Nuclear import and DNA‐binding activity of RFX1

Abstract

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 DNAbinding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent`acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.Keywords: HBV enhancer; nuclear localization signal; nuclear retention; transcription factor; acidic stretch.The RFX family of DNA-binding proteins is highly conserved in evolution and includes members from yeast, Caenorhabditis elegans, mice, and humans, which function in various biological systems [1,2]. RFX1 has been implicated in transcriptional regulation of various viral and cellular genes. Its EP (EF-C) binding site in the enhancers of hepatitis B virus (HBV), polyomavirus, equine infectious anemia virus LTR [3,4], cytomegalovirus and Epstein-Barr virus [5], as well as binding sites in the promoters of MHC class II (X box) [6,7], ribosomal protein rpL30 [8], and interleukin-5 receptor a5 [9] genes, are positively acting regulatory elements in their natural context. In contrast, multimers of the EP site have no stimulatory effect on their own, and can silence the transcriptional activity of different enhancers [3,10]. The EP site was also shown to participate in transcriptional repression induced by p53 [11]. RFX1 binding sites in the intron of c-myc gene [10,12], proliferating cell nuclear antigen promoter [13] and the collagen a2(I) gene [14] were implicated in transcriptional down-regulation. By functional dissection, we demonstrated that RFX1 possesses transcriptional activation and repression domains [15] these domains function independently, and can mutually neutralize each other's effect. Although multiple activities have been assigned for RFX1, in the absence of RFX1-deficient animals or cells, the exact physiological ...

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Regulation of FGF1 Gene Promoter through Transcription Factor RFX1

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