KAP1/TRIM28/TIF1β was identified nearly twenty years ago as a universal transcriptional co-repressor because it interacts with a large KRAB-containing zinc finger protein (KRAB-ZFP) transcription factor family. Many studies demonstrate that KAP1 affects gene expression by regulating the transcription of KRAB-ZFP-specific loci, trans-repressing as a transcriptional co-repressor or epigenetically modulating chromatin structure. Emerging evidence suggests that KAP1 also functions independent of gene regulation by serving as a SUMO/ubiquitin E3 ligase or signaling scaffold protein to mediate signal transduction. KAP1 is subjected to multiple post-translational modifications (PTMs), including serine/tyrosine phosphorylation, SUMOylation, and acetylation, which coordinately regulate KAP1 function and its protein abundance. KAP1 is involved in multiple aspects of cellular activities, including DNA damage response, virus replication, cytokine production and stem cell pluripotency. Moreover, knockout of KAP1 results in embryonic lethality, indicating that KAP1 is crucial for embryonic development and possibly impacts a wide-range of (patho)physiological manifestations. Indeed, studies from conditional knockout mouse models reveal that KAP1-deficiency significantly impairs vital physiological processes, such as immune maturation, stress vulnerability, hepatic metabolism, gamete development and erythropoiesis. In this review, we summarize and evaluate current literatures involving the biochemical and physiological functions of KAP1. In addition, increasing studies on the clinical relevance of KAP1 in cancer will also be discussed.

KRAB domain-associated protein 1 (KAP1), also known as tripartite motif-containing 28 (TRIM28) or transcriptional intermediary factor 1 beta (TIF1β) was identified as an interacting protein for Krüppel-associated box zinc finger proteins (KRAB-ZFPs) in 1996[1-4]. Since then, KAP1 has been reported in regulating multiple aspects of physiology, for examples, cell differentiation, DNA damage response (DDR), virus replication, immune response and tumorigenesis (Figure 1). As KAP1 binds to the conserved KRAB repression domain, which is present in many transcription factors, KAP1 is considered as a critical transcriptional co-repressor[2-4]. For instance, many proteins involved in chromatin remodeling or histone modification, such as heterochromatin-associated protein 1 (HP1), nuclear co-repressor (N-CoR), histone deacetylase (HDAC), chromodomain helicase DNA binding protein 3/nucleosome remodeling deacetylase (CHD3/NuRD), histone methyltransferases (HMTs), have been identified in KAP1-containing complexes[5-9] (Table 1). Consequently, KAP1 epigenetically regulates gene expression through multiple transcriptional co-repressor complexes. In addition to regulating KRAB-ZFPs, the activity of transcription factors lacking KRAB domain, such as c-Myc and E2F1, can also be regulated by KAP1[10-14]. KAP1 is subjected to multiple post-translational modifications (PTMs), including phosphorylation and SUMOylation (Figure 2). We and others have demonstrated that these PTMs coordinately regulate the gene repressive function of KAP1[15-18].

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Figure 1 KRAB domain-associated protein 1 is involved in multiple aspects of cellular physiology. Neurology: Kap1 knockout in mouse forebrain induces higher level of anxiety-like behavior. Developmental Biology: Several conditional kap1 deletions impair normal cell development including embryonic stem cell (ESC) differentiation, spermatogenesis, erythropoiesis, and the development of T-cell and B-cell. Immunology: KAP1 is involved in immune responses by regulating T/B cell activity and immune tolerance. Virology: KAP1 is critical to suppress retroviral activation and prevent HIV integration. Cancer Biology: KAP1 is positively or negatively correlated with prognosis in different cancer types. The roles of KAP1 in maintaining genome stability, mediating DNA damage response and affecting cell proliferation in vitro imply its potential roles during tumorigenesis.

