Identification of the DNA sequence that interacts with the gut-enriched Kruppel-like factor

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Abstract
The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2 zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Krüppel proteins, including the lung Krüppel-like factor (LKLF), the erythroid Krüppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. In the present study we determined the DNA binding sequence of GKLF using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis a DNA with the sequence 5′-G/AG/AGGC/TGC/T-3′ was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the β-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence was capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. Our results establish GKLF as a sequence-specific transcription factor likely involved in regulation of expression of endogenous genes.