Hetero-oligomerization Among the TIF Family of RBCC/TRIM Domain-containing Nuclear Cofactors: A Potential Mechanism for Regulating the Switch Between Coactivation and Corepression

Abstract

The RING-B box-coiled-coil (RBCC) motif (also re-named recently as the tripartite motif (TRIM)) is a widely distributed motif that is hypothesized to be a protein–protein interface. The RBCC/TRIM domain of the corepressor KAP-1 is both necessary and sufficient to interact directly with the transcription repressor KRAB domain. Each subdomain of the KAP-1-RBCC contributes directly to the oligomerization and/or ligand recognition. Little is known about the function or the natural binding ligands for the RBCC/TRIM domain of the other TIF family members. In order to investigate whether hetero-oligomerization might be a biological regulatory mechanism, we have evaluated the hetero-oligomerization potential of the TIF family members including KAP-1, TIF1α, TIF1γ, and the RBCC/TRIM family members including PML1, and MID1. We have reconstituted and characterized the oligomerization for these proteins using baculovirus and mammalian expression systems by biochemical approaches. Our data indicate that the RBCC/TRIM domains of KAP-1, TIF1α and TIF1γ exist in a homo-oligomeric state. However, there is little cross-talk between KAP-1 and other TIF family members, suggesting that a high degree of specificity for oligomerization interface and ligand recognition is intrinsically built into the RBCC/TRIM domain of KAP-1. Finally, we demonstrate that TIF1α interacts with TIF1γ and the coiled-coil region of TIF1γ is necessary for this interaction. The hetero-oligomerization between TIF1α and TIF1γ implies a potential regulatory mechanism for transcriptional regulation.

Introduction

The RING-B box-coiled-coil (RBCC) motif (renamed recently as the tripartite motif (TRIM)) is found in an increasing number of proteins with important roles in cell differentiation, development, oncogenesis, and apoptosis.1., 2., 3. The definitive element of the RBCC/TRIM domain is the RING finger, which is found in the most NH₂-terminal position in the RBCC/TRIM domain. It forms the consensus C3HC4 and binds two molecules of zinc in a unique cross-braced metal ligation scheme. Mutagenesis studies have confirmed the requirement for the RING finger for many of the biological functions of RBCC/TRIM proteins.1., 3., 4., 5. The second signature motif is the B-box, which is also a cysteine+histidine-rich region that binds zinc.⁶ Two B-box motifs are often found immediately COOH-terminal to the RING finger in the RBCC/TRIM domain. The third signature sequence is the coiled-coil motif that displays amphipathic helical character and is required for protein–protein interactions mediated by the RBCC/TRIM domain. Two hallmark properties of RBCC/TRIM domain proteins are: (1) their ability to form higher-order oligomeric structures, and (2) their presence in discrete macromolecular subcellular compartments. Each of these properties appears to require the highly conserved coiled-coil region. It is noteworthy that the arrangement of RBCC/TRIM subdomains, NH₂ to COOH terminus, is conserved throughout evolution, suggesting that the structure may serve as an integrated functional unit. A reasonable hypothesis is that the coiled-coil region may oligomerize the protein, thereby aligning the RING and B-boxes properly for ligand recognition, analogous to the leucine-zipper-basic region arrangement in the Fos, Jun, and Myc proteins.⁵

Several RBCC/TRIM proteins have been defined as oncoproteins and the targets for mutation leading to familial diseases. The PML and TIF1α RBCC/TRIM proteins were discovered due to their fusion to heterologous proteins resulting from chromosomal translocation.7., 8., 9. Perhaps the best understood example so far is the PML protein, whose RBCC/TRIM domain is fused to the retinoic acid receptor alpha (RARα) as a result of a t(15:17) translocation.7., 8., 10. This results in aberrant regulation of RAR target genes and a block in myelocytic differentiation, resulting in acute promyelocytic leukemia.¹¹ The mechanism of this oncogenic activation appears to involve the aberrant recruitment of the N-CoR/HDAC corepressor complex to RA target gene leading to transcriptional repression.12., 13. This abnormal recruitment of N-CoR by PML-RAR requires both the coiled-coil region of PML and its ability to form PML-RAR oligomers. The intact RBCC/TRIM domain is required for the normal functions of PML, including localization to ND10/POD nuclear domains and growth suppression via induction of apoptosis.¹⁴ Thus, it is clear that we need a thorough understanding of the oligomerization properties of the RBCC/TRIM domain in order to explain their disease-causing potential.

