Abnormalities in transcription occur before the starting point of symptoms and so are accompanied by adjustments in histone acetylation, methylation and ubiquitylation [4]C[9]. the complete transcriptome. Using chromatin immunoprecipitation matched with microarray hybridization (ChIP-chip), we interrogated AcH3-gene connections genome-wide in striata of 12-week outdated wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation. Introduction Huntington’s disease (HD) is a progressive neurodegenerative disorder resulting from a trinucleotide CAG repeat expansion in the gene [1]. Pathologically, HD is characterized by a preponderance of neuronal death in the striatum (caudate-putamen). HD patients suffer a triad of movement, cognitive and behavioral issues which steadily worsen throughout the course of the disease [2]. There are currently no effective treatments and the key pathogenic mechanisms that are responsible for the striatal vulnerability leading to the progressive neurodegeneration are unknown. Transcriptional dysregulation is a characteristic of the disease process in human patients and is faithfully recapitulated in multiple animal and cellular models [3]. Abnormalities in transcription occur prior to the onset of symptoms and are accompanied by changes in histone acetylation, ubiquitylation and methylation [4]C[9]. However, whether changes in histone modifications result in the transcriptional abnormalities remains a largely unanswered question. In particular, acetylation of the N-terminal tail of histone H3 is an activating mark for gene expression [10], [11], and increases in histone acetylation precede and facilitate increased transcriptional activity [12], [13]. In HD, levels of acetylated histone H3 (AcH3) associated with downregulated genes are decreased [6]. In an HD cell line and transgenic HD mouse model, mRNA abnormalities were reversed by treatment with inhibitors of histone deacetylases (HDAC), the family of enzymes that remove acetyl groups from histone tails, with concomitant increases in global histone H3 acetylation [6]. Furthermore, decreases in histone acetylation and mRNA levels in the HD cell line can be mimicked in wild-type cells by inhibiting histone acetyltransferases (HATs), enzymes that catalyze the removal of acetyl groups from histone proteins [6]. These results, though limited to a few genes, suggest that decreasing histone acetylation at gene loci is necessary and sufficient for concomitant decreases in mRNA levels. Subsequently, HDAC inhibition is currently Rabbit polyclonal to OSBPL6 being investigated as potential therapeutic intervention for HD as well as other neurodegenerative disorders [14], [15]. However, the relationship between histone acetylation and gene expression has not been studied at the level of the whole genome in the mammalian brain. Furthermore, it is not currently known whether this relationship is altered in the HD brain. While we do know that global levels of histone acetylation do not correspond to histones at specific gene loci [6], it is unknown if the genome-wide distribution of histone acetylation is altered in HD or if the genomic distribution of histone acetylation accounts for gene expression abnormalities. We used a genome-wide approach to capture acetylated histone H3 K9/K14 (AcH3)-DNA interactions and interrogated the chromatin immunoprecipitation products on DNA microarrays (ChIP-chip) to determine genomic locations of AcH3.An individual probe was defined as bound if p0.005, while a gene was defined as bound if at least one probe representing that gene is bound. For quantitative analyses, we determined differentially bound genes between WT and TG samples by calculating a composite score for each gene by subtracting the summation of all WT bound probes from the summation of all TG bound probes as follows: Gene Expression Analysis Using a previously published gene expression microarray dataset [17] from 12-week old R6/2 TG and WT littermates, we used Affymetrix MAS5 analysis to normalize probe intensities and monitor present/absent calls. mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional CB1 antagonist 2 abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates CB1 antagonist 2 that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation. Introduction Huntington’s disease (HD) is a progressive neurodegenerative disorder resulting from a trinucleotide CAG repeat expansion in the gene [1]. Pathologically, HD is characterized by a preponderance of neuronal death in the striatum (caudate-putamen). HD patients suffer a triad of movement, cognitive and behavioral issues which steadily worsen throughout the course of the disease [2]. There are currently no effective treatments and the key pathogenic mechanisms that are responsible for the striatal vulnerability leading to the progressive neurodegeneration are unknown. Transcriptional dysregulation is a characteristic of the disease process in human patients and is faithfully recapitulated in multiple animal and cellular models [3]. Abnormalities in transcription occur prior to the onset of symptoms and are accompanied by changes in histone acetylation, ubiquitylation and methylation [4]C[9]. However, whether changes in histone modifications result in the transcriptional abnormalities remains a largely unanswered question. In particular, acetylation of the N-terminal tail of histone H3 is an activating mark for gene expression [10], [11], and increases in histone acetylation precede and facilitate increased transcriptional activity [12], [13]. In HD, levels of acetylated histone H3 (AcH3) associated with downregulated genes are decreased [6]. In an HD cell line and transgenic HD mouse model, mRNA abnormalities were reversed by treatment with inhibitors of histone deacetylases (HDAC), the family of enzymes that remove acetyl groups from histone tails, with concomitant raises in global histone H3 acetylation [6]. Furthermore, decreases in histone acetylation and mRNA levels in the HD cell collection can be mimicked in wild-type cells by inhibiting histone acetyltransferases (HATs), enzymes that catalyze the removal of acetyl organizations from histone proteins [6]. These results, though limited to a few genes, suggest that reducing histone acetylation at gene loci is necessary and adequate for concomitant decreases in mRNA levels. Subsequently, HDAC inhibition is currently being investigated as potential restorative treatment for HD as well as other neurodegenerative disorders [14], [15]. However, the relationship between histone acetylation and gene manifestation has not been studied at the level of the whole genome in the mammalian mind. Furthermore, it is not currently known whether this relationship is modified in the HD mind. While we do know that global levels of histone acetylation do not correspond to histones at specific gene CB1 antagonist 2 loci [6], it is unfamiliar if the genome-wide distribution of histone acetylation is definitely modified in HD or if the genomic.