Overexpression of the Linker histone-binding protein tNASP affects progression through the cell cycle.
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-DeltaNLS-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-DeltaNLS, progression through the G(1)/S border is delayed. Cells transiently transfected to overexpress the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS) or sNASP were not delayed in progression through the G(1)/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-DeltaHBS 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 G(1)/S border through mediation of DNA-H1 histone binding.
Alekseev
Oleg M
OM
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill 27599-7090, USA.
Bencic
David C
DC
Richardson
Richard T
RT
Widgren
Esther E
EE
O'Rand
Michael G
MG
D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics
D001324:Autoantigens; D018797:Cell Cycle Proteins; C489390:NASP protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; C067535:Nasp protein, mouse; D004247:DNA