As a model for understanding the biochemical and biological functions of RBCC/TRIM domain proteins, we have focussed on the TIF subfamily of RBCC/TRIM proteins. In humans, the TIF family is comprised of three members that display remarkably similar overall architecture (Figure 1).9., 15., 16., 17., 18., 19., 20. Each contains an NH₂-terminal RBCC/TRIM domain, a centrally located TSS sequence of unknown function, and tandem COOH-terminal PHD and bromodomains. KAP-1 and TIF1α also share the pentapeptide, HP1 interaction domain, whereas TIF1α alone contains a nuclear receptor interaction domain (NRID).17., 21., 22.

It is likely that all TIF family members function as transcriptional intermediary factors, either as coactivators or corepressors. TIF1α was discovered as a ligand-dependent co-regulator for nuclear hormone receptors. It binds directly to RXR-RAR proteins via its NRID and mediates silencing of target genes probably via HP1 recruitment.17., 23. TIF1γ was discovered via low-stringency hybridization screens and has no known protein partner for tethering it to DNA.²⁰ Both TIF1α and TIF1γ contain intrinsic transcriptional repression functions that are localized to their COOH-terminal PHD and bromodomains; however, the downstream machinery that mediates gene silencing is not well defined.

KAP-1/TIF1β is the best understood member of the family. We discovered KAP-1 in a screen for proteins that would bind to the KRAB repression domain.¹⁵ The Kruppel-associated box (KRAB) domain is a 75 amino acid residue module found at the NH₂ terminus of almost 200 C2H2 zinc-finger proteins in the human genome.24., 25., 26. This highly conserved domain is composed of two amphipathic charged helices and mediates potent, DNA-binding-dependent repression of transcription in mammalian cells.15., 16., 18., 19., 27. We have characterized the KRAB-KAP-1 interaction extensively and this has provided the first rules for an RBCC/TRIM ligand interaction.28., 29. The KRAB domain binds directly with high affinity to the RBCC/TRIM domain of KAP-1. This binding requires all three subdomains of the RBCC/TRIM domain and mutation of any individual subdomain abolishes binding. The KAP-1 RBCC/TRIM domain oligomerizes as a homo-trimer, and oligomerization is obligate for KRAB binding.²⁹ The stoichiometry of KRAB-KAP-1 complex is 1:3.²⁸ In vivo, the KRAB-mediated gene silencing is absolutely dependent on KAP-1 binding.¹⁵ The gene-silencing mechanism involves recruitment of the NURD histone deacetylase complex, binding to a histone methylase, and recruitment of heterochromatin proteins.21., 22., 30., 31. However, it is unclear how or if KAP-1 trimerization influences these activities.

Thus, the TIF subfamily of RBCC/TRIM proteins share common domains and biological properties. An important unresolved question is whether there is cross-talk among these closely related family members, possibly mediated by hetero-oligomerization. There are many precedents for hetero-oligomerization among proteins with extended coiled-coil regions.5., 32., 33., 34., 35. Hetero-oligomerization provides a mechanism for generating tremendous combinatorial diversity from a small number of subunits, and often produces complexes with unique biochemical and biological properties.36., 37., 38.

In this study, we have performed a comprehensive systematic analysis of the oligomerization properties of the TIF family, PML1, and MID1 RBCC/TRIM proteins using a unique baculovirus co-infection system. Our data indicate that the RBCC/TRIM domains of KAP-1, TIF1α and TIF1γ exist as homo-oligomers. Although the KAP-1 protein did not hetero-oligomerize with TIF1α, TIF1γ, PML1, and MID1, there is significant cross-talk between TIF1α and TIF1γ, which alters the corepression properties of TIF1α.