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Figure 2 KAP1 structure, post-translational modifications and interacting proteins. KAP1 has multi-domains for protein-protein interaction and post-translational modification. RBCC: RING-B1-B2-coiled-coil; TSS: TIF signature sequence; HP1 BD: HP1 binding domain; PHD: Plant homeo domain. Numbers represent the sequence of amino acids; Blue: SUMOylation sites; Red: Serine phosphorylation sites targeted by the indicated kinases (shown in red) or antagonized by phosphatases (shown in green); Orange: Tyrosine phosphorylation sites targeted by the indicated kinase family; KLIYF: PP1 binding site; PxVxL: HP1 binding site; Dotted lines: Protein-protein interaction; SFKs: Src family kinases; HP1: Heterochromatin-associated protein 1; ATM: Ataxia-telangiectasia mutated; PP1: Protein phosphatase 1; CHD3/NuRD: Chromodomain helicase DNA binding protein 3/nucleosome remodeling deacetylase; HDAC: Histone deacetylase.

Deletion of Kap1 in mouse embryo leads to embryonic lethality[19], suggesting that KAP1 is critical during embryonic development and should be involved in a wide-range of biological/physiological processes. Although most of the KAP1 studies have been focusing on its transcriptional functions, emerging evidence suggests that KAP1 also exerts transcription-independent function. Given that KAP1 functions as a scaffold protein to constitute KAP1-containing complexes that regulate chromatin structure, it also plays an important role in maintaining genome stability by facilitating DNA repair in response to DNA damage through chromatin remodeling[20-22]. Moreover, the RING and the plant homeodomain (PHD) domains of KAP1 possess intrinsic enzymatic activity to potentially catalyze SUMOylation and ubiquitylation[15,23-25]. Interestingly, KAP1 resides in distinct cellular compartments, including the pericentric and centromeric heterochromatin, euchromatin and cytoplasm[6,25,26], implicating its essential functions for different cellular activities. Taken together, the diverse function and regulated subcellular localization suggest that KAP1 impacts multiple aspects of biological processes and it warrants more rigorous investigations of this important protein in the future. In this review, we aim to discuss both the transcriptional and non-transcriptional functions of KAP1. The PTMs of KAP1 involved in these processes will be highlighted. Lastly, the clinical relevance of KAP1 in cancer will also be elaborated.

Given that the overall structure of KAP1 has been extensively reviewed previously[27] (and references therein) we will mainly focus on the PTMs of KAP1 and how these PTMs crosstalk to each other and affect the interaction between KAP1 and its partners. KAP1 is a member of TIF1 family, which includes four proteins, TIF1α, TIF1β, TIF1γ and TIF1δ. As the other members in the TIF1 family, KAP1 has an N-terminal tripartite motif (TRIM), RBCC domain, which is composed by a RING finger, 2 B-box zinc fingers and a coiled-coil region. In addition, KAP1 also shares a central TIF1 signature sequence (TSS), an HP1 binding domain (HP1BD), a C-terminal combination of PHD and bromodomain with the other TIF1 members. Different from the other TIF1 proteins, KAP1 does not have a nuclear receptor (NR) box[27] (Figure 2).

The RBCC domain of KAP1 interacts with various KRAB-ZFPs and is considered as an important region for the KAP1 recruitment to KRAB-ZFP binding sites across the genome[28,29]. It has been demonstrated that RBCC domain forms a homotrimer with a single KRAB domain[30]. Interestingly, a recent study suggests that KAP1 can still bind to promoter regions without RBCC domain, suggesting additional mechanisms that might contribute to the KAP1 recruitment to transcription factors on the promoter regions[31]. The TSS domain is adjacent to the RBCC domain, and is required for the transcriptional repressive activity of TIF1γ[32]. However, the function of TSS in KAP1 has yet to been defined.

The hydrophobic PxVxL pentapeptide is located at the central region of KAP1, namely HP1BD. HP1BD interacts with the chromoshadow domain of HP1 proteins and this KAP1-HP1 interaction is critical for the KAP1-mediated gene silencing[5,6,33,34]. It is believed that KAP1-HP1 complex plays a critical role in heterochromatin maintenance and gene silencing. In fact, HP1BD is required for nuclear retention of KAP1 and disrupting KAP1-HP1 interaction reactivates the imprinted gene expression by perturbing histone and DNA methylation levels in vivo[31,35]. However, the detailed mechanism underlying the KAP1 recruitment to HP1 on heterochromatin remains to be elucidated.

