An approximately 2.3-kilobase pair fragment of the nACh receptor β4 subunit 5′-flanking DNA (Fig. 1A) was subcloned into the β-gal expression vector, pSG-MAR (Fuchs et al., 2002) generating the construct pSGB4SH (Fig. 1B). This region of the β4 promoter contains two transcriptional regulatory elements we previously characterized in vitro in the context of a luciferase-based reporter vector. These elements are referred to as a CT box and a CA box based on their nucleotide compositions (Fig. 1C). The 19-base pair CT box contains 3 repeats of the sequence 5′-CCCT-3′ (Hu et al., 1995) and directly interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Purα (Du et al., 1998; Du et al., 1997). The CA box (5′-CCACCCC-3′) directly interacts with the general transcription factors Sp1 and Sp3 (Bigger et al., 1996; Bigger et al., 1997) and the more spatially-restricted factor Sox10 (Liu et al., 1999). To confirm that the transcriptional activity of pSGB4SH, a β-gal-based system, is similar to that of the previous β4/luciferase constructs, transient transfections were carried out using the rat pheochromocytoma cell line, PC12 (Greene and
