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Human Molecular Genetics Advance Access originally published online on April 4, 2007
Human Molecular Genetics 2007 16(11):1293-1306; doi:10.1093/hmg/ddm078
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models

Ghazaleh Sadri-Vakili1, Bérengère Bouzou2, Caroline L. Benn1, Mee-Ohk Kim1, Prianka Chawla1, Ryan P. Overland1, Kelly E. Glajch1, Eva Xia1, Zhihua Qiu1, Steven M. Hersch1, Timothy W. Clark2, George J. Yohrling3 and Jang-Ho J. Cha1,*

1 Department of Neurology, 2 Center for Interdisciplinary Informatics, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, B114-2000, 114 16th Street, Charlestown, MA 02129-4404, USA and 3 Johnson & Johnson Pharmaceuticals, Research & Development, L.L.C., Welsh & McKean Roads, PO Box 776, Spring House, PA 19477, USA

* To whom correspondence should be addressed. Tel: +1 6177241481; Fax: +1 6177241480; Email: cha{at}helix.mgh.harvard.edu

Received February 22, 2007; Accepted March 20, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Transcriptional dysregulation plays a major role in the pathology of Huntington's disease (HD). However, the mechanisms causing selective downregulation of genes remain unknown. Histones regulate chromatin structure and thereby control gene expression; recent studies have demonstrated a therapeutic role for histone deacetylase (HDAC) inhibitors in polyglutamine diseases. This study demonstrates that despite no change in overall acetylated histone levels, histone H3 is hypo-acetylated at promoters of downregulated genes in R6/2 mice, ST14a and STHdh cells, as demonstrated by in vivo chromatin immunoprecipitation. In addition, HDAC inhibitor treatment increases association of acetylated histones with downregulated genes and corrects mRNA abnormalities. In contrast, there is a decrease in mRNA levels in wild-type cells following treatment with a histone acetyltransferase inhibitor. Although changes in histone acetylation correlate with decreased gene expression, histone hypo-acetylation may be a late event, as no hypo-acetylation is observed in 4-week-old R6/2 mice. Nevertheless, treatment with HDAC inhibitors corrects mRNA abnormalities through modification of histone proteins and may prove to be of therapeutic value in HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Huntington's disease (HD) is an autosomal dominant neurological disease characterized by problems with movement, cognition and behavioral functioning. Recent studies have provided strong evidence for transcriptional dysregulation as a major mechanism underlying HD pathogenesis [reviewed in (1,2)]. Transcriptional dysregulation is an early event in HD pathology and is present across multiple HD models. Human HD and transgenic mouse models of HD demonstrate downregulation of specific genes at the level of mRNA expression (3–8). Previous studies have demonstrated alterations of neurotransmitter receptor protein levels and mRNA levels in the R6/2 mouse model (4,9,10). Furthermore, the widespread inquiry of mRNA levels by expression profiling using DNA microarrays of R6/2 brain confirmed the finding that specific genes are downregulated in this HD mouse model (11,12). These results suggest that transcriptional dysregulation is an important mechanism in HD pathogenesis. However, the mechanisms that cause selective downregulation of target genes remain unknown.

Regulation of gene expression has two components that act in concert: binding by transcriptional activators and repressors and alteration of chromatin structure governed by histone modifications. The amino terminal tails of the core histones (H2A, H2B, H3 and H4) are strongly basic and contain specific amino acid residues that are sites for several post-translational modifications such as acetylation, methylation, phosphorylation and ubiquitylation (13,14). In general, acetylation of lysine residues corresponds to transcriptionally active chromatin and promotes transcription (14,15).

Given that changes in acetylation of histones lead to changes in chromatin structure, and in turn gene transcription, we sought to determine if hypo-acetylation of histones is an underlying mechanism of transcriptional dysregulation in HD. One possibility is that there is global hypo-acetylation of histones. Another possibility is that histone hypo-acetylation occurs only at specific genomic loci. To address this question, we utilized the technique of in vivo chromatin immunoprecipitation (ChIP). ChIP allows one to measure changes in histone modifications at the specific gene targets. Our findings demonstrate that, despite no significant overall difference in histone acetylation in HD cell lines and animal models, there is a progressive decrease in acetylation of histones associated with downregulated genes. In addition, hypo-acetylated histones are associated with downregulated genes in a region- and brain-specific manner, with the striatum being the most affected area. Furthermore, administration of HDAC inhibitors, sodium butyrate and phenylbutyrate, increase the association of acetylated histones with downregulated genes and correct mRNA abnormalities in both cell and animal models of HD. Treatment of wild-type cells with curcumin, a histone acetyltransferase inhibitor, results in hypo-acetylation of histones and decreased mRNA expression. Taken together, these results suggest that hypo-acetylation of histones associated with specific genes leads to changes in gene expression in HD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Global histone acetylation in R6/2 mice
The first issue was to determine if there is an overall alteration in histone acetylation in HD. Acetylated histone H3 (AcH3) levels were measured using western blot analysis using an antibody that recognizes H3 acetylated at lysine 9 and lysine 14 in 4-, 8- and 12-week-old R6/2 transgenic HD mice. There was no significant difference in AcH3 levels between wild-type and transgenic mice in any of the brain regions measured: striatum (STR), cortex (CTX), hippocampus (HIP) and cerebellum (CBL) (Fig. 1A and B). Furthermore, there was no significant difference in total histone H3 levels between wild-type and transgenic mice in any of the brain regions (Fig. 1C and D). AcH3 and total H3 levels were also measured in liver and no differences were found between wild-type and transgenic mice (data not shown).


