Characterising the interaction between TRIM28 and members of the Krab-ZFP family in transcription silencing

Abstract

Retroviruses have been infecting their metazoan hosts for tens of millions of years. Endogenous retroviruses (ERVs) arise when a retrovirus infects the host’s germ line and becomes inherited in a Mandelian manner. It is estimated over 50 waves of endogenisation events have occurred in ancestral human lineages and ERVs now comprises around 8–10% of the human and mouse genomes. While ERVs have lost their ability to spread from cell to cell, some have gained the ability to retrotranspose within the host genome causing mutational insertions. Moreover, accumulating evidence indicate that the highly abundant ERV DNA also provides important regulatory elements for the correct expression of host genes. Therefore, ERV elements are under tight control during early embryogenesis, largely through TRIM28-mediated transcription repression. TRIM28 (also known as KAP1 or TIF1β) functions as the co-repressor protein for the largest family of transcription factors, namely the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs). Silencing is initiated by the recruitment of TRIM28 to a target locus by members of the Krab-ZFP. Subsequently, TRIM28 functions as a scaffold protein to recruit chromatin modifying effectors featuring SETDB1 (SET domain bifurcated histone lysine methyltransferase 1), the heterochromatin protein 1 (HP1) protein family, and the nucleosome remodelling and deacetylase (NuRD) complex. Although many protein partners involved in silencing have been identified, the molecular basis of the protein interactions that mediate silencing remains largely unclear. TRIM28 belongs to the tripartite motif (TRIM) protein family with over 80 members in humans. They share a conserved N-terminal TRIM, also known as the RBCC as it consists of a RING domain, one or two Bbox domains and an antiparallel coiled-coil. In this study we aimed to use structural and biophysical techniques to characterise the interaction between TRIM28 and members of the Krab-ZFP family. We have identified the first Bbox domain (B1) as a molecular interface responsible for the formation of higher-order TRIM28 oligomers. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt oligomerisation but have no observed effect on transcriptional silencing, defining a single TRIM28 dimer as the functional unit for silencing. Using assembly-deficient mutants, we employed small angle X-ray scattering and biophysical techniques to characterise binding to members of the Krab-ZFP family. This allowed us to narrow and define the binding interface to the central coiled-coil region of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins.