Evolution of DNA specificity in a transcription factor family produced a new gene regulatory module

Abstract

Complex gene regulatory networks require transcription factors (TFs) to bind distinct DNA sequences. To understand how novel TF specificity evolves, we combined phylogenetic, biochemical, and biophysical approaches to interrogate how DNA recognition diversified in the steroid hormone receptor (SR) family. After duplication of the ancestral SR, three mutations in one copy radically weakened binding to the ancestral estrogen response element (ERE) and improved binding to a new set of DNA sequences (steroid response elements, SREs). They did so by establishing unfavorable interactions with ERE and abolishing unfavorable interactions with SRE; also required were numerous permissive substitutions, which nonspecifically improved cooperativity and affinity of DNA binding. Our findings indicate that negative determinants of binding play key roles in TFs' DNA selectivity and-with our prior work on the evolution of SR ligand specificity during the same interval-show how a specific new gene regulatory module evolved without interfering with the integrity of the ancestral module.

Fig. 1. Evolution of novel specificity occurred via a discrete shift between AncSR1 and AncSR2

(A) Architecture of SR response elements. All SRs bind to an inverted palindrome of two half-sites (gray arrows) separated by variable bases (n). x, sites at which ERE and SREs differ. (B) SR phylogeny comprises two major clades, which have non-overlapping specificity for ligands (stars) and REs (boxes). Preferred half-sites for each clade are shown; bases that differ are underlined. Ancestral and extant receptors are colored by RE specificity (purple, ERE; green, SREs; blue, extended monomeric ERE). Orange box, evolution of specificity for SREs; number of substitutions on this branch and the total number of DBD residues are indicated. Nodal support is marked by the approximate likelihood ratio statistic: unlabeled, aLRS 1 to 10; •, aLRS 10 to 100; ••, aLRS>100. Scale bar is in substitutions per site. (C) AncSR1 specifically activates reporter gene expression driven by ERE (purple bar), with no activation from SRE1 (light green) or SRE2 (dark green); AncSR2’s specificity is distinct. Bar height indicates fold-activation relative to vector-only control. (D) Ancestral binding affinities reflect distinct specificities for ERE vs. SREs. Bars heights indicate the macroscopic affinity (K_A,mac) of binding to palindromic DNA response elements, measured using fluorescence polarization. Colors as in panel C. (E-G) The components of macroscopic binding affinity—affinity for a half-site (K₁) and cooperativity of binding (ω)—by AncSR1 and AncSR2, were estimated by measuring K_A,mac on a full palindromic RE and K₁ on a half-site, then globally fitting the data to a model containing both parameters. Error bars show SEM of three experimental replicates. See Fig. S1; Tables S1-S3.

Fig. 2. Structures of ancestral proteins give insight into the molecular determinants of specificity

(A) X-ray crystal structures of AncSR1 bound to ERE (left); AncSR2 bound to SRE1 (right). Cartoon shows protein dimers; surface shows DNA. Black arrow, beginning of unresolved C-terminal tail. Dotted line, unresolved AncSR1 loop near dimerization interface. Cyan spheres, sites of permissive substitutions. Grey spheres, zinc atoms. (B) Enlarged view of recognition helix in the DNA major groove (black box in A). Sticks, side chains of RH residues making polar contacts with DNA. Dotted lines, hydrogen bonds and salt bridges from protein to DNA. (C) Buried solvent-inaccessible surfaces in Ų at the protein-DNA and protein-protein interfaces in the crystal structures for each protein chain. Parentheses, calculations when residues unresolved in the AncSR1 crystal structure are excluded. See Table S4.

Fig. 3. Genetic basis for evolution of new DNA specificity

(A) AncSR1 and AncSR2 sequences. Substitutions between AncSR1 and AncSR2 are shown. Dots, conserved sites. ˆ, recognition helix (RH) and *, permissive substitutions. Grey box, RH. (B) Effect of RH and 11 permissive (11P) substitutions in luciferase reporter assays. Lower and upper case letters denote ancestral and derived states, respectively. Fold activation over vector-only control is shown, with SEM of three replicates. (C) RH substitutions shift half-site affinity among REs, and permissive substitutions non-specifically increase half-site affinity and cooperativity. The corners of the square represent genotypes of AncSR1, with or without RH and 11P substitutions. At each corner, circle color shows RE preference; numbers are the ratio of the K_Amac for binding to SRE1 (upper) or SRE2 (lower) versus ERE. Along each edge, vertical bar graphs show the effect of RH or permissive substitutions on the energy of association for the dimeric complex (grey background); contributions of effects on half-site binding (beige) and cooperativity (cyan) are shown. Bar color shows effects on binding to ERE (purple), SRE1 and SRE2 (light and dark green, respectively). Graphs in the square’s center show the effect of 11P and RH combined. Mean ± SEM of three experimental replicates is shown. See Figs. S2-S4; Tables S3 and S5.

