The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain

Abstract

Krüppel-associated box (KRAB) zinc finger proteins are a large class of tetrapod transcription factors that usually exert transcriptional repression through recruitment of TRIM28/KAP1. The evolutionary root of modern KRAB domains (mKRAB) can be traced back to an ancestral motif (aKRAB) that occurs even in invertebrates. Here, we first stratified three subgroups of aKRAB sequences from the animal kingdom (PRDM9, SSX and coelacanth KZNF families) and defined ancestral subdomains for KRAB-A and KRAB-B. Using human ZNF10 mKRAB-AB as blueprints for function, we then identified the necessary amino acid changes that transform the inactive aKRAB-A of human PRDM9 into an mKRAB domain capable of mediating silencing and complexing TRIM28/KAP1 in human cells when employed as a hybrid with ZNF10-B. Full gain of function required replacement of residues KR by the conserved motif MLE (positionsA32-A34), which inserted an additional residue, and exchange of A9/S for F, A20/M for L, and A27/R for V. AlphaFold2 modelling documented an evolutionary conserved L-shaped body of two α-helices in all KRAB domains. It is transformed into a characteristic spatial arrangement typical for mKRAB-AB upon the amino acid replacements and in conjunction with a third helix supplied by mKRAB-B. Side-chains pointing outward from the core KRAB 3D structure may reveal a protein-protein interaction code enabling graded binding of TRIM28 to different KRAB domains. Our data provide basic insights into structure-function relationships and emulate transitions of KRAB during evolution.

Mapping the interaction between Trim28 and the KRAB domain at the center of Trim28 silencing of endogenous retroviruses

“…Consistent with the models presented, mutation of conserved amino acids within the KRAB‐A domain have previously been shown to abrogate Trim28‐mediated transcriptional repression 7,35,36 . These studies identified and described highly conserved sequence motifs from ZFP10 and ZFP748 that are shared by the KRAB‐ZFP protein family.…”

“…Consistent with the models presented, mutation of conserved amino acids within the KRAB-A domain have previously been shown to abrogate Trim28-mediated transcriptional repression. 7,35,36 These studies identified and described highly conserved sequence motifs from ZFP10 and ZFP748 that are shared by the KRAB-ZFP protein family. These sequences, including the "DV," "EEW," and "MLE" motifs, disrupt the interaction with Trim28 and transcriptional repression when mutated (Figure S3).…”

Special Issue: Structure, Function and Evolution of Protein Domains

“…CLAMP is an essential C2H2 Zn-finger protein that binds to thousands of sites in the Drosophila chromosome and is involved in multiple functions, including X chromosome dosage compensation, embryonic reprogramming, and chromatin remodeling, reminiscent of the DPF domain of PHF10, described by Chugunov et al [ 3 ]. Whereas Zn-fingers generally bind DNA, the proteins containing Zn-fingers possess other domain(s) that dictate protein–protein interactions, as also mentioned by Lorenz et al [ 6 ]. Likewise, the CLAMP proteins contain a conserved N-terminal homodimerization domain that facilitates long distance interaction between chromosomal loci.…”

“…Lorenz et al [ 6 ] drew our attention to eukaryotic transcription factors, specifically a protein-protein interaction domain known as Krüppel-associated box (KRAB), commonly found together with the Cys2His2 (C2H2) Zn-finger domain. This subclass, referred as KRAB Zn-finger (KZNF) proteins, first appeared ~400 million years ago and is widely and abundantly distributed in all extant species from invertebrates to mammals; humans, for example, possess roughly 400 KZNF genes.…”