Figure 1
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  		Figure 1. Acetylated histone H3 levels in R6/2 mouse brain. (A) Western blot showing levels of AcH3 in histone extracts from 4-, 8- and 12-week-old wild-type (WT) and transgenic (TG) R6/2 striatum (STR), cortex (CTX), hippocampus (HIP) and cerebellum (CBL). (B) Representative densitometry values for AcH3 levels at 4 weeks. There is no significant difference in AcH3 levels in any of the brain regions. Open bars, WT; filled bars, TG; IDV, integrated density value. Error bars: SEM (n = 4). (C) Western blot showing levels of H3 in histone extracts from 4-, 8- and 12-week-old WT and TG STR, CTX, HIP and CBL. (D) Densitometry values for H3 levels at 4 weeks. There is no significant difference in H3 levels between WT and TG in any of the brain regions. Open bars, WT; filled bars, TG; IDV, integrated density value. Error bars: SEM (n = 4). (E) Coomassie gel demonstrates equal protein loading for western blots.

 
Altered association of histones with specific genes in R6/2
Global AcH3 levels do not necessarily represent the status of histones associated with downregulated genes. We thus sought to determine the acetylation status of histones associated with specific genes. Based on expression profiling analysis we are able to interrogate specific downregulated genes. For example, the genes encoding dopamine D2 receptor (gene: Drd2, mRNA: D2), preproenkephalin (gene: Penk1; mRNA: PPE) and brain-derived neurotrophic factor (gene: Bdnf; mRNA: BDNF) are downregulated in HD, while the NR1 subunit of the NMDA receptor (gene: Grin1, mRNA: NR1) and ß-actin (gene: Actb; mRNA: ß-actin) genes are expressed at normal levels (11). We tested the hypothesis that acetylated histones have altered association with downregulated genes using ChIP coupled with real-time PCR. Compared with wild-type, there was decreased association of AcH3 with the Drd2 gene in 12-week-old transgenic R6/2 whole brain as measured by ChIP (Fig. 2A). There was also a non-significant decrease in AcH3 association with Penk1. Furthermore, there was no change in AcH3 association with Actb or Grin1 genes, whose expression is unchanged in HD (Fig. 2A). Next, we investigated whether the altered association of AcH3 with Drd2 is region-specific. There was a significant decrease in AcH3 association with the Drd2 gene in the transgenic striatum, but not in cortex, hippocampus, cerebellum or liver (Fig. 2B). Changes in AcH3 association with the Drd2 gene are tissue- and region-specific, given that there is no change in AcH3 association in the liver or other tested brain regions (Fig. 2B).


Figure 2
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  		Figure 2. Decreased association of AcH3 with downregulated genes in R6/2 mouse brain. (A) There is a significant decrease in AcH3 association with Drd2 and no change in the association with Actb and Grin1 in 12-week-old R6/2 whole brain samples. Bars represent the amount of immunoprecipitated DNA normalized to input DNA. Open bars, WT; filled bars, TG; Error bars, SEM (n = 6–8). (B) There is a significant decrease in AcH3 association with Drd2 gene in 12-week-old TG striatum (STR) only. *P < 0.05. Striatum (STR), cortex (CTX), hippocampus (HIP), cerebellum (CBL), liver (LIV). Open bars, WT; filled bars, TG; error bars, SEM (n = 5–7).

 
In order to determine the time course of histone hypo-acetylation at downregulated genes, AcH3 association was measured in 4-, 8- and 12-week-old R6/2 mice. Overall, there is a progressive increase in AcH3 association with genes in the wild-type striatum in contrast to a relative decrease in AcH3 association with genes in the transgenic striatum. While there was no change in AcH3 association with genes at 4 weeks of age, there was a significant decrease in AcH3 association with the Drd2 and Penk1 genes at 8 weeks in transgenic R6/2 striatum (Fig. 3B). In addition, there was a trend towards a decrease in association of AcH3 with all tested genes by 12 weeks (Fig. 3C). These results suggest a progressive decrease in AcH3 association with genes in transgenic R6/2 striatum (Fig. 3).


