2, are present in the mouse retina (data not shown), indicating that the components for active HDAC3 and HDAC2 complexes are expressed in this tissue.
Modulation of the HAT and HDAC activity balance during neurodegeneration
In healthy cells, HAT and HDAC activities are balanced to regulate transcriptional activity . The disruption of this balance, as demonstrated through the use of HDAC inhibitors, can lead to apoptosis principally in rapidly dividing cells [42, 43]. Disruption of the HAT:HDAC balance also appears to play a role in neurodegenerative diseases, albeit by a mechanism that appears to be different from the lethal imbalances that cause cancer cell death. Rather than decreases, relative increases in HDAC activity contribute to the progression of neuronal apoptosis in several disease models [37, 44, 45]. One of the consequences of this imbalance could be an overall decrease in histone acetylation, which leads to a decrease in gene expression. Studies in which HDAC activity is suppressed by HDAC inhibitors, presumably restoring the HAT:HDAC balance, show that this treatment is able to attenuate neuronal apoptosis. Several groups investigating models of polyglutamine expansion neurodegenerative diseases, such as Huntington's disease, for example, have used HDAC inhibitors to prevent cell loss [38, 46–48]. In these models, the relative increase in HDAC activity has been hypothetically attributed to a decrease in HAT activity resulting from the sequestration and degradation of the acetyltransferase, CREB binding protein (CBP) [38, 46], while HDAC activity levels remain unchanged . This model of neurodegeneration implies that the relative increase in HDAC activity is a passive consequence of the selective loss of CBP. Our data, although consistent with the idea that HDAC activity is relatively increased, suggest that this change is reflective of an active increase in nuclear HDAC levels in dying RGCs, associated with both a modest increase in HDAC gene expression and the translocation of an active protein. In fact, nuclear HAT activity assays show no significant changes in overall acetyltransferase activity in retinas harvested from eyes after ONC (data not shown).
The overall importance of HDAC activity in the process of RGC death can be probed with HDAC inhibitors such as TSA. TSA pretreatment was able to attenuate the down regulation of at least one gene normally expressed in RGCs (Fem1c R3 ). Consistent with the other reports showing a protective effect in models of neurodegeneration, TSA was also able to provide a modest protective effect to RGCs after ONC. The underlying cause for this latter effect is not known and may be due to a variety of factors, such as stabilizing the balance between HAT and HDAC activity or allowing for increased acetylation of factors such as Sp1, which has been shown to be neuroprotective in a model of hypoxic stress . Conversely, it is equally possible that maintaining normal gene expression may have a secondary protective effect to the RGCs. For example, previously we showed that the anti-apoptotic gene BclX was down-regulated after optic nerve crush in rats . Preventing this decrease could result in an increase in RGC resistance to a damaging stimulus by antagonizing the actions of proteins like BAX.
Although several studies have linked an increase in HDAC activity to neuronal death, others have demonstrated that the overexpression of HDACs can be neuroprotective. Chen and Cepko showed that the overexpression of HDAC4 led to increased protein stability of the transcriptional activator HIF1α, which had a protective effect in a model of photoreceptor degeneration . A related study demonstrated that part of the beneficial effect of HDAC4 on HIF1α transcriptional activity was due to HIF1α's increased ability to bind the histone acetyltransferase, p300 , which could be beneficial during neurodegeneration when fewer active HATs are available [37, 44]. In these cases, the protective effect of increased HDAC activity appeared to be restricted to cytoplasmic activity on non-histone substrates.
Irrespective of the principal function of HDAC activity during cell death, the phenomenon of gene silencing is likely going to act as a barrier to regaining normal cell function in neuroprotective strategies. Bax-deficient RGCs, which are completely resistant to apoptosis after ONC, for example, can remain in a genetically silent, heterochromatic state for months following injury (unpublished data). Similarly, inhibition of HDAC activity using TSA, still resulted in some cell atrophy characterized by soma shrinkage. Shrinkage of RGCs after optic nerve damage has been described by others , including in Bax-/- RGCs . This atrophy response may be indicative of the apoptotic volume decrease described in some neuronal cell types , which is considered to be a very early event in the apoptotic program and is regulated by a rapid efflux of intracellular potassium in many neurons, including retinal ganglion cells [56, 57]. The effects we observe in TSA-treated mice suggest that epigenetic changes leading to gene silencing is downstream of the apoptotic volume decrease. Nevertheless, a complete understanding of the early changes in affected ganglion cells remains an important consideration for neuroprotective strategies. Clearly, regaining normal cell function will ultimately require reactivation of normal gene expression, meaning reversal of epigenetic changes associated with silenced genes.
