DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Abstract

NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys 2 -His 2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T -G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently).Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins, we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.

Upregulation of rat Ccnd1 gene by exendin-4 in pancreatic beta cell line INS-1: interaction of early growth response-1 with cis-regulatory element

“…The antisense strand (5′-GCGGGGGTG-3′) of the putative EGR1 sequence perfectly matches a consensus EGR1 binding sequence, 5′-GCG(T/G)(G/A)GG(C/A/T)G-3′ [31]. Specific EGR1 binding to this putative binding site was confirmed by immune-supershift and competition assays.…”

“…Comparing the sequences of the rat (5′-CGCCTCGGC-3′) and human (5′-CGCCCCCGC-3′), the thymine and guanine at −104 and −102 in rats are replaced with cytosine in humans. Specifically, the cytosine residue at −102 has been described as a critical base for EGR1 binding [31]. Thus, the presence of a guanine residue at position −102 of the rat promoter may be unfavourable for EGR1 binding in our EMSA study.…”

End effects in DNA recognition by zinc finger arrays

“…It would appear that at the 3′-border of the Zif268 binding site there is marginally stronger preference for guanine than at the 5′-end (data not shown). This asymmetry in sequence specificity was borne out independently by the informative binding site selection experiments of Swirnoff and Milbrandt (25), which revealed stronger discrimination at the 3′-end of the binding site (guanine selected in 100% of sites) than at the 5′-end, where guanine (93%) and adenine (7%) were selected. The relatively high levels of discrimination indicated for the weak end contacts in these experiments presumably result from amplification of relatively weak binding preferences by the iterative selection protocol.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…Egr-1 binds GC rich regions of the promoter of its target genes, binding the consensus sequence GCGG/TGGGCG with the highest affinity (Swirnoff and Milbrandt, 1995;Paillard et al, 2004). Egr-1 has the highest affinity for sequences that preserve guanine at positions 1,3,6,7, and 9 and cytosine at position 2 of the consensus sequence (Swirnoff and Milbrandt, 1995;Paillard et al, 2004).…”

“…Egr-1 has the highest affinity for sequences that preserve guanine at positions 1,3,6,7, and 9 and cytosine at position 2 of the consensus sequence (Swirnoff and Milbrandt, 1995;Paillard et al, 2004). The three zinc fingers of Egr-1 can recognize more than one base of the binding sequence, allowing for variability of the binding sequence.…”