Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner.
Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers.
Apt
D
D
Institute of Molecular and Cell Biology, National University of Singapore.
Liu
Y
Y
Bernard
H U
HU
D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic; D004848:Epithelium / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics* / Q000494:pharmacology; D027383:Papillomaviridae / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors / Q000235:genetics* / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; C077305:CTF-1 transcription factor; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; C497647:NFIC protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004247:DNA