In summary, this study demonstrates that a change in histone acetylation levels, particularly in the promoter regions of silenced genes, may be part of the underlying mechanism of apoptotic gene silencing. In addition, there is some evidence indicating that HDAC3 may be the predominant HDAC isoform responsible for apoptotic histone deacetylation in dying RGCs. Initial HDAC inhibition studies indicate that TSA can prevent gene silencing and has a neuroprotective effect, suggesting that histone deacetylation may be a critical stage in the apoptotic pathway.
Experimental animals and optic nerve crush
All mice were handled in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals for research. The majority of experiments were conducted on CB6F1 mice, which have been used in the past by our group to quantify cell loss and changes in RGC transcript levels, as a result of ONC. In some experiments, however, ROSA3, a substrain of C57BL/6 mice , were used to utilize the expression of βGEO marker protein as a way to more precisely quantify RGC-specific gene expression. These latter mice express βGEO as the result of transcription of the Fem1c gene, which is predominantly RGC-specific in the retina. They also exhibit similar kinetics of cell loss observed in the CB6F1 strain. For both strains, a random mixture of males and females between the ages of 4-6 months were used. ONC was performed unilaterally as described previously to initiate degeneration of the retinal ganglion cells . Retinas were harvested 1, 3, 5, 7, or 14 days post ONC as indicated.
In some cases, retinal ganglion cells were retrogradely labeled with the tracer dye, fluorogold (Molecular Probes, Eugene, OR). Labeling was performed by first exposing the superior collicli on each side of the brain and placing a small piece of gel-foam, soaked in 0.9% NaCl containing 2% fluorogold, to each exposed surface. ONC surgery was performed 3 days after dye application.
HDAC activity assay and nuclear protein extraction
Nuclear proteins were extracted from whole retinas according to Andrews and Faller (1991). Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL). HDAC activity assays were performed using a Fluor de Lys kit (BIOMOL International, Plymouth Meeting, PA). Triplicate samples containing 4 μg of protein each were loaded in an opaque 96-well plate with Fluor de Lys substrate at a final concentration of 150 μM. Following a 20 minute incubation at room temperature, 1 × Fluor de Lys developer with trichostatin A was added to stop the reaction and develop the fluorescent signal. Plates were read using a CytoFluor plate reader at 360 nm excitation and 440 nm emission wavelengths (Perkin Elmer, Waltham, MA). All samples were corrected to a buffer-only blank and normalized to the HeLa extract controls.
Western blot analysis
Western blot analysis on nuclear and cytoplasmic retinal protein fractions (see above) was performed as previously described  with the following modifications. Each fraction was loaded as a single lane (500 μg) on separate12% polyacrylamide gels and transblotted onto Immobilon P (Millipore, Inc, Billerica, MA). Membranes were stained with Ponceau S, cut into strips, and probed with various antibodies. Rabbit polyclonal antibodies for HDACs 1-5 (Santa Cruz, city CA) and anti-acetyl-histone H4 ChIP grade rabbit antiserum (Millipore, Inc) were used at a 1:100 dilution. Goat anti-rabbit secondary antibodies, conjugated to alkaline phosphatase, were used at a 1:500 dilution (Jackson ImmunoResearch Laboratories Inc, West Grove, PA). Blots were color developed using NBT and BCIP and digitally scanned.
