IDENTIFICATION OF ESSENTIAL NUCLEOTIDES OF THE FP1 ELEMENT RESPONSIBLE FOR ENHANCEMENT OF LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE-TRANSCRIPTION

Low density lipoprotein (LDL) receptor gene is regulated at the transc riptional level by the intracellular level of sterols in animal cells. We have recently identified a 20 bp long region (-145 to -126), desig nated Footprint 1 (FP1), participating in maximal expression of the hu man LDL receptor gene in the absence of sterols in HepG2 cells [Mehta, K. D., Chang, R., Underwood, J., Wise, J. and Kumar, A. (1996) J. Bio l. Chem., 271, 33616-33622]. To determine the minimal FP1 sequence and to define the critical nucleotides required for function, a series of single nucleotide substitutions were introduced in the FP1 region. Tw enty-three independent mutations were analyzed by transfection into He pG2 cells. These studies localize the regulatory region to 14 bp and d emonstrate the requirement for essential guanine nucleotides at positi ons -135 and -136 for FP1 function. Furthermore, transfection studies suggest that the FP1-dependent increase in reporter gene expression is possibly mediated through interaction with the sterol-regulatory elem ent. UV cross-linking and Southwestern blot analysis identified FP1-bi nding factors of similar to 50 and 125 kDa, which we have denoted p50 and p125. Mutations of the critical guanine residues (-135/-136) decre ased the formation of the specific protein-DNA complex with the FP1 se quence and abolished its binding to the p125. We conclude that direct interaction of the p125 factor with these nucleotides of the FP1 eleme nt potentially contributes to FP1-dependent induction of LDL receptor gene expression.