STRESS INDUCTION OF THE MAMMALIAN GRP78 BIP PROTEIN GENE - IN-VIVO GENOMIC FOOTPRINTING AND IDENTIFICATION OF P70CORE FROM HUMAN NUCLEAR EXTRACT AS A DNA-BINDING COMPONENT SPECIFIC TO THE STRESS REGULATORY ELEMENT/

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calciu m sequestration in the endoplasmic reticulum. When the cells are subje cted to endoplasmic reticulum stress, in particular the depletion of s tored calcium and/or the accumulation of abnormal proteins, the rate o f transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95 % conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3 ' half of the grp core, whereas other regulatory elements are constitu tively occupied. This inducible binding to the 3' half of the human gr p78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack o f constitutive in vivo protection at this region is not due to the abs ence of a binding factor in nuclear extracts. Using in vitro gel mobil ity shift assays, we detected a constitutive binding activity which ex hibits specificity and affinity to the stress-inducible region. Throug h sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fract ionation and renaturation analysis, the activity is found in polypepti des with molecular sizes of 65 to 75 kDa. After a three-step purificat ion scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70 CORE is sufficient to form a complex specific to the stress inducible region.