Nanog, Sox2, and Oct4 are transcription factors all essential to maintaining the pluripotent embryonic stem cell phenotype. Through a cooperative interaction, Sox2 and Oct4 have previously been described to drive pluripotent-specific expression of a number of genes. We now extend the list of Sox2-Oct4 target genes to include Nanog. Within the Nanog proximal promoter, we identify a composite sox-oct cis-regulatory element essential for Nanog pluripotent transcription. This element is conserved over 250 million years of cumulative evolution within the eutherian mammals. A Nanog proximal promoter-EGFP (enhanced green fluorescent protein) reporter transgene recapitulates endogenous Nanog mRNA expression in embryonic stem cells and their differentiated derivatives. Sox2 and Oct4 interaction with the Nanog promoter was confirmed through mutagenesis and in vitro binding assays. Electrophoretic mobility shift assays indicate that the Sox2-Oct4 heterodimer forms more efficiently on the composite element within Nanog than the similar element within Fgf4. Using chromatin immunoprecipitation, we show that Oct4 and Sox2 bind to the Nanog promoter in living mouse and human embryonic stem cells. Furthermore, by specific knockdown of Oct4 and Sox2 mRNA by RNA interference in embryonic stem cells, we provide genetic evidence for a link between Oct4, Sox2, and the Nanog promoter. These studies extend the understanding of the pluripotent genetic regulatory network within which the Sox2-Oct4 complex are at the top of the regulatory hierarchy.Nanog is a homeobox-containing transcription factor with an essential function in maintaining the pluripotent cells of the inner cell mass and in the derivation of embryonic stem cells (ESCs) 1 from these (1). Furthermore, overexpression of Nanog is capable of maintaining the pluripotency and self-renewing characteristics of ESCs under what normally would be differentiation-inducing culture conditions (2). Concomitant with this essential function in pluripotent cell maintenance is its restricted expression pattern. Nanog transcripts first appear in the inner cells of the morula prior to blastocyst formation (1, 2), are restricted to the inner cell mass in the blastocyst (3), and are no longer detectable at implantation. Expression of Nanog reappears in the proximal epiblast at embryonic day 6 and remains restricted to the epiblast as development progresses (4). The factors controlling expression of this gene have yet to be described. The POU domain-containing Oct4 and the HMG domaincontaining Sox2 are two other transcription factors known to be essential for normal pluripotent cell development and maintenance (5, 6). Although both have independent roles in determining other cell types (6, 7), at least part of their function in pluripotent cells is via a synergistic interaction between the two to drive transcription of target genes. Currently known targets of Sox2-Oct4 synergy are Fgf4, Utf1, and Fbx15, as well as Sox2 and Pou5f1 (the gene encoding Oct4) themselves (8 -13). Each of these targe...

N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal aminoterminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity. More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (K D ) of 127.5 6 45 nM, which was within the range of the IC 50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized K D .Keywords: NR2B NMDA receptor; protein refolding; circular dichroism; dissociation constant; dynamic light scattering N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that exhibit voltage-dependent Mg 2+ channel block. Under physiological conditions, NMDA receptors play vital roles in excitatory synaptic transmission, synaptic plasticity, learning, and memory formation as well as neuronal development (Dingledine et al. 1999). However, overactivation of these receptors can cause excessive Ca 2+ influx into the neurons, leading to excitotoxic neuronal cell death associated with ischemic stroke (Dirnagl et al. 1999;Lee et al. 1999) and head trauma (Obrenovitch and Urenjak 1997). The causative role of NMDA receptors has also been implicated in seizures (Meldrum et al. 1999). As such, NMDA receptors can be potential targets for therapeutic intervention in these pathological situations.The NMDA receptor consists of a complex of four or five subunits that include an obligatory NR1 subunit together with the NR2A-D or NR3A-B subunits (Dingledine et al. 1999). The architecture of each 4 These authors contributed equally to this work. Reprint requests to: Chian-Ming Low, Department of Pharmacology, MD2 #04-22, Faculty of Medicine, National University of Singapore, 18 Medical Drive, S117597, Singapore; e-mail: phclowcm@nus.edu.sg; fax: +(65) 68737690.Abbreviations: NMDA, N-methyl-D-aspartate; ATD, amino-terminal domain; CD, circular dichroism; DLS, dynamic light scattering; K D , dissociation constant; LIVBP, leucine/isoleucine/valine-binding protein; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; MS, mass spectrometry.Article and publication are at

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