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

Abstract

The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2zinc 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 beta-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.

Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells

“…Whether this difference is specified by the Sp2 DNA-binding domain or is a consequence of our use of full-length protein for binding site selection studies remains to be determined. It is worth pointing out that although the Sp2 consensus sequence differs from those previously derived for Sp/XKLF family members, similar to the consensus sequences of zinc finger proteins, such as NGFI-A, NGFI-C, Zif268, and Egr3, the consensus Sp2-binding site is flanked by thymidine residues (35,39,47).…”

Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation

“…A consensus DNA binding sequence was empirically determined for KLF4 [11]. This sequence is present in many gene promoters and includes the CACCC element and the basic transcription element (BTE).…”

Characterization of the Human Lung CYP2F1 Gene and Identification of a Novel Lung-specific Binding Motif

“…A 1.3-kbp sequence from the CYP2B promoter was shown to drive lung-specific reporter gene expression in a transgenic mice, and it was shown that C/EBP␣ and C/EBP␦ regulate CYP2B pulmonary expression during differentiation (19). In addition, the promoter contains a BTE site that has been demonstrated in numerous studies (35,36,47) to regulate CYP1A1 transcription. The BTE site of CYP1A1 has been recognized to bind a number of transcription factors from the Sp/XKLF family of factors, which are involved in both constitutive and tissuespecific regulation (48).…”