A High-Throughput Mutational Scan of an Intrinsically Disordered Acidic Transcriptional Activation Domain

Abstract

Transcriptional activation domains are essential for gene regulation, but their intrinsic disorder and low primary sequence conservation have made it difficult to identify the amino acid composition features that underlie their activity. Here, we describe a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features-acidity, hydrophobicity, intrinsic disorder, and short linear motifs-by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4 reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most-active variants keep their aromatic residues exposed to the solvent. Our results illustrate how the function of intrinsically disordered proteins can be revealed by high-throughput rational mutagenesis.

Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains

“…The ability of GCN4 to interact with MED15 and activate gene expression has been attributed to specific hydrophobic patches and aromatic residues in the GCN4 AD (Drysdale et al, 1995; Staller et al, 2018; Tuttle et al, 2018). We created a mutant of GCN4 in which the 11 aromatic residues contained in these hydrophobic patches were changed to alanine (Figure 7C).…”

A high‐throughput method to identify trans‐activation domains within transcription factor sequences

“…Library complexity can be minimized by the use of defined, chemically synthesized DNA fragments restricted to the TFs’ native (+1) reading frames, thereby reducing the number of candidates by a factor of six (for single‐amino acid resolution) or—at the expense of resolution—multiples of six (note however that synthesized fragments have typically rather short maximum lengths and are fairly expensive). The use of chemically synthesized DNA fragments also allows the assessment of variant sequences with defined mutations to probe the functional importance of certain peptide motifs or individual amino acids, as has been recently done for the tAD of the yeast TF Gcn4 with a method similar to tAD‐seq (Staller et al , , published while this manuscript was under review). We further note that the strategy used here should also be applicable to equivalent screens for other protein‐domain functions that can be coupled—directly or indirectly—to the expression of a selectable marker such as GFP.…”

Classifying disease-associated variants using measures of protein activity and stability

“…High-throughput genome sequencing combined with assays specific to the function of the protein of interest enable highly parallel assessments of the functionality of each individual variant 7 in so called MAVEs (multiplexed assays of variant effects) 8 . Briefly explained, a MAVE involves creating a large library of variants and subsequently selecting for a property of interest [9][10][11][12] . MAVEs have been applied to a substantial number of proteins and domains, overall indicating a surprising mutational tolerance as many missense variants retain wild type-like function 13,14 .…”