The N terminus of KAP1 contains the RBCC domain, a high affinity protein interaction domain stretching from amino acids 20 to 377. The RBCC domain is necessary and sufficient for interaction of KAP1 with the KRAB repression module of KRAB-ZNFs; all three RBCC subdomains contribute to interaction with KRAB modules (6). Although all TIF1 family members contain an RBCC domain, only KAP1 can bind to the KRAB repression modules of KRAB-ZNFs (6). A number of biochemical and biophysical experiments have revealed that the KAP1 RBCC domain binds as a homotrimer to a single KRAB domain. This oligomerization promotes folding of the KRAB domain and encapsulates it in a protease-resistant core (27). The Ring subdomain is a double zinc-binding C3HC4 motif found in >200 proteins that are components of macromolecular complexes with diverse functions in oncogenesis, RNA transport, cell cycle, ubiquitination, and signal transduction (28). The B-box is a cysteine-rich zinc-binding motif of the form CHC3H2 and, together with the coiled-coil domain, provides an extended hydrophobic α-helical region that presents a strong interface for protein-protein interactions (27). Adjacent to the RBCC is the TSS domain; deletion of this domain abrogates transcriptional repression mediated by TIF1γ (8).

The central region of KAP1 includes the HP1-binding domain, which is a hydrophobic PxVxL pentapeptide located between amino acids 486 and 497 (29). The interaction of KAP1 with the chromoshadow domain of HP1 family members is required for repression of reporter genes (30), discussed in greater detail below. Immunofluorescence studies show that the majority of KAP1 has the same staining pattern as HP1γ, which is present throughout the nucleoplasm but excluded from nucleoli. A small percentage of KAP1 is concentrated into dot-like structures that are regions of pericentric heterochromatin, and an even smaller percentage is found in heterochromatic foci and nucleoli in regions co-occupied by HP1β. These studies are consistent with multiple roles for KAP1-HP1 complexes in silencing euchromatic and pericentric heterochromatic regions (26). The remaining central region of KAP1 is least conserved among all of the TIF1 family members and is rich in prolines, glycines, and serines. No well defined structure has been assigned to this domain; rather, the entire central region of KAP1 is in a highly extended and flexible conformation (29). Perhaps this region provides KAP1 with the adaptability needed for interaction with a multitude of protein complexes.

The C-terminal tandem PHD and bromodomain (called the PB domain) of KAP1 lie between amino acids 618 and 835 and function as a highly cooperative unit for transcriptional repression, with both domains being required to obtain maximum levels of repression (31). The PHD finger of KAP1 is a 60-amino acid domain with a C4HC3 arrangement consisting of two zinc atoms cross-braced between antiparallel β-sheets. The bromodomain of KAP1 is a 100-amino acid stretch consisting of four helices bundled in a unique left-turn topology (32). Typically, bromodomains are found in transcriptional activators and are involved in the recognition of acetylated histone tails (33). Similar to other bromodomain-containing proteins, the bromodomain of KAP1 has a conserved hydrophobic core and recognizes the backbone of histone tails. However, unlike the other proteins, KAP1 has lost its ability to contact acetyllysine residues (31). The PB domain of KAP1 can also interact with two chromatin-modifying enzymes: Mi2α, an isoform of the Mi2 protein found in the NuRD (nucleosome remodeling and histone deacetylation) complex, and SETDB1 (SET domain, bifurcated 1), an H3K9me3-specific histone methyltransferase. Because the addition of an inhibitor of histone deacetylases (HDACs) only partially relieves KAP1-mediated repression of reporter genes and because only a small fraction of KAP1 stably associates with Mi2α in vivo, it is thought that Mi2α may play only a minor role in KAP1-mediated repression (31, 34). In contrast, trimethylation of histone H3 at Lys-9 by SETDB1 creates high affinity genomic binding sites for the KAP1-HP1 complex (because of the ability of HP1 to bind to H3K9me3). This, along with the observation that KAP1 and SETDB1 colocalize at thousands of genomic sites in the human genome (35), suggests that SETDB1 may play an important role in KAP1-mediated repression.

