DOI: 10.5061/dryad.18931zd7m
title: Data from: The structure of an ancient genotype-phenotype map shaped the functional evolution of a protein family

description: Mutation is more likely to produce some phenotypes than others, but the
 causal role of these production propensities in the evolution of
 phenotypic diversity remains unclear. There are two major challenges: it
 is difficult to separate the effect of the genotype-phenotype (GP) map
 from that of natural selection when analyzing natural diversity, and most
 extant phenotypes evolved long ago in species whose GP maps cannot be
 recovered. Using reconstructed ancestral transcription factors, we created
 libraries containing all possible amino acid combinations at historically
 variable sites in the proteins’ DNA binding interface (the genotypes) and
 measured their capacity to specifically bind DNA elements containing all
 possible combinations of nucleotides at historically variable sites (the
 phenotypes). The two ancestral GP maps were strongly anisotropic (the
 distribution of phenotypes encoded by genotypes is highly nonuniform) and
 heterogeneous (the phenotypes accessible around each genotype vary
 dramatically among genotypes), but the extent and direction of these
 properties differed dramatically between the maps. In both cases, these
 properties steered evolution toward the lineage-specific phenotypes that
 evolved during history. Our findings establish that ancient properties of
 the GP relationship were causal factors in the evolutionary process that
 produced present-day patterns of functional conservation and diversity.
description: # Data from: The structure of an ancient genotype-phenotype map shaped the
 functional evolution of a protein family [Access this dataset on
 Dryad](https://doi.org/10.5061/dryad.18931zd7m) This dataset contains the
 raw read counts and the estimated mean fluorescence for all protein
 genotypes across all 16 response elements for three experimental
 replicates. We used fluorescence-activated cell sorting (FACS) to separate
 cells based on their GFP expression. We performed two rounds of sorting:
 an initial “enrichment sort” to enrich for GFP+ variants in the full
 libraries, and a second, higher resolution “binned sort” on the enriched
 libraries to generate quantitative fluorescence estimates for each
 variant. To normalize fluorescence to cell volume, GFP gates were drawn to
 have a slope of 1.5 on a log(FSC-A)-log(GFP) plot. We sorted 2.5×10^7
 cells per library in the enrichment stage. The binned sort was performed
 to yield three replicates per library. Binned sorting followed the
 enrichment sort protocol but used four GFP bins instead of two, with
 ~1.6×10^8 cells collected per replicate. Sequencing libraries were
 constructed from plasmids extracted from the enrichment sort GFP–
 population and the four binned sort populations. Replicate 1 of the binned
 sort libraries was sequenced on a NextSeq High Output run. The remaining
 replicates were sequenced on a NovaSeq S1 run. Quantitative estimates of
 mean fluorescence per protein-DNA complex were estimated from the
 distribution of reads across the four fluorescence bins. ### Corresponding
 author information `Name: Joseph W. Thornton Affiliation: Department of
 Ecology and Evolution, and Department of Human Genetics, University of
 Chicago, Chicago, IL, USA email: joet1@uchicago.edu` ### Related
 publication `Herrera-Álvarez, S., Patton, J.E.J., Thornton, J.W.
 (ACCEPTED; May-2025) The Structure of an Ancient Genotype-Phenotype Map
 Shaped the Functional Evolution of a Protein Family. Nature Ecology and
 Evolution.` ## Files and variables File: raw_read_counts.tar.gz
 *Description:* The compressed directory `raw_read_counts.tar.gz`, contains
 a total of 5 directories and 908 `csv` files. Each file contains raw read
 counts for every protein-RE complex in the enrichment and binned sorts
 across three experimental replicates (includes counts from NextSeq and
 NovaSeq runs). See Supplementary Information “Details on replicate
 sorting, sequencing and processing” for further details. When applicable,
 file names in folders `binned_REP1`, `binned_REP2` ,`binned_REP3` and
 `binned_REP4` follow the pattern:
 `AncSR(01)_REBC(02)_BB(03)_REP(04)_AA_var_count.csv` ``` - `(01)` denotes
 the DBD protein background - AncSR1 - AncSR2 - `(02)` denotes the Response
 Element Barcode (REBC). There are 16 RE elements, therefore there are 16
 REBCs - `(03)` denotes the Bin Barcode (BB). There are four fluorescent
 bins, therefore there are 4 BBs. - `(04)` denotes the experimental
 replicate ``` When applicable, file names in folder `Debulk` follow the
 pattern `AncSR(01)_REBC(02)_GFPneg_Debulk_AA_var_count.csv` ``` - `(01)`
 denotes the DBD protein background - AncSR1 - AncSR2 - `(02)` denotes the
 Response Element Barcode (REBC). There are 16 RE elements, therefore there
 are 16 REBCs ``` For each `csv` file, the following columns are included:
 ``` - `AA_var`: The amino acid genotype at the four RH sites - `Count`:
 Number of reads - `REBC`: Identifier of the protein background and REBC -
 `BinBC`: Identifier of the BB (BB1-BB4). If file is from `Debulk` sort,
 the identifier is `GFPneg` - `REP`: Identifier of the experimental
 replicate (REP1-REP4). If file is from `Debulk` sort, the identifier is
 `Debulk` ``` File: NovaSeq_DMS_datasets.tar.gz *Description:* The
 compressed directory `NovaSeq_DMS_datasets.tar.gz`, contains a 5 `csv`
 files. Each file contains the estimated fluorescence of ech protein-DNA
 complex from the read counts (includes counts from NextSeq and NovaSeq
 runs). **Note**: estimated fluorescence only applies for the binned sort
 replicates. For each `csv` file, the following columns are included: ``` -
 `AA_var`: The amino acid genotype at the four RH sites - `REBC`:
 Identifier of the protein background and REBC - `Count_b1`: Number of
 reads in Bin 1 - `Count_b2`: Number of reads in Bin 2 - `Count_b3`: Number
 of reads in Bin 3 - `Count_b4`: Number of reads in Bin 4 - `Count_total`:
 Total read count across 4 bins - `REP`: Identifier of the experimental
 replicate (REP1-REP4) - `cellCount_b1`: Estimated number of cells with RH
 genotype in Bin 1 - `cellCount_b2`: Estimated number of cells with RH
 genotype in Bin 2 - `cellCount_b3`: Estimated number of cells with RH
 genotype in Bin 3 - `cellCount_b4`: Estimated number of cells with RH
 genotype in Bin 4 - `meanF`: Estimated mean fluorescence of protein-DNA
 complex ``` ## Code/Software All the reproducible code to use and run the
 data can be found at
 [https://github.com/JoeThorntonLab/RH-RE_scanning](https://github.com/JoeThorntonLab/RH-RE_scanning)