HDAC inhibition imparts beneficial transgenerational effects in Huntington's disease mice via altered DNA and histone methylation

Abstract

Increasing evidence has demonstrated that epigenetic factors can profoundly influence gene expression and, in turn, influence resistance or susceptibility to disease. Epigenetic drugs, such as histone deacetylase (HDAC) inhibitors, are finding their way into clinical practice, although their exact mechanisms of action are unclear. To identify mechanisms associated with HDAC inhibition, we performed microarray analysis on brain and muscle samples treated with the HDAC1/3-targeting inhibitor, HDACi 4b. Pathways analyses of microarray datasets implicate DNA methylation as significantly associated with HDAC inhibition. Further assessment of DNA methylation changes elicited by HDACi 4b in human fibroblasts from normal controls and patients with Huntington's disease (HD) using the Infinium HumanMethylation450 BeadChip revealed a limited, but overlapping, subset of methylated CpG sites that were altered by HDAC inhibition in both normal and HD cells. Among the altered loci of Y chromosome-linked genes, KDM5D, which encodes Lys (K)-specific demethylase 5D, showed increased methylation at several CpG sites in both normal and HD cells, as well as in DNA isolated from sperm from drug-treated male mice. Further, we demonstrate that first filial generation (F1) offspring from drug-treated male HD transgenic mice show significantly improved HD disease phenotypes compared with F1 offspring from vehicle-treated male HD transgenic mice, in association with increased Kdm5d expression, and decreased histone H3 Lys4 (K4) (H3K4) methylation in the CNS of male offspring. Additionally, we show that overexpression of Kdm5d in mutant HD striatal cells significantly improves metabolic deficits. These findings indicate that HDAC inhibitors can elicit transgenerational effects, via cross-talk between different epigenetic mechanisms, to have an impact on disease phenotypes in a beneficial manner.

Keywords: epigenetic; histone; neurodegenerative; therapeutic; transgenerational.

Fig. 1.

Differential DNA methylation due to HDAC inhibitor treatment. Data for CpG sites differentially methylated between HDACi 4b-treated and vehicle-treated (delta beta > 0.2) in normal and HD human fibroblasts on the 27K DNA methylation array. Yellow denotes a decrease in methylation due to HDACi 4b treatment; black denotes an increase in methylation. The total number of DMPs in each cell type is shown along the y axis. Chr., chromosome, SRY, sex-determining region of Chr Y; SYNPR, synaptoporin; TSPY4, testis-specific protein, Y-linked 1; TTTY14, testis-specific transcript, Y-linked 14.

Fig. 2.

Effect of HDACi 4b on DNA methylation at the Kdm5d locus in mouse sperm DNA. The DNA methylation array revealed two regions increased in methylation in the Kdm5d locus with HDACi 4b treatment. Two pairs of primers were designed to verify each region in ±100 bp of the methyl-array probe site. Sites 1 and 2 are designed to detect a promoter region of 250 bp upstream of the Kdm5d transcription start site. Sites 3 and 4 are designed to detect a region 30.18 kb downstream of the Kdm5d transcription start site. (A) HDACi 4b treatment significantly increased the methylation signals in the promoter “site 1” region of Kdm5d but not in the “site 2” region. (B) Both “sites 3 and 4” increased the methylation signal with HDACi 4b treatment. (C) Promoter CpG region of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is a negative control, and Intracisternal A-particle (IAP) and H19 imprinted maternally expressed transcript (H19) are maternally imprinted positive controls for DNA methylation. All relative methylation was normalized to the methylation level of the Gapdh promoter region. Statistical differences were determined by a two-tailed Student t test: *P < 0.05 vs. vehicle group (n = 6).

Fig. 3.

Comparisons of disease phenotypes in offspring of male N171-82Q transgenic mice treated with HDACi 4b (F1HDTg-4b) relative to offspring of N171-82Q transgenic mice treated with vehicle (F1HDTg-veh). (A) Effects of HDACi 4b exposure on the body weights of N171-82Q transgenic mice. (B) Rotarod performance of offspring of vehicle- and HDACi 4b-exposed N171-82Q transgenic mice. Males and females are shown separately. Two-way ANOVA revealed significant differences between male F1HDTg-4b mice compared with male F1HDTg-veh mice, whereas female mice did not show statistically significant differences. N.S., not significant. (C) Number of correct choices of first 10 trials in the alternating T-maze test. One-way ANOVA revealed significant differences between vehicle-exposed WT and N171-82Q transgenic mice (**P < 0.001) and a significant difference between vehicle-exposed and HDACi 4b-exposed N171-82Q mice (*P < 0.05). Both male and female mice were combined in this assay. (D and E) Effects of HDACi 4b exposure on open-field activity of N171-82Q transgenic mice over a 10-min test period. Two-way ANOVA revealed significant differences between HDACi 4b-exposed and vehicle-exposed N171-82Q transgenic mice for distance, ambulatory time, stereotypic time, and average velocity only in male mice, as indicated.

Fig. 4.

(A) Real-time qPCR results showing altered expression of Kdm5d in cortex of male offspring of vehicle-treated or HDACi 4b-treated male mice. Values shown are the mean ± SEM expression value from n = 4–5 mice per group. One-way ANOVA was used to determine significant differences, and these are indicated by asterisks: **P < 0.001; ***P < 0.0001. (B) Western blot analysis showing elevated Kdm5d protein and reduced H3K4me expression in cortex of male offspring of vehicle-treated (F1HD-veh) and HDACi 4b-treated (F1HD-4b) male HD transgenic mice. Representative blots are shown, although n = 8 F1HD-veh and n = 9 F1HD-4b samples were analyzed in total. (Right) Bar graph quantification of band signals is shown. Student t test was used to determine significance, as indicated by asterisks: *P < 0.05. (C) Correlation between Kdm5d expression levels and H3K4me levels. Pearson correlation analysis of Western blot data for Kdm5d and H3K4me was used, with each point representing a different sample (R = −0.502, P = 0.045). (D) Improvement of the Htt-elicited metabolic deficits in STHdh^Q111 striatal cells elicited by KDM5D overexpression. Cells were transfected with human KDM5D cDNA using FuGENE transfection reagent and XTT assays performed after 24 h. Significant differences were determined by one-way ANOVA, followed by a Dunnett’s posttest comparing to the mock-transfected control: *P < 0.05; ***P < 0.0001.