The C-terminal PHD and bromodomain of KAP1 (PB domain) recognize histone tail and are also required for the KAP1-mediated gene silencing by recruiting histone modifiers. Specifically, KAP1 interacts with CHD3/NuRD complex, and histone methyltransferase, SET domain, bifurcated 1 (SETDB1)[8,9]. These observations suggest a model for KAP1-dependent recruitment of histone modifiers for histone methylation and heterochromatin formation to achieve gene silencing[27].

Notably, the carboxyl-terminus of KAP1 is subjected to multiple types of PTM. Several studies have revealed that KAP1 is SUMOylated and the SUMOylation of KAP1 is required for its repressive function[15,16,18]. Within or adjacent to the PB domain, six lysines of 554, 575, 676, 750, 779 and 804 have been validated as SUMOylation sites, and the distinct SUMOylation combinations differentially affect the interaction between the bromodomain with SETDB1 and CHD3[15,16,18,28]. KAP1 also undergoes auto-SUMOylation[15]. The magnitude of KAP1 SUMOylation is balanced by deSUMOylases, sentrin specific peptidase 1 (SENP1), SENP7 and the phosphorylation status at serine-824 of KAP1[15,17,36,37].

The serine-824 of KAP1 is primarily phosphorylated by phosphoinositide 3 kinase-like protein kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), and DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs)[38]. This specific serine 824-phosphorylation is crucial for DDR in different aspects, for examples, the ATM-mediated KAP1 serine-824 phosphorylation is responsible for activating DNA damage checkpoints and chromatin relaxation[17,20], whereas ATR or DNA-PKcs presumably compensates for ATM-deficiency during DDR[39]. Our laboratory has demonstrated that protein phosphatase 1 (PP1) interacts with KAP1 through the PP1-binding motif in the coiled-coil region of KAP1 to dephosphorylate KAP1 at serine-824[36]. In addition, protein phosphatase 4 (PP4) also mediates the KAP1 dephosphorylation at serine-824 and another phosphorylation site at serine-473 upon DNA damage[40,41].

Many DNA damage-inducing agents render both KAP1 serine-824 phosphorylation and serine-473 phosphorylation through ATM-Chk2 or ATR-Chk1 pathways[42]. KAP1 serine-473 phosphorylation is also involved in efficient DNA repair and cell survival upon DNA damage[42-44]. However, unlike the phosphorylation at serine-824, the KAP1 serine-473 phosphorylation is diffusely localized in the nucleus instead of accumulating at damage sites and forming foci[44,45]. It remains to be established whether different KAP1 phosphorylation sites play distinct roles in response to DNA damage. Interestingly, the KAP1 serine-473 phosphorylation regulates cell cycle progression. A study demonstrated that PKCδ phosphorylates KAP1 at serine-473 during S phase of cell cycle. This event dampens the KAP1 and HP1γ interaction and de-represses cyclin A2 to promote S phase progression[46]. Conceivably, KAP1 serine-473 phosphorylation perturbs its association with HP1γ, thereby rendering pan-nuclear distribution. This may be due to the structural alteration of PxVxL domain, which is the HP1 binding site of KAP1. KAP1 mutant that loses its binding ability with HP1 indeed translocates to cytoplasm[31]. This implies that KAP1 serine-473 phosphorylation could function beyond gene regulation in cytoplasm. Results from several studies also suggest that KAP1 serine-473 phosphorylation is associated with immune response. First, KAP1 is constitutively phosphorylated at serine-473 but not serine-824 upon T cell receptor activation in thymocytes[47,48]. Second, Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) infection induces phosphorylation at KAP1 serine-473 in endothelial cells[49]. Many KAP1’s partners, such as STAT1/3, NF-κB and IRF5/7[24,49-52], are involved in inflammation and immune response. Further exploring the role of KAP1 serine-473 phosphorylation in the immune responses induced by viral infection or other types of stress could be rewarding. More recently, Kubota et al[53] reported that tyrosines-449, 458 and 517 of KAP1 are phosphorylated by Src family kinases (SFKs). The phosphorylation of these tyrosine residues also interferes the interaction between KAP1 and HP1. As SFKs are involved in regulating a wide-range of oncogenic processes, including cell growth and differentiation, this finding implies that KAP1 plays a role in SFK-mediated oncogenic transformation.