The ability of specific KRAB-ZNFs to bind to the RBCC domain of KAP1 has been studied mainly using artificial assays. Using a mammalian two-hybrid system, the RBCC domain of KAP1 was tested for interaction with KRAB domains from 61 different KRAB-ZNFs (48). The majority of these KRAB-ZNFs could bind KAP1 and were dependent on this interaction for their transcription-repressive abilities. One of the tested KRAB-ZNFs was KOX1. The KRAB domain of KOX1, which was initially used to purify KAP1, is often used as a positive control for KAP1 protein interactions and when testing KAP1-mediated repression (1, 2, 35, 49–51). Interestingly, when full-length KOX1 was tested for its ability to bind endogenous KAP1, it showed a much weaker interaction than was previously observed in vitro, suggesting that, although the KRAB domains of many KRAB-ZNFs are capable of binding KAP1 in vitro, this does not necessarily indicate that the KRAB-ZNF is a major KAP1 interaction partner in the cell (48). KAP1 can also interact with KRAB domains that are not associated with zinc finger domains. For example, the KRAB-O (KRAB only) protein serves as a bridge between the DNA-binding protein SRY (sex-determining region Y) and KAP1, recruiting KAP1 to SRY-binding sites (52, 53). Similarly, others have identified a protein called VHLaK (pVHL-associated KRAB-A domain-containing protein), which serves as a bridge between KAP1 and the von Hippel-Lindau tumor suppressor protein (54). Interestingly, both KRAB-O and VHLaK are alternatively spliced versions of KRAB-ZNF genes that produce proteins containing the KRAB domain but lacking the DNA-binding zinc fingers. In certain cell types, KAP1 has also been shown to associate with the transcription factors MM1, E2F1, MDM2, STAT (signal transducers and activators of transcription) family members, HNRNPAB, TEL/ETV6, CCAAT/enhancer-binding protein β, and NGFI, and through these interactions, KAP1 takes part in numerous processes such as intestinal homeostasis, epithelial-mesenchymal transition, and the immediate-early stress response (18, 19, 24, 55–59).

To determine which, if any, of the KRAB-ZNFs and/or other DNA-binding proteins recruit KAP1 to the genome, KAP1-binding sites were identified using ChIP, followed by microarrays (ChIP-chip) or by sequencing (ChIP-seq), which allows the identification of binding sites for a protein to be identified on a genome-wide scale (60–62). More than 7000 binding sites for KAP1 were identified in NTera2 cells by performing whole genome ChIP-chip experiments (63). Subsequently, using a combination of ChIP-chip and ChIP-seq, KAP1 targets were identified in numerous normal and tumor cells. In all cell types, KAP1 displays a unique genomic localization pattern (Fig. 2A). The strongest KAP1-binding sites are the 3′-coding exons of ZNF genes, whereas the other KAP1-binding sites are either near transcription start sites or in intragenic regions. To determine whether KRAB-ZNFs are involved in recruitment of KAP1 to the target sites, ChIP-seq experiments were performed using a series of mutant KAP1 proteins. These studies showed that KAP1 deleted for the RBCC domain was no longer recruited to the 3′-coding exons of ZNF genes, thus providing strong in vivo support for KRAB-ZNF-mediated recruitment of KAP1 (64). Further studies revealed that ZNF274 colocalizes with KAP1 at 3′-coding exons of ZNF genes (35). It has not yet been possible to demonstrate that other KRAB-ZNFs that show positive in vitro interactions with KAP1 bind to KAP1 genomic sites.⁴ However, the association of KAP1 with specific KRAB-ZNFs may be highly cell type-specific. Interestingly, KAP1 deleted for the RBCC domain can still bind to promoter regions, indicating that KAP1 is recruited to these sites by a novel mechanism independent of a KRAB-ZNF. Thus, there are at least two mechanisms (Fig. 2B) for recruiting KAP1 to the genome, one involving KRAB-ZNFs and one involving other DNA-binding proteins (64). Although the factor that recruits KAP1 to promoters has not yet been identified, mutational analyses suggest that KAP1 may be recruited to promoter targets through protein-protein interactions that occur in the central domain stretching from amino acids 380 to 618 but outside of the HP1 box (64).

Most of the evidence supporting a role for KAP1 as a transcriptional regulator comes from experiments in which an isolated KRAB domain has been fused to a DBD of another transcription factor. For example, the KOX1 KRAB domain fused to the PAX3 DBD can recruit KAP1 and repress a stably integrated PAX3 site-containing promoter, and a Gal4-KRAB fusion protein can repress the activity of a reporter-promoter with five Gal4 sites (34, 39). Others have used inducible KRAB fusion proteins to demonstrate KAP1-mediated repression (38, 66, 67). Studies of mutant KRAB domains also provide support for a role for KAP1 in repression; substitution mutations in the KRAB domain of various KRAB-ZNFs at two highly conserved residues that are critical for interaction with KAP1 result in diminished repression activity (1, 49, 68, 69). Additionally, KAP1 can repress transcription of reporter genes when directly tethered to DNA as a Gal4-KAP1 fusion protein (39). All of these studies reinforce the idea that KRAB-ZNFs mediate repression in a KAP1-dependent manner, which leads to the creation of a heterochromatic epigenetic profile at the targeted locus (Fig. 3A). KAP1 has also been associated with transcriptional activation; using reporter-promoter assays, KAP1 was shown to function as a coactivator for NGFI-B and CCAAT/enhancer-binding protein β (19, 55, 58). The mechanisms by which KAP1 can activate transcription are not known, but perhaps KAP1-associated HDACs and histone methylases function to modify and release repressor proteins bound to the target promoters.