Figure 3
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  		Figure 3. Progressive decrease in AcH3 association with downregulated genes in R6/2 mice. (A) There is no change in the association of AcH3 with genes in 4-week-old tg R6/2 striatum (STR) compared with wt. (B) There is a significant relative decrease in AcH3 association with Drd2 and Penk1 but notActb, Grin1 and Bdnf in the TG R6/2 STR at 8 weeks. (C) There is a significant relative decrease in AcH3 association with Drd2, Penk1 and Actb genes in the 12-week-old TG R6/2 STR. *P < 0.05. Striatum (STR), cortex (CTX), hippocampus (HIP), cerebellum (CBL), liver (LIV). Open bars, WT; filled bars, TG; error bars, SEM (n = 4–7).

 
Altered histone acetylation in cell models of Huntington's disease
The phenomena most relevant to HD pathogenesis should be present in different models. The R6/2 mouse is a very useful model, but it has limitations due to the fact that this transgenic model contains only exon 1 of the HD gene (9). We have therefore measured AcH3 association with downregulated genes in two different cell lines, inducible ST14a cells and STHdh cells.

Total AcH3 and H3 protein levels were measured in the parental ST14a (12.4), wild-type huntingtin (Htt)- (HD19) or mutant Htt- (HD43) inducible cells in the absence or presence of the inducer (0.5 µg/ml doxycycline) by western blot. Parental ST14a cells are a neural progenitor cell line derived from the striatum of embryonic rats with HD19 cells expressing 26 glutamines and HD43 cells expressing 105 glutamines (16). There was no significant difference in global AcH3 or H3 levels in the wild-type Htt expressing cells (HD19) or the mutant Htt expressing cells (HD43) compared with the parental cells (Fig. 4A). We also tested whether downregulated genes are associated with hypo-acetylated histones in ST14a cells. Cannabinoid receptor 1 (gene: Cnr1; mRNA: CB1) and PPE mRNA levels were significantly decreased following mutant Htt expression in HD43 cells as assessed by reverse transcription real-time PCR (RT-qPCR) (Fig. 4B). We next measured AcH3 association with both downregulated and unchanged genes in ST14a cells. There was decreased association of AcH3 with Cnr1, a gene that is downregulated in HD43 cells compared with the parental and HD19 cells (Fig. 4C). There was a trend towards a decrease in the association of AcH3 with the Penk1 gene, a gene that is downregulated in the HD43 cells (Fig. 4C). However, there was no change in AcH3 association with the Grin1 and Actb genes in the presence of mutant Htt. These findings suggest that alterations in histone acetylation occur at specific downregulated genes and are dependent on the presence of mutant and not wild-type Htt.


Figure 4
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  		Figure 4. Decreased AcH3 association with downregulated genes in inducible ST14a cells. (A) Western blots show that there is no difference in AcH3 or H3 levels between cell lines. Open bars, untreated cells; filled bars, doxycycline treated; IDV, integrated density value; error bars, SEM (n = 3). (B) RT-qPCR demonstrates a significant decrease in cannabinoid receptor 1 (CB1) and preproenkephalin (PPE) mRNA levels in the HD43 cells when compared with HD19 and parental cell lines. Hatched bars, parental cells; open bars, HD19 cells; filled bars, HD43 cells. Error bars, SEM (n = 3–4). (C) There is a significant decrease in AcH3 association with Cnr1 in the HD43 cells. *P < 0.05. Hatched bars, parental cells; open bars, HD19 cells; filled bars, HD43 cells; error bars, SEM (n = 4); Cnr1, cannabinoid receptor 1; Penk1, preproenkephalin; Grin1, NR1 subunit of NMDA receptor; Actb, ß-actin.

 
Next, we measured AcH3 levels in the STHdh cells, which differ from R6/2 mice and ST14a cells in that they express full-length Htt and have been prepared from the striatal tissue of knock-in mice (17). STHdh7/7 cells express wild-type Htt, while STHdh111/111 cells express two mutant copies containing 111 glutamines. Overall levels of AcH3 were not decreased in the STHdh111/111 cells; in fact, there was a significant increase in AcH3 (Fig. 5A). In addition, STHdh111/111 cells exhibited a significant decrease in the total H3 levels (Fig. 5A). This antibody specifically recognizes unmodified histone H3, therefore the decrease in H3 levels could be explained by an increase in AcH3 levels.


