Research Report Role of upstream stimulating factors in the transcriptional regulation of the neuron-specific K–Cl cotransporter KCC2 Marika Markkanen 1 , Pavel Uvarov 1 , Matti S. Airaksinen ⁎ Neuroscience Center, Viikinkaari 4, 00014 University of Helsinki, Finland ARTICLE INFO ABSTRACT Article history: Accepted 6 August 2008 Available online 13 August 2008 The neuron-specific K–Cl cotransporter (KCC2) maintains a low intracellular Cl - concentration in neurons and is necessary for fast hyperpolarizing responses to GABA and glycine. The mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. Expression of the major isoform, KCC2b, is strongly upregulated during neuronal maturation, and is modulated by neuronal activity, trauma, and neurotrophic factors. In the present study, we have focused on the regulatory influence of the upstream stimulating factors USF1 and USF2 via an E-box control element in the KCC2b promoter (E-box KCC2b ). Electrophoretic mobility shift assay in cell lines and chromatin immunoprecipitation in neurons demonstrated binding of endogenous USF1 and USF2 to the E-box KCC2b element. Mutation of the E-box KCC2b site resulted in reduced KCC2b promoter activity in cell lines and cortical neurons. Overexpression of a dominant-negative form of USF confirmed the involvement of endogenous USF proteins in the regulation of the KCC2b gene. The results suggest that binding of USF proteins to the E-box KCC2b may contribute to the upregulation of KCC2b gene expression in developing brain. © 2008 Elsevier B.V. All rights reserved. Keywords: KCC2 USF1 USF2 Promoter regulation EMSA ChIP Potassium–Chloride cotransport Neuronal chloride homeostasis Transcription factor 1. Introduction The neuron-specific K–Cl cotransporter KCC2 (for review, see Mercado et al., 2004; Payne et al., 2003) is necessary for the maintenance of the low Cl - concentration in mature neurons and thus required for hyperpolarizing actions of neurotrans- mitters GABA and glycine (Hubner et al., 2001; Rivera et al., 1999). We have recently reported the existence of two neuronal splice variants of KCC2, with alternative promoters and first exons (Uvarov et al., 2007). The main isoform, now termed KCC2b, is expressed exclusively in CNS neurons (Kanaka et al., 2001; Payne et al., 1996; Rivera et al., 1999). During embryonic development KCC2b mRNA expression is strongly upregulated and follows neuronal maturation (Li et al., 2002; Stein et al., 2004). KCC2b developmental upregulation seems to depend on neurotrophic factors (Aguado et al., 2003) and on neuronal activity (Kanold and Shatz 2006; Liu et al., 2006; Rivera et al., 2004). A significant reduction in KCC2b mRNA expression is observed after various forms of neuronal injury and patholo- gical neuronal activity (Nabekura et al., 2002; Rivera et al., 2002, 2004) and the resulting depolarizing shift in GABAergic transmission may contribute to the pathogenesis of temporal epilepsy and neuropathic pain (Cohen et al., 2002; Coull et al., 2003; Rivera et al., 2002). For this reason, it is important to study transcriptional mechanisms and regulatory elements in the KCC2b promoter. BRAIN RESEARCH 1236 (2008) 8 – 15 ⁎ Corresponding author. Fax: +3589 191 57620. E-mail address: matti.airaksinen@helsinki.fi (M.S. Airaksinen). Abbreviations: USF, upstream stimulating factor; bHLH, basic helix–loop–helix; EMSA, electrophoretic mobility shift assay; Egr4, early growth response 4; DIV, days in vitro; ChIP, chromatin immunoprecipitation 1 Contributed equally to this work. 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.007 available at www.sciencedirect.com www.elsevier.com/locate/brainres