Purification and characterization of nucleolin and its identification as a transcription repressor.

Abstract

Expression of the acute-phase response genes, such as that for alpha-i acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.The initiation of transcription in eukaryotes is an intricately controlled process. Short sequence motifs in the promoter regions of genes interact in a specific manner with DNAbinding transcription factors. These bound factors interact with general transcription factors and thereby result in gene transcription. Not only transcriptional activators but also repressors are important in the controlled regulation of gene expression. For a given gene, the combinations of cis elements and the trans-acting factors are major determinants of transcriptional activity. Protein-protein interactions and posttranslational modifications are important for regulating the activities of these factors. An array of transcriptional activators and repressors have been identified and characterized (9,12,14,15,18,24,35,37).Tissue injury and infection produce significant alterations of the host metabolic and immune homeostasis (19). It has become increasingly clear that many of these changes result from a complex cascade of mononuclear phagocyte-derived endogenous mediators, in particular, cytokines. Injection of purified lipopolysaccharide (LPS) into laboratory animals leads to the development of many biological activities with similarities to those that follow tissue injury and infection. These can range from an acute-phase response to shock with lethal outcome. The well-studied biological activities of LPSinduced liver gene expression are mediated by multiple cytokines, including interleukins 1, 6, and 11, leukemia inhibitory factor, and tumor necrosis factor alpha (13,19,30,31). We used LPS-induced transcription of the alpha-1 acid glycoprotein (AGP) gene as a model for studying the regulation of gene expression during the acute-phase response (9, 24). Transcription of the AGP gene in response to LPS treatment is regulated by both a positive factor, AGP/EBP (C/EBP-P), and a negative factor, factor B (24). During the acute-phase * Corresponding author. Mailing address: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taip...

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