Figure 5
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  		Figure 5. Decreased AcH3 association with downregulated genes in STHdh cells. (A) Western blots show that there is a significant increase in AcH3 levels and a significant decrease in H3 levels in STHdh111/111 cells. Error bars: SEM (n = 4). (B) RT-qPCR confirms microarray findings and demonstrates a significant decrease in VDR, NCAM1, Inhibin b, NADPH deh and TCF7 mRNA levels in STHdh111/111 cells. Open bars, STHdh7/7 cells; filled bars, STHdh111/111 cells. Data normalized to STHdh7/7. Error bars, SEM (n = 3–4). (C) There is a significant decrease in AcH3 association with Vdr, Ncam1, Inhbb, Dhrs4 and Tcf7 in STHdh111/111 cells. Open bars, STHdh7/7 cells; filled bars, STHdh111/111 cells. Error bars, SEM (n = 4–6). *P < 0.05.

 
In order to determine if gene expression was altered in STHdh cells, we carried out microarray expression profiling analysis using Affymetrix gene chips, and confirmed the results by RT-qPCR. The analysis demonstrated that Drd2, Penk1, Cnr1 and Grin1 were not expressed in these cell lines. However, gene expression profiles for STHdh7/7 and STHdh111/111 cells were different, and of genes that were expressed in both cell lines, there were more genes downregulated in STHdh111/111 cells. The analysis of the gene ontology categories of the genes (695) with decreased expression in the STHdh111/111 cells shows that DNA replication, DNA metabolism, cell cycle, cell division, chromosome segregation and mitosis (Table 1) were the categories that are the most significantly enriched. In contrast, the same gene ontology analysis of the genes (561) with upregulated expression in the STHdh111/111 cells showed that signaling, immune response, defense response, collagen and extracellular matrix (Table 2) were the categories that are the most significantly enriched.


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  		Table 1. Functional clustering of downregulated genes in STHdh111/111 cells

 


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  		Table 2. Functional clustering of upregulated genes in ST Hdh111/111 cells

 
On the basis of the expression profiling analysis we identified specific downregulated genes in STHdh cells. For example, vitamin D receptor (gene: Vdr; mRNA: VDR), inhibin beta-b (gene: Inhbb; mRNA: Inhibin b), transcription factor 7 (gene: Tcf7; mRNA: TCF7), neuronal cell adhesion molecule (gene: Ncam1; mRNA: NCAM1) and NADPH dehydrogenase (gene: Dhrs4; mRNA: NADPH deh), were genes downregulated in STHdh111/111 cells, while Actb, glyceraldehyde-3-phosphate dehydrogenase (gene: Gapdh; mRNA: GAPDH) and microtubule-associated protein 2 (gene: Mtap2; mRNA: MAP2) were expressed at wild-type levels. Upon confirmation of the microarray findings by RT-qPCR (Fig. 5B), we measured changes in AcH3 association with these downregulated genes in the STHdh cells. There was a significant decrease in AcH3 association with these downregulated genes in STHdh111/111 cells, as assessed by ChIP, similar to the findings in R6/2 mice and ST14a cells (Fig. 5C). Accordingly, there was no change in AcH3 association with genes that are normally expressed (Fig. 5C).

Effect of histone deacetylase inhibitors on gene expression in Huntington's disease models
It is critical to establish whether changes in histone acetylation lead to transcriptional dysregulation in HD or are simply a consequence thereof. Eight-week-old transgenic R6/2 mice and their wild-type littermates were treated with the HDAC inhibitor phenylbutyrate (400 mg/kg) for 7 days. Following phenylbutyrate treatment there was a significant increase in the association of AcH3 with the downregulated genes Drd2 and Penk1 (Fig. 6A). Furthermore, there was a correction of mRNA levels for D2 and PPE in the R6/2 striatum following phenylbutyrate treatment (Fig. 6B). This study demonstrates that HDAC inhibitor treatment in vivo corrects mRNA abnormalities by having a direct effect on histone acetylation. In a parallel set of experiments, STHdh cells were treated with the histone deacetylase inhibitor sodium butyrate (10 µM, 24 h). AcH3 association with all genes was increased following sodium butyrate treatment of STHdh111/111 cells (Fig. 6C). In addition, treatment of STHdh111/111 cells with sodium butyrate also increased mRNA levels of downregulated genes (Fig. 6D).


Figure 6
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  		Figure 6. HDAC inhibitor treatment increases AcH3 association with genes and reverses mRNA abnormalities. (A) There is a significant increase in AcH3 association with Drd2 and Penk1 genes in the R6/2 striatum following phenybutyrate (400 mg/kg, seven days) treatment. Error bars, SEM (n = 5–6). (B) There is a significant increase in D2 and PPE mRNA levels in the R6/2 STR following phenylbutyrate treatment. Error bars, SEM (n = 5–6). (C) There is an increase in AcH3 association with Vdr, Inhbb, Dhrs4, Tcf7 genes in the STHdh111/111 cells following sodium butyrate (10 µM, 24 h) treatment. Error bars, SEM (n = 4–5). (D) There is a significant increase in VDR, Inhibin b, NADPH deh, TGF7 mRNA levels following sodium butyrate treatment. Error bars, SEM (n = 4–5). *P < 0.05. Open bars, WT or STHdh7/7; filled bars, TG or STHdh111/111 cells. Data for cell lines normalized to STHdh7/7.

