Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene Jingang Gui a , Yan Song a , Nian-Lin R. Han a , Shu-Feng Zhou c , Fwu-Shan Sheu a,b, * a Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore b The University Scholars Program, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore c Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore Received 8 April 2006 Available online 25 April 2006 Abstract Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5 0 -deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258 bp 5 0 -flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Neurogranin; GC-rich sequence; Sp1; Sp3; Transcription factors; Basal transcription activity; EMSAs; AT-rich motif; Promoter binding proteins Neurogranin/RC3 (Ng) is a postsynaptic protein that binds to calmodulin (CaM) in the absence of or in low lev- els of Ca 2+ [1,2]. It regulates the availability of Ca 2+ /CaM complex and modulates the homeostasis of intracellular calcium in neurons [3–5]. The Ng level in the central ner- vous system (CNS) is regulated by thyroid hormone, reti- noic acid, and vitamin A [6–10], whose insufficient levels are concomitantly linked with an age-related decrease in Ng level [11–14]. Ng gene transcription could be induced by triiodothyronine (T3) in hypothalamic GT1-7 cells [15]. Ng gene is composed of four exons and three introns and the first two exons encode the Ng protein. A thyroid hormone responsive element was identified in the first intron of the human Ng gene homolog [16,17]. Ng promot- er lacks the canonical TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites and the bas- al promoter activity could be up-regulated by phorbol ester and phorbol ester-binding PKCs in HEK 293 cells [18]. Sequence analysis on rat Ng promoter showed that a GC-rich sequence (+11 to +40) is probably an overlapping binding site for transcriptional factors and may be impor- tant for basal transcription activity of Ng [18]. Several pos- sible cis-regulatory elements including GR, GCF, SP-1, AP-1, AP-2, and PEA3-binding sites were predicted to reside in the 5 0 -flanking region of Ng gene [18,19].A 20 kDa chromosomal non-histone high-mobility-group protein (HMG) that binds to AT-rich regions of Ng pro- moter was revealed to be a PKC substrate whose binding affinity to Ng promoter was dramatically reduced upon phosphorylation [18,20]. Currently, only very sparse information is available for the roles of cis-regulatory elements and trans-factors in the transcription regulation of Ng. The goal of the 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.04.054 * Corresponding author. Fax: +65 67792486. E-mail address: dbssfs@nus.edu.sg (F.-S. Sheu). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 345 (2006) 124–132 BBRC