LETTERS 76 VOLUME 35 | NUMBER 1 | SEPTEMBER 2003 NATURE GENETICS Huntingtin protein is mutated in Huntington disease 1 . We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild- type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE 3,4 . In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE- containing genes involved in the maintenance of the neuronal phenotype 5 . Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes. Huntington disease is a dominantly inherited disorder caused by a CAG expansion in the 5′ coding region of the gene HD (encoding huntingtin), resulting in the progressive neuronal death of striatal and cortical neurons. Various lines of evidence indicate that transla- tion of the trinucleotide repeat expansion into glutamine endows the protein with a newly acquired toxic activity 6 , but impairment of wild- type huntingtin function may also contribute to the disease 7 . Wild- type huntingtin sustains the production of cortically derived BDNF, a survival factor for the striatal neurons that die in the disease. In par- ticular, loss of huntingtin-mediated BDNF production leads to reduced trophic support to the striatum 2 . Wild-type and mutant huntingtin differently modulate BDNF production mostly by acting at the level of BDNF promoter II transcription 2,8,9 . We previously found that wild-type but not mutant huntingtin increases the activity of the 1.1-kb BDNF promoter II containing an NRSE 2 , a silencer of gene expression typical of several neuronal genes 3–5 . To elucidate the mechanisms by which wild-type but not mutant huntingtin stimulates transcription of BDNF, we first transfected parental ST14A neural cells 10 and their cellular derivatives overex- pressing full-length wild-type or mutant huntingtin 11 with a cat con- struct containing 300 bp of BDNF promoter II encompassing the NRSE. cat activity was higher in cells expressing full-length wild-type huntingtin but lower in cells expressing full-length mutant huntingtin than in parental cells (Fig. 1a). Transfection of the same cells with a construct containing the NRSE in the BDNF promoter alone fused to the heterologous thymidine kinase promoter (tkNRSE-cat) gave simi- lar results (Fig. 1b). Mutation of the NRSE abolished the effects of both wild-type and mutant huntingtin on BDNF promoter II activity (Fig. 1c). We conclude that the effects of wild-type and mutant hunt- ingtin on the activity of BDNF promoter II are mediated by the NRSE. We carried out the same experiment in other neural cell lines derived from knock-in mice in which a CAG expansion was inserted into the endogenous mouse gene Hdh 12 . Transfection of heterozygous and homozygous huntingtin knock-in cells with the tkNRSE-cat construct resulted in a progressive decrease in promoter activity, proportional to the loss of endogenous wild-type huntingtin (Fig. 1d). We also found progressively lower levels of BDNF exon II mRNA in heterozygous and homozygous knock-in cells compared with controls (Fig. 1e). Analogously, the level of BDNF exon II mRNA was approximately 65% lower in the cerebral cortex of heterozygous huntingtin knock-out mice 13 as compared with wild-type littermates, suggesting that tran- scription from promoter region II is influenced by the levels of wild- type huntingtin (Fig. 1f). Overexpression of wild-type huntingtin 1 Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy. 2 Centre for Molecular Medicine and Therapeutics Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. 3 Institute of Biotechnology, University of Helsinki, Helsinki, Finland and Department of Gene Technology, Tallinn Technical University and National Institute of Chemical Physics and Biophysics, Tallinn, Estonia. 4 These authors contributed equally to this work. Correspondence should be addressed to E.C. (elena.cattaneo@unimi.it). Published online 27 July 2003; doi:10.1038/ng1219 Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes Chiara Zuccato 1,4 , Marzia Tartari 1,4 , Andrea Crotti 1 , Donato Goffredo 1 , Marta Valenza 1 , Luciano Conti 1 , Tiziana Cataudella 1 , Blair R Leavitt 2 , Michael R Hayden 2 , Tõnis Timmusk 3 , Dorotea Rigamonti 1 & Elena Cattaneo 1 © 2003 Nature Publishing Group http://www.nature.com/naturegenetics