Single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimeric transcription factor

Abstract

The Jun-Fos heterodimeric transcription factor is the terminal link between the transfer of extracellular information in the form of growth factors and cytokines to the site of DNA transcription within the nucleus in a wide variety of cellular processes central to health and disease. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TGACTCA cis element and all possible single nucleotide variants thereof encountered widely within the promoters of a diverse array of genes. Our data show that Jun-Fos heterodimer tolerates single nucleotide substitutions and binds to TGACTCA variants with affinities in the physiologically relevant micromolar to submicromolar range. The energetics of binding are richly favored by enthalpic forces and opposed by entropic changes across the entire spectrum of TGACTCA variants in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific TGACTCA variants in a preferred orientation. Our 3D atomic models reveal that such orientational preference results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues R263 and K148 located within the basic regions of Jun and Fos, respectively. Taken together, our data suggest that the single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimer and that such behavior may be a critical determinant of differential regulation of specific genes under the control of this transcription factor. Our study also bears important consequences for the occurrence of single nucleotide polymorphisms within the TGACTCA cis element at specific gene promoters between different individuals.

Figure 1

Protein and DNA sequences. (a) Subdivision of bZIP domain into its respective N-terminal basic region (BR) and the C-terminal leucine zipper (LZ) for Jun and Fos transcription factors. The BR and LZ subdomains are colored blue and brown, respectively. The five signature leucines (L1-L5) characteristic of LZ subdomains are boxed and bold faced. The basic residues within the BR subdomains that hydrogen bond with specific DNA bases are labeled by vertical arrows. (b) Nucleotide sequence of 15-mer dsDNA oligo containing the TGACTCA motif. The TGACTCA motif is capitalized whilst the flanking nucleotides are shown in small letters. The numbering of various nucleotides relative to the central C/G base pair (assumed to be at zero position) in both strands is indicated. The TGA and TCA half-sites within this motif are also marked. (c) Nucleotide sequence of dsDNA oligo containing the T→A mutation at −3 position and herein referred to as the A-3 oligo. The variant nucleotides in both strands are underlined. (d) Nucleotide sequence of dsDNA oligo containing the A→T mutation at +3 position and herein referred to as the T+3 oligo. The variant nucleotides in both strands are underlined. Note that the A-3 and T+3 oligos shown in (c) and (d) are examples of a pair of symmetrically related dsDNA oligos in that they contain identical half-sites in opposite directions— these half-sites are indistinguishable upon the rotation of the variant motif by 180° in the plane of the paper (two-fold symmetry).

Figure 2

Representative ITC isotherms for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos containing the promoter sites TGACTCA (a), TTACTCA (b) and TGAGTCA (c). Note that the variant motif TGAGTCA is related to the wildtype TGACTCA motif by 2-fold symmetry. The position of the variant nucleotide in each of the sites relative to the consensus sequence TGACTCA is underlined. The solid lines represent the fit of the data points in the lower panels to a function based on the binding of a ligand to a macromolecule using the Microcal ORIGIN software (41).

Figure 3

Differential thermodynamic signatures for the binding of bZIP domains of Jun-Fos heterodimer to various pairs of dsDNA oligos containing TGACTCA variants with similar affinities: (a) TGACGCA relative to TGACACA; (b) AGACTCA relative to TGTCTCA; (c) TGACTGA relative to CGACTCA; (d) TGACTCC relative to TGACTCT; (e) TGACTCG relative to TGACTAA; and (f) TTACTCA relative to GGACTCA. The position of the variant nucleotide in each of the sites relative to the consensus sequence TGACTCA is underlined. ΔΔH, TΔΔS and ΔΔG were calculated from the relationships ΔΔH=ΔH_x-ΔH_y, TΔΔS=TΔS_x-TΔS_y and ΔΔG=ΔG_x-ΔG_y, where the subscripts x and y denote the corresponding thermodynamic parameters for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligo x relative to oligo y, respectively (Table 1).

Figure 4

Analysis of relative binding affinities of symmetrically related pairs of dsDNA oligos containing TGACTCA variants to bZIP domains of Jun-Fos heterodimer. Relative binding affinity is defined as the ratio of the binding affinity of one dsDNA oligo to Jun-Fos heterodimer over that of the other. The nomenclature for the various dsDNA oligos shown is the same as that indicated in Table 1.

Figure 5

3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TGACGCA motif in two possible orientations I (a) and II (b). The two orientations are related by a 180°-rotation of the Jun-Fos heterodimer about the dyad axis of symmetry. The backbones of LZ and BR subdomains of the bZIP heterodimer are colored brown and blue, respectively. The backbone of dsDNA is shown in yellow. The sidechains of R263 in Jun and K148 in Fos are colored green. The guanine at position +1 (G+1) in the sense strand TGACGCA and thymine at position +1 (T+1) in the antisense strand TGCGTCA are colored red. Insets show close-up views of contacts between specific bZIP residues with DNA bases.