Characterization of the NASP promoter in 3T3 fibroblasts and mouse spermatogenic cells

NASP (nuclear autoantigenic sperm protein) is a histone H1 binding protein expressed in all cells undergoing division [Richardson, R.T., Batova, I.N., Widgren, E., Zheng, L.-X., Whitfield, M., Marzluff, W.F., O'Rand, M.G., 2000. Characterization of the histone H1 binding protein, NASP, as a cell cycle regulated, somatic protein. J. Biol. Chem. 275, 30378-30386]. We have previously reported the sequence for the mouse NASP gene and analyzed its proximal promoter region in silico to determine putative regulatory regions [Richardson, R.T., Bencic, D.C., and O'Rand, M.G., 2001. Comparison of mouse and human NASP genes and expression in human transformed and tumor cell lines. Gene 274, 67-75]. In this report we describe various factors regulating the transcription of NASP. Luciferase assays using 3T3 fibroblasts show that the region + 9 to − 135 nt (PR1C) provides the core transcriptional activity for NASP and that extending this region out to − 976 nucleotides partially represses activity. However, when luciferase reporter assays were done in transfected pachytene spermatocytes, the cells that exhibit the highest NASP expression, a different gene regulation picture was revealed. In spermatogenic cells, PR1C is still a relatively strong core promoter, but unlike 3T3 cells, if the construct is extended to − 3002 nucleotides there is marked enhancement of transcription. Electrophoretic mobility shift assays with 3T3 nuclear extracts were used to study the PR1C core promoter in greater detail. In the region immediately upstream of the transcription initiation site we identified two closely associated Sp1 binding sites and a binding site for an Ets family member. Supershift assays further confirmed the presence of Sp1 bound to their respective sites suggesting that Sp1 and Ets are the primary activators of the NASP promoter.