Tyrosine hydroxylase (TH), the biosynthetic enzyme of catecholamine, is synthesized specifically in catecholaminergic neurons. Thus, it is possible that neuronal cell type-specific expression of this gene is coordinately regulated. One of the neuron-specific transcription regulators, neuron-restrictive silencer factor (NRSF)/repressor element I (REI) silencing transcription factor (REST), represses the expression of neuronal genes in non-neuronal cells. To elucidate the molecular mechanisms that control catecholaminergic neuronal expression of human TH, we initially characterized the 5' regulatory region. Previous studies have shown that a 3174 bp fragment of the human TH promoter confers specific expression to the reporter gene in dopaminergic neuron-like cell lines. Within this 5' regulatory region, three putative neuron-restrictive silencer elements (NRSE)/RE1 were identified, which bound NRSF/REST in a sequence-specific manner, as confirmed using EMSA and ChIP assays. In transient transfection assays, deletion or mutation of NRSE/RE1 elements led to a 7-fold increase in activity of the 3.2 kb TH promoter in human neural stem cells (NSCs), but had no major effects on differentiated neuron-like cells. Suppression of NRSF/REST functions with either the historic deacetylase inhibitor, trichostatin, or DN-NRSF induced TH promoter activity. Our data strongly suggest that NRSF/REST functions as a repressor of TH transcription in NSCs via a mechanism dependent on the TH NRSE/RE1 sites. (c) 2006 Elsevier Inc. All rights reserved.