Rational evolution for altering the ligand preference of estrogen receptor alpha

Abstract

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Keywords: Saccharomyces cerevisiae; directed evolution; estrogen receptor; nuclear receptors; synthetic biology; tamoxifen.

FIGURE 1

Saccharomyces cerevisiae platform for evolving nuclear receptors toward designer chemicals. (a). The pSRI1‐HIS3 pSRI2‐URA3 pSRI‐yemCheRRy pPGI1‐YeCitrine (HURRY) strain has the dual survival and fluorescent reporter cassettes under ERα control. Reporter cassettes are composed of the SRi enhancer region and synthetic core promoters 4, 9, or minimal CYC1 promoter resulting in pSRI1, pSRI2, or pSRI. (b) Evaluation of the estrogen responsive growth of the S. cerevisiae HURRY strain versus its parental strains that do not contain one or more reporter cassettes. These strains all express the S. cerevisiae codon optimized ERα (yERα) under control of the constitutive TEF1 promoter from the ARS208 locus, with the exception of the pTEF1‐yERα strain that contains this cassette in the CAN1 locus. The vehicle condition serves as negative control. The curves are fitted using non‐linear regression (Prism version 9.3.1). The error bars indicate the standard error of the mean of three biological replicates. (c) Characterization of the estradiol responses of yemCherry fluorescence of the S. cerevisiae HURRY strain versus its parental clones described in the panel. yemCherry fluorescence is corrected by the yeCitrine fluorescence and expressed relative to the vehicle condition. The curves are obtained as described for panel a.

FIGURE 2

Methodology for engineering receptor proteins through rational evolution. (a) Schematic representation of the three sections of the rational evolution pipeline: (1) in silico identification of the amino acid positions in the human ERα‐LBD to be mutated, (2) simultaneous diversification of defined amino acid locations, and (3) characterization of NR variants for designer phenotypes. Rational evolution starts from double stranded plasmid DNA encoding the gene expression cassette of interest, including promoter (green arrow), CDS (orange region), terminator (red T), and 120 bp homology arms to the target site for genomic integration (red), near ARS208a. The plasmid DNA is singularized by a nicking endonuclease (at the purple dot) and exonuclease III. Next, the single stranded DNA plasmid is bound by a 5′‐biotinylated‐ (indicated by a red B) and a 3′‐3′dT boundary oligonucleotide and multiple 5′‐phosphorylated mutagenic primers with degenerate codons (red X) at the in silico determined putative sites. Non‐complementary overhangs extending the homology arms are indicated in red and tilted. Next, primers are extended and ligated in an isothermal assembly reaction. The assembled 5′‐biotinylated DNA strands are isolated by paramagnetic streptavidin‐coated beads (beige beads) and purified by alkali washing prior to PCR using outnested priming sites. This library of dsDNA mutant gene expression cassettes is stably integrated near the ARS208a locus of the S. cerevisiae HURRY strain by addition of a Cas9 expression vector that also transcribes the accommodating sgRNA during transformation. Every transformant stably expresses one unique receptor variant. To isolate the active XR fraction, the transformant pool is plated on medium lacking uracil and histidine but containing the chemical of choice. Transformants expressing a receptor variant that is activated by the chemical of choice are able to express URA3 and HIS3 and survive. If more than 1000 unique colonies are obtained, further selection by fluorescence‐based cell sorting using yemCherry as a proxy for receptor activity can be applied to isolate transformants expressing the highest yemCherry fluorescence signal in presence of the chemical of interest. From the sorted pool or immediately after growth selection, a maximum of 1000 colonies are subjected to clonal screening. Screening by overnight incubation with the native ligand or the chemical of interest, followed by detection of yemCherry and yeCitrine fluorescence intensities, can identify XRs that lost their responsiveness to the native ligand and improved the response to the chemical of choice (indicated in teal). After genotyping, the desired isolate can serve as a new starting point for subsequent rational evolution rounds. (b) Identification of hot spot amino acid positions for computed binding by the chemical of interest, tamoxifen in this study. Positions are chosen based on the individual conservation (right hand side y‐axis) and the energetic contribution to the ligand interface (left hand side y‐axis) of the human estrogen receptor α with estradiol or tamoxifen. Residues with high conservation scores and minimal contribution to tamoxifen binding are selected for the first rational evolution round to promote tamoxifen responsiveness (orange). Positions with conservation scores between 5 and 8 that show high estradiol binding energies are evolved to impair estradiol response (light blue).