The artificial recruitment experiments clearly demonstrate that KAP1 can influence transcriptional activity in reporter assays. However, whether KAP1 regulates the expression of endogenous cellular genes is less clear. Most cellular promoters bound by KAP1 are not bound by SETDB1 or H3K9me3 (46, 64). However, KAP1 may use alternative methods, perhaps changes in histone acetylation (30), to regulate transcription at cellular promoters. For example, two complexes, one containing KAP1, MM1, and Myc and another containing KAP1 and ZNF160, have been shown to repress transcription from cellular promoters in an HDAC-dependent manner in specific cell types (59, 70). Also, KAP1 can interact with STAT3 (57, 71), and reduction of KAP1 levels results in modest increases in the levels of several STAT3-regulated cellular RNAs. However, the mechanism by which KAP1 mediates these effects is not known. No studies were performed to examine KAP1 occupancy of the regulated promoters; therefore, it is not known if KAP1 directly binds to these promoters. In fact, evidence was presented that KAP1 influences the subnuclear localization of STAT3, suggesting that the effects of KAP1 may have been through protein-protein interactions that occur off the DNA. KAP1 can also bind to the E2F1 protein, and ChIP assays showed KAP1 and E2F1 binding at an E2F target promoter (56). However, it was only in the presence of overexpressed proteins that binding of KAP1 could be detected on the target promoter; further studies in which additional promoters are analyzed are required to address the possibility that E2F1 may recruit KAP1 to the genome. Interestingly, KAP1 seemed to increase the interaction of E2F1 with HDAC1 and to decrease the acetylation on the E2F1 protein, suggesting that KAP1 may affect the regulation of E2F target genes by decreasing the activity of the E2F1 protein.

KAP1 has been suggested to regulate apoptosis in a manner independent of its transcriptional activities. KAP1 acts cooperatively with MDM2, a ubiquitin E3 ligase that binds to p53 and marks it for degradation, by recruiting HDAC1 to the MDM2-p53 complex, leading to deacetylation and degradation of p53. Although MDM2 is the major ubiquitin ligase for p53, KAP1 is independently capable of promoting p53 ubiquitination, suggesting that it may encode or recruit a ubiquitin E3 ligase (24). Recently, MAGE proteins, which are highly expressed in various cancers, were shown to be cofactors in KAP1-mediated suppression of p53 activity. MAGE proteins bind to KAP1 and enhance formation of the KAP1-MDM2-p53 complex, leading to suppression of p53-mediated apoptosis and promotion of tumor cell survival (77).

KAP1 has also been implicated in DNA repair. DNA damage such as double-strand break formation induces ATM, which phosphorylates KAP1 (78). It has been proposed that ATM-mediated phosphorylation of KAP1 in response to genotoxic stress results in loss of sumoylated KAP1, leading to derepression of KAP1 target genes involved in promoting cell cycle arrest and apoptosis (79). The KRAB-ZNF ZBRK1 has been shown to repress the GADD45 (growth arrest and DNA damage clone 45) gene in a KAP1-dependent manner (80); perhaps depression of such genes due to a switch from sumoylated to phosphorylated KAP1 is critical for DNA repair. Recent findings implicate protein phosphatase 1β (PP1β) in the recovery of KAP1 repressive function after DNA damage-induced phosphorylation (81). PP1β can interact with the coiled-coil domain of KAP1 and dephosphorylate KAP1, promoting sumoylation of KAP1 and return of its repressive function. Thus, KAP1 exists in a balance between a phosphorylated and a sumoylated state, which influences its repressive abilities (79). Such studies suggest that investigation of the role of KAP1 in regulating the transcriptome should perhaps be repeated under DNA-damaging conditions. However, KAP1 is also thought to have a non-transcriptional role in regulating the DNA damage response (Fig. 4). Upon DNA damage, there is a rapid localization of phosphorylated KAP1 to DNA damage foci, where it colocalizes with numerous DNA damage response proteins (78). Loss of phosphorylated KAP1 renders cells hypersensitive to DNA damage and leads to loss of DNA damage-induced chromatin decondensation, suggesting that KAP1 must play an active role in this process (73, 82). Although phosphorylation of KAP1 is required for the ATM-mediated global chromatin decondensation in response to double-strand breaks (65, 82), the mechanism by which phosphorylated KAP1 mediates this response is still unknown. Perhaps the switch to its phosphorylated form can cause the local chromatin decondensation required for access of DNA repair proteins, and return to its sumoylated form can assist in re-forming condensed chromatin after the DNA is repaired.