Evaluation of transcripts in the retina by quantitative PCR
Primers for ChIP and qPCR analysis
Size of product (bp)
Primers for qPCR mini array analysis
Size of product (bp)
Immunofluorescence and quantification of AcH4
Indirect immunofluorescence on 5 μm thick retinal cryosections was done as described previously . Primary antibodies included γH2AX (monoclonal, Millipore, Inc), HDAC2, HDAC3, and AcH4 (previously mentioned) and were used at a 1:100 dilution. Secondary antibodies used were goat anti-rabbit with a Texas Red label (1:100) and goat anti-mouse with a FITC label (1:100) (both from Jackson ImmunoResearch Laboratories). The images were obtained using a Zeiss Axioplan 2 Imaging microscope with Axiovision 184.108.40.206 software (Carl Zeiss MicroImaging, Inc., Thornwood, NY) and viewed in Adobe Photoshop.
To quantify AcH4 staining, nuclear pixel intensity was measured in the ganglion cell (GCL) and inner nuclear layers (INL) using the outline function of the Zeiss Axiovision software. A minimum of 99 cells was counted in each layer and the mean pixel intensity per nucleus was calculated as a function of the nuclear area (in μm2). The calculated pixel intensity of GCL nuclei was normalized to the calculated pixel intensity of nuclei of the INL and the final calculated intensity was expressed as a ratio of crush:control.
Chromatin immunoprecipitation (ChIP) assays
Acetyl-histone H4 ChIP assays were performed as outlined by the manufacturer (Millipore). In each assay, 6 retinas were pooled and half of each sample was mixed with either AcH4 antibody or normal rabbit serum for a control (Jackson ImmunoResearch Laboratories). The supernatant obtained from the normal serum samples following immunoprecipitation was regarded as the input. DNA from immunopreciptates was unlinked from protein complexes and purified further by phenol/chloroform extraction. Samples were analyzed in triplicate using qPCR as described above. The data obtained from qPCR were analyzed by subtracting the normal serum samples from the AcH4 immunoprecipitated samples and converting this to a percentage of the total input. These numbers were then expressed as a ratio of crush:control and normalized to the day 0 values.
HDAC inhibitor studies
To inhibit HDAC activity in the retina, TSA was injected either intraperitoneally (1 mg/kg in DMSO) 24 hours prior to ONC surgery or intravitreally (1 μl of 20 μM TSA in DMSO) immediately after ONC surgery. Vehicle injections consisted of an equal volume of DMSO. The effects of TSA on gene expression were conducted on Fem1c R3/+ mice, which express the βGEO enzyme predominantly in RGCs in the retina. The level of βGeo expression was analyzed using two methods. Firstly, the level of βGEO activity in individual retinas was quantified by β-Galactosidase solution assay (Promega, Madison, WI) 5 days post ONC. The plates were read with an ELX800 microplate reader (Bio-Tek Instruments Inc., Winooski, VT). Duplicate samples of each eye was measured and total activity was calculated after subtraction of the β-Galactosidase activity measured in wild-type mice and corrected to the amount of total protein loaded in each sample. Secondly, βGEO expressing cells were identified histochemically, 5 days post ONC, by staining retinal preparations with X-Gal, followed by whole mounting as previously described . To assess the effects of TSA on RGC loss, a 1 mg/kg intraperitoneal injection of TSA was administered 24 hours prior to ONC of adult CB6F1 mice. Two weeks after ONC, cells in the GCL were Nissl-stained and counted and compared to controls .
Data was collected from a minimum of 3 independent samples in all experiments, and shown as the mean ± Standard Error in graphs. All statistical analyses were performed using the Student's t-test with statistical significance set at P ≤ 0.05.
This work was funded by grants from the National Eye Institute (R01 EY12223 to R.W.N. and CORE grant P30 EY016665), an unrestricted research grant from Research to Prevent Blindness, and the RPB Wasserman Award (to R.W.N.). The authors would also like to thank Dr. Shiming Chen for providing assistance with the HDAC inhibitor treatment protocol, Joel Dietz for his assistance in conducting qPCR experiments, Katherine Janssen and Dr. Sheila Semaan for assistance with the β-Galactosidase assays, and Drs. Donald Zack and Zhiyong Yang for providing quantitative data for ganglion cell specific gene expression from their microarray studies.
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