FIGURE 3

Rational evolution of the estrogen receptor α (ERα) toward tamoxifen as an agonist. (a) Indication of hot spot residues for simultaneous randomization to facilitate tamoxifen binding (PDB ID:1QKU for representation) (b) Screening of the 913 rationally evolved NR variants in the HURRY strain for their activities in presence of 10 nM estradiol and 10 μM tamoxifen through flow cytometry. Fluorescence intensities are normalized to the 10 nM estradiol‐stimulated wild‐type yERα, which is set to 100%. Each data point represents the average reading of 10,000 single cells of that unique strain. (c) Evaluation of the activity of rationally evolved top isolates in mammalian HEK 293 T cells in the absence of a ligand (vehicle shown in black), in the presence of estradiol (pink), tamoxifen (blue), or combined ligands (green). For the combined ligands, 10 nM estradiol is selected as it represents the highest observed physiological concentration in metazoans. Physiological estrogen concentration ranges are indicated by gray shading. Luciferase values in presence of vehicle are set at 1. The error bars indicate the standard error of the mean from three biological replicates. The curves are fitted using non‐linear regression (Prism version 9.1.3). (d) Structural determination of putative amino acid locations to impair estradiol binding starting from the most tamoxifen‐selective identified variant of the first rational evolution round (preTERRA) (PDB ID:1QKU for representation) (e) Profiling of the 649 PreTERRA‐derived NR variants, accommodating mutations aimed at impairing estradiol binding, in the HURRY strain. Fluorescence intensities are normalized to the 100 nM estradiol‐stimulated wild‐type yERα, which is set to 100%.

FIGURE 4

Harnessing the TERRA variant into a viable genomic tool. (a) Dual tamoxifen‐inducible Cre recombinase expression cassette composed of the CAG promoter, the Cre recombinase CDS fused both up‐ and downstream by either the TERRA‐LBD or the ERT2‐LBD and the poly A signal. The HEK293‐loxP‐GFP‐loxP‐RFP cells maintain the loxP‐GFP‐loxP‐RFP reporter cassette composed of the CMV promotor, the GFP CDS with poly A signal flanked both up‐ and downstream by loxP sites followed by the RFP CDS and poly A signal. (b) Dose–response curve of the TERRA‐Cre‐TERRA and ERT2‐Cre‐ERT2 fusion proteins for estradiol and tamoxifen after stimulation for 24 h in mammalian HEK293‐loxP‐GFP‐loxP‐RFP cells. Physiological estrogen concentration ranges are indicated by gray shading. Error bars represent the standard error of the mean from three biological replicates. (c) Scheme for in vivo test of Cre‐TERRA. At the age of 8 weeks, mT/mG mice received an intraperitoneal injection of recombinant AAV9 encoding Cre‐TERRA or Cre‐ERT2. At 10 weeks of age, the mice were given either vehicle or tamoxifen via oral gavage. Image was created with Biorender.com. (d) One week thereafter, GFP and dTom fluorescence in the heart of the mice were quantified via microscopic analysis and were used to calculate the % of GFP‐positive area within the heart of the mouse. At least 2 mice were tested per condition. The results are shown as scatter plot with the mean ± SD as line and whiskers; statistical analysis was performed by an Ordinary Two‐way ANOVA with Sidak's multiple comparisons test. (e) Representative microscopic images of heart tissue of Cre‐TERRA or Cre‐ERT2 transduced mT/mG mice. The dTom and GFP fluorescent signals in these overlays are shown in red and green, respectively.