Identification of essential nucleotides of the FP1 element responsible for enhancement of low density lipoprotein receptor gene transcription

Punita Dhawan, Ruixin Chang, Kamal D. Mehta

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Low density lipoprotein (LDL) receptor gene is regulated at the transcriptional level by the intracellular level of sterols in animal cells. We have recently identified a 20 bp long region (-145 to -126), designated Footprint 1 (FP1), participating in maximal expression of the human LDL receptor gene in the absence of sterols in HepG2 cells. 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. Twenty-three independent mutations were analyzed by transfection into HepG2 cells. These studies localize the regulatory region to 14 bp and demonstrate the requirement for essential guanine nucleotides at positions -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 element. UV cross-linking and Southwestern blot analysis identified FP1-binding factors of ~ 50 and 125 kDa, which we have denoted p50 and p125. Mutations of the critical guanine residues (-135/-136) decreased the formation of the specific protein-DNA complex with the FP1 sequence and abolished its binding to the p125. We conclude that direct interaction of the p125 factor with these nucleotides of the FP1 element potentially contributes to FP1-dependent induction of LDL receptor gene expression.

Original languageEnglish (US)
Pages (from-to)4132-4138
Number of pages7
JournalNucleic acids research
Volume25
Issue number20
DOIs
StatePublished - Oct 15 1997

Fingerprint

LDL Receptors
Sterols
Nucleotides
Hep G2 Cells
Transfection
Southwestern Blotting
Genes
Gene Expression
Mutation
Guanine Nucleotides
Nucleic Acid Regulatory Sequences
Guanine
Reporter Genes
DNA
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Identification of essential nucleotides of the FP1 element responsible for enhancement of low density lipoprotein receptor gene transcription. / Dhawan, Punita; Chang, Ruixin; Mehta, Kamal D.

In: Nucleic acids research, Vol. 25, No. 20, 15.10.1997, p. 4132-4138.

Research output: Contribution to journalArticle

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