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Nuclear Factor I (NFI) proteins are DNA binding proteins functioning as transcription and replication factors. As part of a study of the diversity of the Nuclear Factor I protein family, we isolated and sequenced seven NFI cDNA clones from a chicken promacrophage library. Five of these clones are derived from the NFI-A gene, the other two from the NFI-C gene. Comparison of the deduced chicken NFI amino acid sequences with mammalian NFI sequences reveals that there are corresponding vertebrate isoforms, indicating a strong conservation of NFI structure during evolution. This finding leads to the prediction that more mammalian isoforms will be discovered corresponding to the chicken NFI variants reported here.

Co-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.

Polymerase II transcription of a human gene for the small nuclear RNA U2 is dependent on two different promoter elements: a TATA-equivalent proximal sequence element and a distal enhancer element, which has been shown to contain Sp1- and octamer-binding sites. We have investigated the functional interplay between these transcription factor-binding sites of the enhancer, following transfection of U2 maxigene constructions into HeLa cells. There is a functional non-additive co-operation between the octamer-binding factor and Sp1, which is not dependent on the evolutionally conserved steric arrangement of these binding sites. We demonstrate that the conserved Sp1-binding site of the U2 enhancer can be fully substituted by a nuclear factor I (NFI) binding site, and that the octamer-binding factor functions in stimulating transcription in conjunction with either Sp1 or NFI. Since the octamer-binding factor is most probably the same protein as nuclear factor III (NFIII), the results imply that the NFI/NFIII complex, involved in adenovirus DNA replication, also can function as an efficient activator of transcription.

Employing an exonuclease III protection assay we detected a protein in crude HeLa nuclear extracts binding, with apparent sequence specificity, to molecular ends of adenovirus type 2 (Ad2) DNA. This protein, designated nuclear factor IV (NFIV), was purified to homogeneity and was shown to be a hetero-dimer of 72,000 and 84,000 Mr. Binding to terminal Ad2 sequences was strongly enhanced by the presence of either of the sequence-specific DNA-binding proteins nuclear factor I and nuclear factor III. These proteins appeared to function as blockades for translocation of NFIV on DNA, thus producing apparent sequence specificity. In the absence of such a blockade, NFIV moved freely, without energy input, on any double-stranded DNA forming a regular DNA-multimeric protein complex as shown by methidiumpropyl EDTA footprinting and electron microscopy. Binding is completely dependent upon the presence of molecular ends. Evidence was obtained for a two-step mechanism in which termini are recognized by NFIV and used as a starting point for subsequent translocation. The possible functions of the protein in adenovirus DNA replication and in cellular processes requiring DNA termini are discussed.