Up-regulation of upstream stimulatory factors by protein malnutrition and its possible role in regulation of the IGF-binding protein-1 gene

Protein malnutrition drastically induces the expression of the IGF-binding protein-1 (IGFBP-1) gene. We have previously shown that the region between -77 and -112 bp upstream of the rat IGFBP-1 gene contributes to the respons e of this gene to amino acid limitation. In an attempt to elucidate the bas is of the responsiveness of this putative amino acid response unit (AARU), we searched the nucleus of the rat liver for a trans-acting factor whose bi nding to AARU was dependent on protein nutrition. Liver nuclear extracts of rats fed a protein-free diet and of those fed a control diet were compared by EMSA using the AARU as probe. One of the protein-probe complexes underw ent a drastic increase after dietary protein deprivation. Assays using spec ific antibodies and several competitor oligonucleotides led to identificati on of the protein composing the complex as upstream stimulatory factor-1 (U SF) and USF-2. The binding site of the USF proteins in the AARU turned out to be a CACGGG sequence that was homologous to the consensus USF-binding se quence (E box; CANNTG). Further, Western blot analyses showed that a protei n-free diet caused significant increases in USF-1 and USF-2 levels. Thus, e levated expression of the IGFBP-1 gene under protein malnutrition can be at tributable to increased binding of USF to its promoter, which results from increased USF levels. The data suggest that the changes in these ubiquitous ly distributed transcription factors play an important role in the nutritio nal regulation of expression of mammalian genes.