 
HDAC inhibitor treatment alters other histone modifications
There is cross-talk between different histone modifications, but it is not clear if changes in histone acetylation are linked to other histone modifications, such as ubiquitylation. Monoubiquitylation of histone H2A (uH2A) at lysine 119 promotes gene silencing and repression. Kim et al., found that genes downregulated in transgenic R6/2 brain have increased uH2A association at their promoters. This increase in uH2A appears to contribute to transcriptional repression in HD (Mee-Ohk Kim, manuscript in preparation). We therefore measured changes in histone H2A ubiquitylation in STHdh cells following sodium butyrate treatment (10 µM, 24 h).

While there was no change in total histone H3 levels in either cell line, there was a significant increase in AcH3 levels in both STHdh7/7 and STHdh111/111 cells following sodium butyrate treatment (Fig. 7). Sodium butyrate treatment decreased uH2A levels in the STHdh7/7 cells (Fig. 7). These findings suggest that histone acetylation functions upstream of histone monoubiquitylation in HD.


Figure 7
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  		Figure 7. HDAC inhibitor treatment decreases uH2A levels in STHdh cells. Western blots show that there is a significant increase in uH2A levels in STHdh111/111 cells. There is a significant decrease in uH2A levels in STHdh7/7 cells following sodium butyrate (100 µM, 24 h) treatment. There is a significant increase in AcH3 levels in both cell lines following sodium butyrate treatment. There is no change in histone H3 levels. Error bars, SEM (n = 3). *P < 0.05; The ‘hash’ symbol indicates a significant increase in uH2a levels in untreated STHdh111/111 cells when compared with untreated STHdh7/7 cells P < 0.05.

 
The histone acetyltransferase inhibitor, curcumin, decreases mRNA expression in STHdh7/7 cells
In order to determine if hypo-acetylation of histones is sufficient to cause a decrease in mRNA levels, STHdh7/7 cells were treated with curcumin, an anti-inflammatory agent and anti-oxidant that has been shown to be a specific inhibitor of histone acetyltransferases (HAT) (18,19) and changes in mRNA levels were measured. There was a significant decrease in AcH3 levels following curcumin (100 µM, 24 h) treatment as measured by western blots (Fig. 8A). However, there was no change in histone H3 levels. In addition, curcumin treatment of STHdh7/7 cells significantly decreased mRNA levels of all genes as measured by RT-qPCR (Fig. 8B).


Figure 8
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  		Figure 8. HAT inhibitor treatment decreases AcH3 levels and mRNA expression in STHdh7/7 cells. (A) Western blots show that there is a significant decrease in AcH3 levels and no change in H3 levels in STHdh7/7 cells following curcumin (100 µM, 24 h) treatment. Error bars, SEM (n = 3). (B) RT-qPCR demonstrates a significant decrease in VDR, NCAM1, Inhibin b, NADPH deh, TCF7, ß-actin, MAP2 and GAPDH mRNA levels in STf cells following curcumin treatment. Error bars, SEM (n = 4). *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In human HD brain, as well as numerous animal and cell models of HD, mRNA levels are altered, suggesting that transcriptional dysregulation is a pathogenic mechanism in HD (2,20). Therefore, it is imperative to determine the underlying molecular events. Chromatin remodeling through modification of histone proteins is a mechanism that regulates gene expression. For example, a well-characterized histone modification is the acetylation of lysine residues that corresponds to transcriptionally active chromatin and activation of gene transcription. Although the overall status of histone modifications is largely unknown in HD, recent studies support the importance of histone acetylation in HD. In cell models of HD, polyglutamine decreases histone acetylation, and HDAC inhibitors have been shown to reduce polyglutamine-induced toxicity (21–24). HDAC inhibitors also improve the phenotype of transgenic Drosophila and mouse models of HD (23–26). Therefore, correction of histone modification abnormalities may be of therapeutic benefit in HD, and clinical trials with HDAC inhibitors have begun. The underlying mechanisms whereby HDAC inhibitors prove therapeutic remain unknown. Specifically, HDAC inhibitors may improve phenotype by either upregulating those genes that have been repressed by mutant Htt. Another possibility is that HDAC inhibitors upregulate expression of other beneficial genes.

Both phenylbutyrate and sodium butyrate have been shown to increase lifespan in HD mice and to increase global levels of histone acetylation (25,26). But the effect of these HDAC inhibitors on histones associated with specific genes was not known; these compounds may exert their benefit by upregulating other genes. In the current study, treatment of R6/2 mice and STHdh cells not only increased association of acetylated histones with downregulated genes but also corrected aberrant mRNA expression. These findings demonstrate a viable molecular mechanism by which HDAC inhibitors improve disease phenotype. This is the first study to demonstrate that HDAC inhibitor treatment in vivo corrects mRNA abnormalities by having a direct effect on histone acetylation.

