Structure and functional mapping of the KRAB‐KAP1 repressor complex

Abstract

Transposable elements are a genetic reservoir from which new genes and regulatory elements can emerge. However, expression of transposable elements can be pathogenic and is therefore tightly controlled. KRAB domain‐containing zinc finger proteins (KRAB‐ZFPs) recruit the co‐repressor KRAB‐associated protein 1 (KAP1/TRIM28) to regulate many transposable elements, but how KRAB‐ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB‐ZFP, ZNF93. Structure‐guided mutations in the KAP1‐KRAB binding interface abolished repressive activity in an epigenetic transcriptional silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome‐wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB‐KAP1 molecular interface, which is critical for a central transcriptional control axis in vertebrates. In addition, the structure‐based prediction of KAP1 recruitment efficiency will enable optimization of KRABs used in CRISPRi. image KRAB‐ZFPs recruit the co‐repressor KAP1 to control transcription at thousands of genomic loci and to mediate programmable gene repression by CRISPRi. Here the crystal structure of a KRAB‐KAP1 complex enables the identification and functional validation of the binding interface between the KRAB domain of ZBF93 and KAP1. The KRAB‐KAP1 structure reveals the conserved binding interface between KRAB‐ZFPs and KAP1. Binding interface mutations abolish binding and transcriptional repression by KAP1. Disruption of KAP1‐KRAB binding causes H3K9me3 loss at thousands of genomic loci. Analysis predicts KAP1 recruitment to different KRABs and thereby allows optimization of KRABs used in CRISPRi