KAP1 is also demonstrated to be acetylated and the level of KAP1 acetylation is downregulated by HDAC10[54]. Although HDAC10 regulates KAP1 transcriptional co-repressor activity, there is no direct evidence showing whether the acetylation affects KAP1-mediated transcriptional control. Studies on the regulation of KAP1 acetylation and whether KAP1 acetylation crosstalks with other types of KAP1 lysine-PTM are expected to provide additional insights into the roles of PTMs in governing KAP1 function.

KAP1 was originally identified to be associated with KRAB-ZFPs, suggesting a role of KAP1 in transcriptional control. In addition to the RBCC domain, there are also other domains allowing KAP1 to interact with a wide variety of proteins such as histone acetylases (HATs), HDACs, HMTs and DNA methyltransferases (DNMTs). Thus, KAP1 has the flexibility to regulate transcription through multiple mechanisms, possibly depending on its PTMs, location and specific binding partners (Figure 2, Tables 1 and 2).

While extensive efforts have been made in understanding how KAP1 regulates transcription, less is known about its non-transcriptional functions. In fact, KAP1 has a critical, transcription-independent role in DNA repair processes. Additionally, emerging evidence shows that KAP1 possesses enzymatic activity required for multiple cellular processes. In the following sections, we aim to discuss the role of KAP1 as a signaling scaffold protein in DNA repair and its novel roles as SUMO and ubiquitin E3 ligases.

KAP1 is involved in many aspects of cellular physiology (Figure 1). First, KAP1 exerts critical function during embryonic development because global Kap1-knockout causes embryonic lethality in mice due to the inability to undergo gastrulation[19] (Table 3). The function of KAP1 in maintaining pluripotency of embryonic stem cells (ESCs) has been demonstrated[78,93]. In addition, KAP1 is also required for ESC differentiation[94-97]. Studies using conditional Kap1-knockout mice show that KAP1 plays pivotal roles in different cellular maturation processes such as spermatogenesis, erythropoiesis, and the development of T-cell and B-cell (Table 3)[71,94,98-101]. Beyond regulating T-cell and B-cell differentiation, KAP1 also functions in immune response by additional mechanisms. For example, KAP1 is present in FOXP3-containing complex and facilitates the suppressor activity of regulatory T cells[74]. Moreover, KAP1 is involved in immunoglobulin class switch recombination[102]. Recent studies demonstrate that KAP1 associates with STAT1 and STAT3, master regulators of immune response, to negatively regulate their downstream signaling. These results suggest the involvement of KAP1 in immune response[50,51,82].

Using a mouse model, Jakobsson et al[103] show that conditional deletion of Kap1 in adult forebrain caused higher level of anxiety-like activity, suggesting that Kap1 is a regulator for behavioral stress response. Although the molecular mechanism remains elusive, this could be due to the function of Kap1 in regulating gene expression in hippocampus. It is worth noting that among these dysregulated genes in hippocampus, some are imprinted genes[103]. We speculate that Kap1 is also required for the maintenance of a set of specific imprinted genes because many recent studies have demonstrated that KAP1 mediates DNA methylation by recruiting DNMTs to ICRs during early embryogenesis[59,61,62].

More than maintaining normal cellular physiological functions, KAP1 also regulates several pathways in response to different stresses. As mentioned earlier, several studies have focused on the role of KAP1 in DDR. KAP1 is known to be phosphorylated at serine-824 by ATM upon DNA damage and to transduce downstream signaling[20,38]. For instance, ATM-mediated KAP1 phosphorylation leads to de-repression of p21, Gadd45α, Bax, Puma, and Noxa, causing cell cycle arrest and apoptosis[16,17,36]. Additionally, KAP1 is an ATM downstream mediator during DSB-induced heterochromatin relaxation, which facilitates DNA repair[20,21,83]. KAP1 also cooperates with MDM2 to suppress p53 signaling by promoting p53 degradation[75,104]. Thus, PTMs of KAP1 might be important in regulating p53 activity as well.

In contrast to its co-repressor function, KAP1 can also be a co-activator in specific circumstances. It has been reported that mouse Kap1 interacts with Nrf2 to enhance Nrf2-meidated-cytoprotective function in NIH3T3 cells in response to oxidative stress[76]. However, whether KAP1 exerts similar co-activator activity in human cells is not clear.

