Widespread disruption of repressor element-1 silencing transcription factor/neuron-restrictive silencer factor occupancy at its target genes in Huntington's disease

Abstract

Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

Figure 1.

Endogenous REST/NRSF binding at RE1/NRSEs is increased in HD leading to repression of RE1/NRSE gene transcription. a, Left, ChIP analysis on six RE1/NRSE selected loci in neural cells established from homozygous (Hdh^109/109) knock-in mice and wild-type littermates (Hdh^7/7). Data represent the average of three different experiments. Right, mRNA levels of the same RE1/NRSE-controlled genes in Hdh^7/7 and Hdh^109/109 cells by real-time PCR. Data are the average of three different experiments. *p < 0.05; **p < 0.01 versus Hdh^7/7; ANOVA test. b, Left, ChIP on cortical lysates from R6/2 transgenic mice at 12 weeks of age and corresponding littermates. Right, mRNA levels by real-time PCR of the same RE1/NRSE genes in R6/2 transgenics and controls at the same age. ChIP and mRNA data represent the average of five R6/2 transgenics and five controls. *p < 0.05; ** p < 0.01 versus littermates, ANOVA test. c, Left, ChIP for REST/NRSF on cortical lysates from Hdh^109/109 at 5 months. Data represent the average of three Hdh^109/109 and three Hdh^7/7 mice. *p < 0.05 versus Hdh^7/7 mice; ANOVA test. Right, REST/NRSF binding at the NRSE^bdnf in Hdh^109/109 at 1 and 5 months. Data are the average of three Hdh^109/109 and three Hdh^7/7 mice at 1–5 months. *p < 0.05 versus Hdh^7/7 mice; ANOVA test. d, ChIP for REST/NRSF on liver lysates from three R6/2 mice and three controls at 12 weeks of age. *p < 0.05 versus wild-type mice; ANOVA test.

Figure 2.

Loss of wild-type huntingtin increases REST/NRSF binding at the RE1/NRSE. a, Left, ChIP for REST/NRSF binding at the RE1/NRSE^Bdnf on chromatin lysates from Hdh+/+, Hdh+/−and Hdh−/− ES cells. Right, REST/NRSF mRNA levels in Hdh+/+, Hdh+/− and Hdh−/− ES cells. Data are the average from three independent experiments. *p < 0.05 versus Hdh+/+, ^#p < 0.05 versus Hdh+/−; ANOVA test. b, Left, Real-time PCR for total Bdnf mRNA levels in Hdh+/+, Hdh +/−, and Hdh−/− embryonic stem cells. Data are the average from three independent experiments. *p < 0.05 versus Hdh+/+; ^#p < 0.05 versus Hdh+/−; ANOVA test. Right, Semiquantitative radioactive RT-PCR for Bdnf exon II, III, and IV mRNA levels. β-actin was used as control. Graphs show densitometric analyses of the expression levels of Bdnf mRNA II, III, and IV compared with levels of β-actin. Values represent the percentage of arbitary units (a.u.) relative to Hdh+/+ cells to which 100% was assigned. Data are the average from eight independent PCR experiments. *p < 0.05 versus Hdh+/+; ^#p < 0.05 versus Hdh+/−; ANOVA test. c, ChIP for REST/NRSF on cortical samples from three null Hdh conditional knock-out mice cre Hdh flox/− (indicated as Hdh−/−) and three Hdh flox/− (indicated as Hdh+/−) and wild-type mice. REST/NRSF binding was analyzed at the RE1/NRSE of Bdnf, Syn1, Chrm4, Drd3, Penk1, and Chrnb2. *p < 0.05 versus Hdh+/+ mice; ^#p < 0.05 versus Hdh+/− mice; ANOVA test. d, Bdnf levels in cortical samples from null Hdh conditional knock-out mice. Left, Total Bdnf mRNA levels by real-time PCR. Data are the average from three independent experiments. *p < 0.05; **p < 0.01 versus Hdh+/+ mice; ^#p < 0.05 versus Hdh+/− ANOVA test. Right, Semiquantitative radioactive RT-PCR for Bdnf exon II, III, and IV mRNA levels. β-actin was used as control. A quantitative evaluation of Bdnf mRNA II, III, and IV is shown in supplemental Figure 7 (available at www.jneurosci.org as supplemental material).