The simplest possibility to explain the benefit of HDAC inhibitors is that there is a global hypo-acetylation of histones in HD models. In fact, we found no evidence of global hypo-acetylation, with an increase in histone acetylation in STHdh111/111 cells. These findings leave open the possibility that histone hypo-acetylation occurs at specific genomic loci. There was decreased association of AcH3 specifically with downregulated genes in R6/2 mice, as well as in ST14a and STHdh cell lines. In R6/2 mice, histone hypo-acetylation is both brain- and region-specific in that the striatum is the only region where a significant decrease in the association of AcH3 with genes was detected. Thus, despite widespread expression of the Htt protein, histones appear to be altered selectively in the brain, providing insight to why HD is a neurological disease. Furthermore, HDAC inhibitor treatment of both R6/2 and STHdh cells reverses mRNA abnormalities and increases the association of AcH3 with genes. Confirmation of the R6/2 results with that of the ST14a and STHdh cell lines bolsters the validity of these observations. Taken together, these results show that decreased association of AcH3 with genes is both necessary and sufficient to cause mRNA downregulation.

Decreased AcH3 association with genes in the R6/2 striatum is detectable at 8 weeks of age, affecting a small number of genes and progresses to affect more genes by 12 weeks, including genes that are expressed normally. It is likely that changes in histone acetylation precede changes in mRNA levels. Thus, the progressive nature of these events raises the possibility that more genes would be downregulated at later time points. However, microarray analysis of R6/2 striatum has only been performed in mice as old as 12 weeks (11). Therefore, we measured changes in ß-actin and NR1 mRNA levels, genes whose expression is not changed but are associated with hypo-acetylated histones at 12- and at 14-weeks of age to address this issue. Indeed, both ß-actin and NR1 transcripts were downregulated in R6/2 striatum at 14 weeks of age (Supplementary Material, Fig. S1), arguing that hypo-acetylation of histones precedes decreases in mRNA levels. There is no change in acetylated histone association with genes in R6/2 mice at 4 weeks of age, and significant changes are progressive and detectable at 8 weeks of age. Previous analysis of mRNA levels in the R6/2 striatum demonstrate that there is a decrease in transcript levels starting as early as 6 weeks (11). It is possible that histone hypo-acetylation is a compensatory response to an earlier cellular insult caused by mutant Htt.

How does mutant Htt lead to decreased acetylation of histones at promoters of downregulated genes? One possibility is that mutant Htt interacts with histone proteins. To date, there is no evidence that Htt interacts with histone proteins. However, Htt aggregates have been found to contain histones H3 and H4 (27). Many Htt-interacting proteins possess histone-modifying activity. Fragments of mutant Htt interacts with CREB-binding protein (CBP). CBP contains an acetyltransferase domain and is a co-activator at a number of promoters (28). CBP and other transcription factors have been shown to be sequestered into Htt aggregates in transgenic mice and HD brains (28,29). Therefore, one possibility is that mutant Htt can disrupt histone acetylation through its interactions with histone acetyltransferases (HAT) such as CBP. Curcumin is an anti-inflammatory as well as anti-oxidant agent and has been shown to be a specific inhibitor of p300/CBP HAT activity (18,19). It has been shown to specifically repress the p300/CBP HAT activity-dependent transcriptional activation of chromatin (18). In addition, curcumin effectively blocks histone hyperacetylation in both PC3-M prostate cancer cells and peripheral blood lymphocytes induced by the histone deacetylase inhibitor MS-275, thus identifying curcumin as a novel p300/CBP-specific HAT inhibitor (19). Our results demonstrate that following curcumin treatment, there is a decrease in acetylated histones as well as a general decrease in mRNA levels. However, this pattern of gene downregulation is different from what is observed in HD models. In both animal and cellular models of HD, there is an initial selective decrease in specific mRNA species. Thus, while decreases in CBP HAT activity may be partially involved in gene downregulation in HD, it is clear that selectivity is conferred by an alternative molecular mechanism. It is also possible that curcumin inhibits other HATs. One possibility is that Htt exerts specific effects as a result of its direct interactions with DNA (Caroline Benn, manuscript in preparation).