Some studies demonstrate that KAP1 mediates viral gene expression and plays a role during viral latency. Specifically, KAP1 is associated with KSHV lytic gene promoter to suppress lytic gene expression and in turn maintains virus latency[57]. Similar observation has also been made in retroviruses such as Murine Leukemia Virus (MLV) and human T-cell lymphotropic virus-1 (HTLV-1), that KAP1 restricts pro-viral gene activation[105-108]. Interestingly, recent studies show that retroelements derived from endogenous retroviruses (ERVs), which are extensively present in mammalian genome, can also be silenced by KAP1 to protect genome integrity in embryonic stem cells[109-112]. In addition, KAP1-HDAC1 complex deacetylates and inhibits human immunodeficiency virus (HIV) integrase activity, thereby reducing HIV infectivity and its integration to host genome[113].

The clinical relevance of KAP1 in diseases remains elusive. Most reports were based on studies in cancers. Higher expression of KAP1 has been linked to pro-metastatic cervical cancer[114]. Moreover, the up-regulation of KAP1 could be a potential marker in colorectal cancer patients and higher level of KAP1 is correlated with poorer overall survival in patients with gastric cancer and thyroid carcinoma[115-119]. These clinical studies suggest that higher KAP1 level is linked to a poor prognosis in certain cancers. However, opposite conclusions were also reported. KAP1 overexpression is associated with better overall survival in early-stage lung cancer[14]. Furthermore, a mouse model showed that Kap1-depletion in liver increases male-predominant hepatic adenoma[120] (Table 2).

Based on the studies focusing on transcriptional regulation, KAP1 suppresses p53 transcription activity and has been proposed to be a target for anti-cancer therapy[75,104]. Nonetheless, KAP1 also suppresses activity of oncogenic transcription factors HIF-1α and STAT3[72,82]. In vitro studies using cancer cell lines also show divergent results. KAP1 has been shown to restrain cell growth in breast and lung cancer cells whereas it promotes melanoma cell growth and enhanced KAP1 activity mediates cervical cancer invasion[14,80,114]. Taken together, it is still inconclusive how KAP1 regulates tumorigenesis. Because KAP1 is a multi-faceted protein, it might have tissue-specific function. More study is definitely required to understand how KAP1 contributes to tumorigenesis in particular tissue and whether KAP1 could be a target for cancer therapy.

The functions of KAP1 in diverse cellular physiology have been studied for 18 years. However, the mechanisms of KAP1 in regulating these different cellular processes are still largely unclear. The best-characterized role of KAP1 is its co-repressor function in regulating KRAB-ZFP-mediated or other transcription factor-associated gene silencing. In the future, the identification of more KAP1-interacting transcription factors may help to elucidate how KAP1 regulates gene expression under specific condition. Clearly, KAP1 is critical for maintaining genome integrity as KAP1 modulates the dynamics of hetero- and euchromatin maintenance. In addition, DNA damage induces a robust and transient KAP1 serine-824 phosphorylation and KAP1 is involved in DNA repair pathway choice. However, the molecular mechanism for KAP1-mediated DNA repair requires more rigorous investigation.

KAP1 is subjected to several types of PTM. The phosphorylation at serine-824 and SUMOylation at several lysine residues of KAP1 have been linked to gene regulation. Whether these lysine residues are subjected to additional PTMs and if yes, what is the associated functional consequences need to be further elucidated. Moreover, it would be interesting to look at other PTM sites, such as the phosphorylation of serine-473. Whether the serine-473 phosphorylation affects the transcriptional repressor activity of KAP1 is still under investigation. Another interesting question is how KAP1 shuttles among different cellular compartments by PTMs. The serine-824 phosphorylation has distinct nuclear localization pattern from the serine-473 phosphorylation, thus suggesting that KAP1 phosphorylation may affect its subcellular localization. Intriguingly, KAP1 is not only present in nucleus, and has been observed to translocate from nucleus to cytoplasm[25]. How PTM affects its nucleus-cytoplasmic shuttling and potential chromatin extraction of KAP1 deserve additional investigation. Finally, considering that KAP1 possesses SUMO and ubiquitin E3 activities, it might regulate the function or fate of its SUMO- and ubiquitin-targets. In the future, studying the non-transcriptional function of KAP1 might help to further decipher its unrecognized face.