Fig. 4. Recognition helix substitutions change DNA specificity by altering negative interactions

(A) In MD simulations, RH substitutions reduce hydrogen bonds to ERE but do not increase hydrogen bonds to SREs. Bars show mean number of direct hydrogen bonds from all 10 RH residues to DNA (Purple, ERE; light green, SRE1; dark green, SRE2), each sampled across three MD trajectories, with SEM. (B) RH substitutions reduce packing efficiency at the protein-DNA interface on ERE, but do not improve packing on SREs. Bars show the mean number of atoms in the 10 RH residues within 4.5 Å of a DNA atom. (C) Ancestral residue glu25 (sticks) shifts position due to steric clashes with T-4 and T-3 of SRE1. A representative sample frame from MD trajectories is shown for AncSR1 with ERE (purple) or SRE1 (green). DNA is shown as surface, with atoms in the variable bases -4 and -3 shown as lines; methyls of T-4 and T-3 are spheres. (D-F) Repositioning of glu25 by SREs causes Lys28 to shift, reducing hydrogen bonds to DNA. (D) The average position of these residues in MD trajectories of AncSR1 with various REs is shown when all atoms in the protein-DNA complex are aligned. Distance of lys28 from hydrogen bond acceptor G2 on ERE is shown in black. (E) Displacement of glu25 and lys28 of AncSR1 on SREs relative to their position on ERE. The mean positions of all atoms in each MD trajectory were calculated, the DNA atoms in these “mean structures” were aligned in pairs: bars shows the average distances from the atoms in complexes with SRE1 (dark green) or SRE2 (light green) to the corresponding atom in ERE were calculated. Purple bars, distances between pairs of atoms from independent ERE trajectories. Displacement toward the center of the palindrome was scored as positive, away as negative. Each bar shows the distance averaged across atoms in a residue and three pairs of trajectories with SEM. (F) Lys28 forms fewer hydrogen bonds to DNA on SREs than on ERE. Points show the mean number of hydrogen bonds formed by each RH residue to different REs, with SEM for three MD trajectories. (G,H) Effect of introducing e25G and other RH substitutions on half-site binding affinity (G) and transcriptional activation (H). See Figs. S6-S7, and Table S3. (I) Summary of mechanisms by which ancestral RH excludes SREs. Ancestral glu25 and conserved residue Lys28 form hydrogen bonds (black dotted lines) with ERE bases. These side chains would sterically clash with methyl groups of SRE1 and SRE2, so they are repositioned and are unable to form hydrogen bonds to DNA, leaving unpaired donors (blue) and acceptors (red) at the DNA-RH interface. The RH substitutions resolve the steric clash and remove the unfulfilled donor on e25, increasing SRE affinity. See Figs. S5-S6.

Fig. 5. Permissive substitutions do not improve protein stability or dimerization in the absence of DNA

(A) Crystal structure of AncSR2 bound to SRE1. Sites of permissive substitutions are shown as Cα spheres; red, cyan, and orange indicate clustered groups of sites. Only one residue in the C-terminal group is shown). (B) Permissive substitutions (11P) do not increase protein stability. ΔG_H2O, calculated Gibbs free energy of chemically induced unfolding; m, slope of the unfolding transition; C_M, denaturant concentration at which 50% of protein is folded. (C,D) Permissive substitutions do not increase protein dimerization in the absence of DNA, measured by analytical ultracentrifugation. Distribution (C) and best-fit values (D) of sedimentation velocity coefficients (S_20,w) for AncSR1 (left) or AncSR1+11P (right) at 0.5 mM. The fraction of the total signal under the dominant peak (% total), the estimated molecular weight of that peak (MW) and the expected molecular weight of the monomeric protein (MW_theo) show that AncSR1 and AncSR2 are both predominantly monomeric. RMSD, root mean square deviation of the data from the model; f/f₀, total shape asymmetry. Signal at higher MW peaks may reflect aggregation due to high protein concentration.

Fig. 6. Evolution of a new regulatory module

(A) After duplication of AncSR1, the ancestral specificity for estrogens (purple stars) and ERE (purple box) was maintained to the present in the ER lineage. In the lineage leading to AncSR2, ancestral specificity for both DNA and hormone was lost, and novel sensitivity evolved for SREs (green box) and nonaromatized steroids (green star). A new set of target genes (light grey) was thus activated in response to different stimuli. Green hashes mark the branch on which these events occurred. (B-D) Other potential evoutionary trajectories for evolving new functions would interfere with the ancestral signaling network. (B) Evolution of new specificity for DNA or ligand would cause activation of old targets by new stimuli, or activation of new targets in response to ancestral stimuli. (C-D) Evolution of promiscuity in one or both domains would cause similar effects. (E) The shift in specificity from ERE (purple helices) to SREs (green helices) in AncSR2 involved losing favorable interactions (orange arrows) to ERE, losing unfavorabl negative interactions (red bars) to SRE, and gaining unfavorable interactions to ERE. Offsetting the loss of positive interactions in the DNA major groove, AncSR2 evolved favorable non-specific DNA contacts (blue arrows) and protein-protein interactions (white arrows in dimer interface) that increased cooperativity.