Other molecules can change histone acetylation. For example, nuclear co-repressor (NCoR) interacts with Htt and can recruit histone deacetylases (30). Thus, the recruitment of histone deacetylases through the interaction of mutant Htt with NCoR could lead to hypo-acetylation of histones at specific genes. Ataxin-3, the disease-causing protein in spinocerebellar ataxia type 3, another polyglutamine disease, interacts with the transcriptional co-repressor NCoR (31). Ataxin-3 was associated with increased deacetylase activities and deacetylation of histone H3, leading to repression of gene transcription via histone deacetylation (31). In addition to changes in HDAC activity, mutant Htt interacts with other histone-modifying enzymes to alter acetylation and possibly histone methylation. Huntingtin yeast partner B (HYPB) has recently been identified as a histone methyltransferase (32). Acetylation and methylation of lysine residues are mutually exclusive. Therefore, increased methylation would cause decreased acetylation of specific lysine residues. One possibility is that mutant Htt increases methylation of histones through its interactions with HYPB and thereby decreases acetylation at specific lysine residues. Indeed, Gardian and colleagues have shown that there is an increase in methyl lysine 9 histone H3 in the striatum of N171-82Q mice (24).

Previous studies in cell models of HD have shown that polyglutamine expression decreases histone acetylation, and HDAC inhibitors have been shown to reduce polyglutamine-induced toxicity (21–23). Furthermore, HDAC inhibitors have been shown to improve the phenotype of transgenic Drosophila and mouse models of HD [reviewed in (33)]. Our findings that HDAC inhibitors normalize mRNA levels in R6/2 mice and STHdh111/111 cells strengthens previous findings by demonstrating a direct effect of HDAC inhibitors on transcription. Taken together, these results suggest that although decreased association of AcH3 with specific promoters may be a compensatory mechanism, correction of histone modification abnormalities proves to be of therapeutic benefit and helps to ameliorate disease progression in HD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Transgenic R6/2 mice
Brains from R6/2 transgenic mice and wild-type littermate controls were used in experiments. R6/2 transgenic mice contain exon 1 of the HD gene with an expanded CAG repeat (150–200) under control of the human HD promoter (9). Four-, eight-, and twelve-week-old R6/2 transgenic and wild-type littermate controls were sacrificed, and brains were rapidly removed and frozen in liquid isopentane. Brains were stored at –80°C until use. The guidelines for animal care and use were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (SRAC).

Phenyl butyrate treatment
Eight-week-old R6/2 wild-type and littermate controls received daily intraperitoneal injections of phenyl butyrate (400 mg/kg, Triple Crown USA Inc., Perkasie, PA, USA) or vehicle [phosphate-buffered saline (PBS)] for seven consecutive days.

Cell lines
Striatal cell lines were established from wild-type and Hdh (Q111) knock-in embryonic mice (17). STHdh cell lines express full-length versions of either wild-type (containing seven glutamines) or mutant (111 glutamines) huntingtin. Two different cell lines were used, corresponding to wild-type (STHdh7/7), and homozygous (STHdh111/111) genotypes. STHdh cells were used in passages 5–16. Cells were kept in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Gaithersburg, MD, USA) plus 10% fetal bovine serum at 33°C for propagation, and were placed at 39°C for 48 h to stop their division.

ST14a cells were previously derived from rat embryonic striatum and used to generate inducible lines expressing the N-terminal fragment of huntingtin (16). The cells proliferate at 33°C but become post-mitotic at 39°C. Cells were grown in DMEM and supplemented as described in (34), unless otherwise specified. Tet-free fetal calf serum (FCS; Clontech, Mountain View, CA, USA) was used to supplement the medium for the inducible subclones (Tet-free medium).

Western blotting
For western blot analysis of acetylated histone levels, histone extracts from dissected R6/2 mouse brain, STHdh cell lines or ST14a cell lines were used. From these histone extracts, 1 µg of protein is resuspended in sample buffer. The resuspended samples were boiled at 95°C for 5 min, and fractionated on a 10–20% tricine gel (Invitrogen, Carlsbad, CA, USA) for 90 min at 120 V. Proteins were transferred to PVDF membranes in transfer buffer (3% Tris base, 14.4% glycine, 20% MeOH) at 400 mA x 1 h, and the PVDF was then blocked with 3% milk in PBS before immunodetection with anti-diacetyl lysine 9 and lysine 14 histone H3 (AcH3) antibody (Upstate, Lake Placid, NY, USA) at a dilution of 1:1500 and anti-histone H3 antibody (Upstate) at a dilution of 1:500 overnight (4°C). Primary antibody incubation was followed by four washes (10 min, RT) in diH2O before incubation with the secondary antibody (HRP-conjugated goat anti-rabbit IgG; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), four washes and visualization using the ECL detection system (NEN, Boston, MA, USA). Coomassie gels were used to ensure equal protein loading for western blots.

Chromatin immunoprecipitation assay
We have adapted the ChIP technique for the analysis of brain tissue and have recently published detailed methodology for performing ChIP experiments (35,36). Antibodies used were directed against histone H3 or AcH3 (Upstate). Negative controls included no antibody mock and IgG (Jackson, West Grove, PA, USA).

