A Dissection of Oligomerization by the TRIM28 Tripartite Motif and the Interaction with Members of the Krab-ZFP Family

Highlights

• Trim28 is member of the tripartitie motif protein family, and the universal co-repressor for the Krab-zinc finger proteins.

• The structure of the first Bbox domain reveals a novel dimer interface resulting in the formation of higher-order assemblies.

• The coiled-coil region of TRIM28 mediates the interaction with members of the Krab-ZFP family.

• Mutations in the coiled-coil block binding to the Krab domain, an interaction central to TRIM28 function and the silencing of transcription.

Abstract

TRIM28 (also known as KAP1 or TIF1β) is the universal co-repressor of the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs), the largest family of transcription factors in mammals. During early embryogenesis, TRIM28 mediates the transcriptional silencing of many endogenous retroviral elements and genomic imprinted sites. 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, HP1 and the 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. In the present study, we identified the first Bbox domain (T28_B1 135–203) as a molecular interface responsible for the formation of higher-order oligomers of TRIM28. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt the formation of oligomers 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 characterize binding to member of the Krab-ZFP family. This allows us to narrow and define the binding interface to the center of the coiled-coil region (residues 294–321) of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins.

Introduction

Endogenous retroviruses (ERVs) arise when a retrovirus infects the host's germ line and becomes inherited in a Mandelian manner. It is estimated that over 50 waves of endogenization have occurred in ancestral human lineages with ERVs now comprising approximately 8% of the human genome [1]. ERVs form a class of retrotransposons that can move around the host genome causing mutational insertions. To protect genome integrity, humans and other mammalian hosts undergo targeted silencing of ERVs during embryogenesis via TRIM28-mediated repression of transcription. The deletion of TRIM28 in mouse embryonic stem (ES) cells leads to substantial de-repression of a range of murine ERVs [2], [3]. In addition to genome protection, the TRIM28/Krab-ZFP system also enables the host to domesticate the transcription potential of ERVs for regulating the expression of host genes and developing gene networks [1], [4].

TRIM28 (or KAP1 or TIF1β) belongs to the tripartite motif (TRIM) protein family with over 70 members in the human genome [5]. The family shares a conserved N-terminal domain organization, termed the RBCC or tripartite motif, consisting of a RING domain, followed by one or two B-box domains and a coiled-coil region. The C-terminal domains of TRIM proteins are variable and have been used to classify the family into 11 subfamilies [6]. TRIM28 belongs to the TIF1 subfamily, along with TRIM24 and TRIM33, that share a C-terminal PHD/Bromo domain. The PHD/Bromo domain of TRIM24 and TRIM33 are both histone code readers [7], [8]. In contrast, the PHD/Bromo of TRIM28 does not recognize histone tails but acts as an intramolecular E3 SUMO ligase [9], [10]. Auto-SUMOylation in the PHD/Bromo domain is a major determinant of TRIM28's repressive activity, as mutants lacking SUMOylation sites display an almost complete loss of repression [10], [11].

To target a specific site, TRIM28 itself does not bind DNA, rather it acts as the universal co-repressor for the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs). The Krab-ZFPs are the largest family of transcription factors in mammals with over 350 members in humans and other mammals [12], [13]. The expansion of Krab-ZFPs in mammalian genomes is thought to be due to a requirement to silence ERVs during bursts of viral endogenization [14], [15]. Indeed many Krab-ZFPs recognize ERV loci [13], [16], [17]. The most thoroughly characterized system is the targeting of both endogenous and exogenous murine leukemia virus by ZFP809. ZFP809 recognizes the PBS^Pro primer binding site region of the integrated virus during early stages of embryo development, preventing transcription from the retroviral promoter, facilitating immunity against the integrated virus [18].

Members of the Krab-ZFP family contain discrete N- and C-terminal domains each with a specific function. An array of between 2 and 40 zinc-finger domains are located at the C-terminal end of the protein [12]. Each zinc-finger recognizes three consecutive nucleotides [19] mediating recognition of specific DNA sequences. The N-terminal Krab domain is reported to be intrinsically disordered and is responsible for recruiting TRIM28 [20], [21], [22], [23], [24]. The Krab domain can be further divided into two subdomains encoded by separate exons. The more conserved N-terminal Krab-A box (~ 42 amino acids) is necessary and sufficient for TRIM28 binding and repression [24], [25], [26]. The Krab-B box (~ 30 amino acids), although not required for repression, can potentiate Krab-A mediated repression in an unknown mechanism [24], [27], [28]. Some Krab-ZFPs lack the Krab-B box altogether [12]. Structural studies into Krab domains indicate that it is mostly intrinsically disordered and likely folds upon interaction with TRIM28 [29], [30].

Following recruitment to the host chromatin, TRIM28 is auto-SUMOylated [9], [11] and recruits the histone methyltransferase SETDB1, the histone deacetylase-containing NuRD complex, and heterochromatin protein 1 (HP1). Collectively, these proteins mediate the conversion of euchromatin to heterochromatin, thereby silencing transcription [31], [32].

The coiled-coil domain of TRIM proteins forms an antiparallel dimer that separates the N-terminal RING and Bbox domains onto opposite ends of the approximately 170 Å coiled-coil [33], [34]. It has been shown that many TRIM proteins further assemble into functionally important higher-order assemblies. The RING domains of TRIM23 and TRIM32 form homodimers to activate their E3 ubiquitin ligase activity [35], [36]. The HIV restriction factor TRIM5α can trimerize via the Bbox 2 domain, facilitating the assembly of the protein into a hexagonal lattice on the retroviral capsid upon viral entry [37], [38]. This assembly also brings the adjacent RING domains in close proximity to facilitate activity as an E3 ubiquitin ligase [38], [39], [40]. It has previously been reported that TRIM28 can form higher-order assemblies [41]; however, the detail and biological relevance of this has not been explored. In this study we identify the first Bbox domain (B1) of TRIM28 as the assembly interface for higher-order assembly. We have determined the structure of this domain and shown that B1 dimerization is not a requirement for Krab binding and auto-SUMOylation activity in vitro, and dimerization-null mutants were able to rescue the expression of an endogenous retroviral reporter in TRIM28 KD hESC cells. We have also shown that the Krab domain interacts with the central region of TRIM28 coiled-coil with a defined 2:1 stoichiometry and identified a single-point mutation in this region that abolishes the interaction with the Krab domain.