Overexpression of the linker histone-binding protein tNASP affects progression through the cell cycle

Abstract

NASP is an H1 histone-binding protein that is cell cycle-regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells, and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386). When full-length tNASP fused to green fluorescent protein (GFP) is transiently transfected into HeLa cells, it is efficiently transported into the nucleus within 2 h after translation in the cytoplasm, whereas the NASP nuclear localization signal (NLS) deletion mutant (NASP-ΔNLS-GFP) is retained in the cytoplasm. In HeLa cells synchronized by a double thymidine block and transiently transfected to overexpress full-length tNASP or NASP-ΔNLS, progression through the G1/S border is delayed. Cells transiently transfected to overexpress the histone-binding site (HBS) deletion mutant (NASP-ΔHBS) or sNASP were not delayed in progression through the G1/S border. By using a DNA supercoiling assay, in vitro binding data demonstrate that H1 histone-tNASP complexes can transfer H1 histones to DNA, whereas NASP-ΔHBS cannot. Measurement of NASP mobility in the nucleus by fluorescence recovery after photobleaching indicates that NASP mobility is virtually identical to that reported for H1 histones. These data suggest that NASP-H1 complexes exist in the nucleus and that tNASP can influence cell cycle progression through the G1/S border through mediation of DNA-H1 histone binding.