Figure 3.

Expression of DN:REST derepressed RE1/NRSE-regulated transcription in HD. a, Bdnf, synapsin-1, and M4 muscarinic cholinergic receptor mRNA levels were measured in native Hdh^7/7 and Hdh^109/109 cells after delivery of DN:REST or empty vector. Bdnf, synapsin-1, and M4 muscarinic cholinergic receptor mRNA levels were restored in Hdh^109/109 cells infected with DN:REST compared with untransfected cells and cells transfected with empty vector (− and E, respectively). mRNA levels of REST/NRSF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), genes whose transcription is not regulated by the RE1/NRSE silencer, are not affected by DN:REST expression. Data are expressed as percentage of controls (100% was assigned to Hdh^7/7 cells) and represent the average of four independent experiments. *p < 0.05 versus Hdh^7/7 untreated cells (−); ^#p < 0.05 versus Hdh^109/109 untreated cells and cell with empty vector (− and E, respectively). b, Left, Bdnf exon II, III, and IV mRNA levels in Hdh^7/7 and Hdh^109/109 cells by semiquantitative radioactive RT-PCR. Right, Quantitative analyses of the BDNF mRNA levels. The peak densitometric areas relative to Bdnf II, III, and IV were normalized over the peak densitometric area of the β-actin band. Data are expressed as percentage of controls (100% was assigned to Hdh^7/7 untreated cells) and represent the average of three independent experiments. *p < 0.05 versus Hdh^7/7 cells; ^#p < 0.05 versus Hdh^109/109 untreated cells and cells with empty vector (− and E, respectively). c, Bdnf protein levels determined by ELISA on lysates from Hdh^7/7 and Hdh^109/109 cells and cells infected with DN:REST and empty vector. Data are the average of three independent experiments. *p < 0.05 versus Hdh^7/7 untreated cells (−). ^#p < 0.05 versus Hdh^109/109 untreated cells and cells with empty vector (− and E, respectively).

Figure 4.

REST/NRSF binding at the RE1/NRSE is increased in HD patients. a, ChIP scanning at the human BDNFlocus. ChIP analysis of cortical tissue from human HD (3723 and 2866) and control (3899 and 3688) samples. Anti-REST/NRSF-immunoprecipitated genomic DNA was assessed by real-time PCR using six pairs of oligonucleotide primers designed to produce amplicons covering the RE1/NRSE site in the BDNF gene and flanking sequences located 758 bp upstream the RE1/NRSE and 431, 1222, 2279, and a distal 19350 bp downstream of the RE1/NRSE. Relative positions of these primer pairs are indicated in the schematic diagram (green arrows). White columns represent the average of REST/NRSF enrichment in controls. Gray columns indicate the average of REST/NRSF enrichment in HD subjects. The scanning ChIP assay shows a peak of REST/NRSF occupancy centered at the RE1/NRSE site of the BDNF gene. No enrichment is seen at distal regions, and no enrichment is seen at the coding region of BDNF and β-actin gene, which is not regulated by REST/NRSF. b, REST/NRSF was immunoprecipitated from chromatin extracted from the cerebral cortices of five HD patients (from left to right: 3484, 3723, 3176, 2866, 6062) and five age-matched controls (from left to right: 3899, 3688, 3932, 3888, 3746). REST/NRSF enrichment over a sham antibody (preimmune serum) was calculated by quantitative PCR at the RE1/NRSEs of five REST/NRSF target genes, BDNF, SYN1, CHRM4, CHRNB2 and SNAP25. c, Conventional ChIP and ChIP on chip indicated that REST/NRSF target genes showing increased occupancy in HD subjects can be assigned to nine functional groups.