Input and IP samples were interrogated with gene promoter-specific primers in triplicate reactions (Supplementary Material, Table S1) in real-time PCR analysis as previously described (36,37). Threshold amplification cycle numbers (Tc) using iCycler software were used to calculate IP DNA quantities as percentage of corresponding inputs. Real-time PCR primer sequences are provided in Supplementary Material, Table S1.

RNA extraction and reverse transcription
RNA was extracted from R6/2 dissected brain regions, STHdh cells and ST14a cells using RNeasy kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. Reverse transcription reactions were performed using the Superscript First Strand Synthesis System for RT–PCR reactions using specific primers to quantitate the amount of gene expression.

mRNA gene expression profiling
Total RNA was isolated from STHdh7/7and STHdh111/111cell lines by extraction with TRI-Reagent (Sigma-Aldrich, St Louis, MO, USA) and purified further over RNeasy columns (Qiagen). Total RNA of 15 µg was pooled for each sample and used to generate labeled cRNA probes according to the Affymetrix GeneChip protocol. Biotinylated cRNA probes were hybridized to MOE40.3K oligonucleotide microarrays using the Affymetrix Fluidics Station 400 according to the manufacturer's standard protocol.

Gene expression profiling analysis
Microarray analysis
Chips were developed, scanned and normalized using global scaling. All quality control parameters calculated by Affymetrix GCOS Software were monitored (38). The images of the chips were analyzed to find spotted or damaged array regions and to ensure data quality (39). The Presence/Absence calls from Affymetrix MAS5 were monitored and compared throughout the different samples.

All data were normalized using the GeneChip Robust MultiChips Analysis (GCRMA) algorithm (40,41), performed with R in BioConductor.

The processed images of the arrays were analyzed for quality control, we found all the samples to be of equally very good quality, with an average of 40.5 ± 1.44% Present calls with a range of 38–42.4%, (the background, noise, spike in controls, housekeeping genes presented a variability over the eight samples <7% pre-normalization). In addition, 31.55% of the probe sets were called Present or Marginal in all the eight samples and 44.6% were called Absent in all the eight samples, showing a good consistency of the quality between the samples and between the groups compared.

Selection of differentially expressed probes
Genes with low intensity in all samples were filtered out (log2 intensity <3), and we kept only the probes called Present or Marginal in all the samples. The average of the groups STHdh111/111 (4) and STHdh7/7 (4) was used to calculate the expression for each gene. Tests were computed with two-tailed unpaired t-test for unknown variance and Multiclass analysis using Spotfire DecisionSite 8.0 (42), and unpaired two classes analysis using the Significance Analysis of Microarrays (SAM) algorithm has been computed with the SAM 2.0 plugging for Excel (43).

We defined genes as being regulated when the ratio STHdh111/111/STHdh7/7>was either up- or down-regulated at least two-folds, the difference between the two groups was statistically significant (P ≤ 0.005 and Q < 0.05).

Clustering and self organizing maps
K-means clustering and self-organizing maps (SOM) (44) were used to cluster the previously found genes by similarity of profiles on the normalized averages of the subgroups of samples; and hierarchical clustering on median-normalized samples using cosine correlation with complete linkage was performed on all samples using SpotFire DecisionSite for Functional Genomics 8.0 (45,46).

Functional analysis
Functional annotation analysis has been performed for the up- and down-regulated lists of genes previously found, using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 2006 [http://david.abcc.ncifcrf.gov/home.jsp]. Functional analysis of the three GO categories, of the KEGG, Biocarta and GenMapp pathways, as well as functional clustering using the EASE enrichment score (Fisher Exact with a method that identifies functional categories over-represented in a gene list relative to the representation within the proteome of a given species) for co-occurrence of groups of genes have been performed on both up- and down-regulated lists of genes (47,48).

For each functional group, the enrichment score (EASE method), the geometric mean and the median of the P-values from all the categories clustered together are represented, and for each category the percent of genes, the number of genes and the P-value are represented; categories in italic have high P-values. Databases used are Gene Ontology Biological Process (GO-BP), Molecular Function (GO-MF), Cellular Compartment (GO-CC), Smart Name (SN), Interpro Name, KEGG, Pir Superfamily Name (PIR_SFN), Sp Pir Keywords (SP-PIR), Up Seq Feature (UP_SEQ).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Dr Elena Cattaneo (University of Milano, Italy) for providing us with the ST14a cell lines and Dr Marcy E. MacDonald (Massachusetts General Hospital, Boston, MA, USA) for providing us with STHdh cell lines. This work was supported by NINDS NS38106 (J.-H.J.C.), NS045242 (J.-H.J.C. and S.M.H.), NS045806 (S.M.H.), Glendorn Foundation (J.-H.J.C.), Hereditary Disease Foundation (J.-H.J.C.), HighQ Foundation (G.S.V.) and HDSA Coalition for the Cure (J.-H.J.C. and S.M.H.).

Conflict of Interest statement. None declared.


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 REFERENCES
 

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