Nuclear import and DNA-binding activity of RFX1 - Evidence for an autoinhibitory mechanism

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 thi s conserved region mediates dimerization, and is followed by a unique C-ter minal tail, containing a highly acidic stretch. In HL-60 cells nuclear tran slocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuc lear localization signal (NLS) at the extreme C-terminus. The adjacent 'aci dic region', which showed no independent NLS activity, potentiated the func tion of the NLS. Subcellular fractionation showed that the tight associatio n of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanc ed by the dimerization domain. In contrast, the acidic region inhibited nuc lear association, by down-regulating the DNA-binding activity of RFX1. Thes e data suggest an autoinhibitory interaction, which may regulate the functi on of RFX1 at the level of DNA binding. The C-terminal tail thus constitute s a composite localization domain, which on the one hand mediates nuclear i mport of RFX1, and on the other hand inhibits its association with the nucl eus and binding to DNA. The participation of the acidic region in both acti vities suggests a mechanism by which the nuclear import and DNA-binding act ivity of RFX1 may be coordinately regulated by phosphorylation by kinases s uch as PKC.