ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease

Abstract

Chromatin remodeling and transcription regulation are tightly controlled under physiological conditions. It has been suggested that altered chromatin modulation and transcription dysfunction may play a role in the pathogenesis of Huntington's disease (HD). Increased histone methylation, a well established mechanism of gene silencing, results in transcriptional repression. ERG-associated protein with SET domain (ESET), a histone H3 (K9) methyltransferase, mediates histone methylation. We show that ESET expression is markedly increased in HD patients and in transgenic R6/2 HD mice. Similarly, the protein level of trimethylated histone H3 (K9) was also elevated in HD patients and in R6/2 mice. We further demonstrate that both specificity protein 1 (Sp1) and specificity protein 3 (Sp3) act as transcriptional activators of the ESET promoter in neurons and that mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic, interferes with the DNA binding of these Sp family transcription factors, suppressing basal ESET promoter activity in a dose dependent manner. The combined pharmacological treatment with mithramycin and cystamine down-regulates ESET gene expression and reduces hypertrimethylation of histone H3 (K9). This polytherapy significantly ameliorated the behavioral and neuropathological phenotype in the R6/2 mice and extended survival over 40%, well beyond any existing reported treatment in HD mice. Our data suggest that modulation of gene silencing mechanisms, through regulation of the ESET gene is important to neuronal survival and, as such, may be a promising treatment in HD patients.

Fig. 1.

Increased trimethylation of histone H3 [TMH-H3 (K9)] and altered ESET/SETDB1 expression are found in striatal neurons of HD patients. Immunocytochemical staining for TMH-H3 (K9) (A–C) and ESET (D–F) is markedly increased in the caudate nucleus of HD grades 2 (B and E) and 3 (C and F) brains compared with control brains displaying weak expression of TMH-H3 (K9) (A) and ESET (D). (Scale bars, 100 μm.) (G) Immunoreactivity of ESET is colocalized with NeuN immunoreactivity in caudate neurons in brains from HD patients. (H) Western blots show increased TMH-H3 (K9) and ESET protein expression in the caudate tissue from HD patients (Left). Densitometric analysis of ESET protein levels in control (n = 5) and HD (n = 5) (Right). ∗, Significantly different from control at P < 0.05.

Fig. 2.

ESET promoter activity is up-regulated by Sp3 transcription factor and suppressed by mithramycin. (A) Mouse ESET promoter activity was determined by using a series of deletion reporter constructs that include Sp binding elements (−450/+80, −300/+80, −150/+80, −130/+80, −115/+80, −100/+80, −90/+80, and −10/+80). (B) ESET promoter (−130/+80) activity is up-regulated by Sp3. (C) Sp3-induced ESET promoter activity is down-regulated by mithramycin. (D) Mithramycin (−150/+80) suppresses basal ESET promoter activity in a dose-dependent manner. (E) Cystamine did not suppress ESET promoter activity. (F) Combined mithramycin and cystamine inhibited ESET promoter activity. ∗, Significantly different from 0 dosage at P < 0.05. (G) Distamycin, an anthracycline analogue, did not suppress ESET promoter activity. The fold change of ESET promoter activity was normalized to pGL3-B vector value (D–G). The error bars indicate the SE of three combined experiments.

Fig. 3.

Pharmacological treatment regulates gene expression and protein levels of ESET/SETDB1 in R6/2 mice. (A) Combined mithramycin and cystamine treatment significantly decreased the expression of the ESET gene in R6/2 mice. RT-PCR was performed by using total RNA. The expression level of ESET was normalized to 18S RNA. ∗, Significantly increased compared with WT at P < 0.05; ∗∗, significantly decreased compared with vehicle-treated R6/2 mice at P < 0.01. (B) Combined treatment reduced the protein level of ESET in R6/2 mice. The expression level of ESET was normalized to α-tubulin (α-Tub). ∗, Significantly increased compared with WT at P < 0.05; #, significantly decreased compared with vehicle-treated R6/2 mice at P < 0.05. (C) ESET immunoreactivity in striatal neurons was markedly increased in R6/2 mice (b) compared with WT mice (a). The polytherapy attenuated the ESET immunoreactivity (c). (Scale bar, 50 μm.)

Fig. 4.

Pharmacological treatment modulates the level of TMH-H3 (K9) in R6/2 mice. (A) The TMH-H3 (K9) immunoreactivity in striatal neurons was markedly increased in R6/2 mice (b) compared with WT mice (a). Polytherapy attenuated the TMH-H3 (K9) immunoreactivity (c). (Magnification, ×10; scale bar, 200 μm.) (B) Combined mithramycin and cystamine treatment reduced mtHtt aggregates and sequestration of TMH-H3 (K9) by mtHtt in R6/2 mice. (a, d, and g) MtHtt (green). (b, e, and h) TMH-H3 (K9) (red). (c, f, and i) Overlay images. (C) Mithramycin and cystamine combinational treatment significantly reduced the level of TMH-H3 (K9) in R6/2 mice. The level of TMH-H3 (K9) was normalized to total histone H3 level. (D) Densitometric analysis of TMH-H3 (K9) protein levels from the data in C. ∗, Significantly increased compared with WT at P < 0.05; #, significantly decreased compared with vehicle-treated R6/2 mice at P < 0.05.

Fig. 5.

Combined mithramycin and cystamine treatment extends survival and improves the clinical and neuropathological sequelae in R6/2 mice. (A) Kaplan–Meier probability of survival analyses of mithramycin and cystamine alone and combined treatment in R6/2 mice. (B) Effects of polytherapy on body weight in R6/2 mice. (C) Effects of combined treatment on rotarod performance. The motor performance in R6/2 mice was significantly improved throughout the temporal sequence of the experiment. ∗, P < 0.01. (D) Photomicrographs of coronal sections from the rostral neostriatum through the level of the anterior commissure in a WT littermate mouse (a), a PBS-treated R6/2 mouse (b), and a mithramycin and cystamine-treated R6/2 mouse (c) at 90 d. There was gross atrophy of the brain in the PBS-treated R6/2 mouse along with ventricular hypertrophy (b) as compared with the WT littermate control mouse (a). In contrast, the mithramycin and cystamine-treated R6/2 mouse brain (c) showed reduced gross brain atrophy and ventricular enlargement compared with the PBS-treated R6/2 mouse (b). Corresponding Nissl-stained tissue sections from the dorsomedial aspect of the neostriatum in a WT littermate control (d), PBS-treated R6/2 mouse (e), and mithramycin and cystamine-treated R6/2 mouse (f) are also shown. There was significantly less neuronal atrophy (P < 0.01) in the mithramycin and cystamine-treated R6/2 mouse compared with the PBS-treated R6/2 mouse. Reduction of mtHtt immunoreactivity in the mithramycin and cystamine-treated R6/2 mice (i). (Scale bars: Da, 2 mm; Df, 100 μm.)