ErbB3-binding protein 1 encodes two alternatively spliced EBP1 isoforms, p48 EBP1 and p42 EBP1. The p48 EBP1 isoform is 54 amino acids longer than p42 EBP1 at its N-terminus. Both EBP1 isoforms are constitutively expressed in all tissues and cells, including cells that do not express the ERBB3 receptor, and only p42 EBP1, and not p48 EBP1, binds to ERBB3 (1, 2). Embryonic development inherently involves many distinct cellular activities, including cell proliferation, survival, and differentiation (2-6), and unlike p42 EBP1, p48 EBP1 is expressed throughout embryonic tissues including brain, contributing to epigenetic control by suppressing the gene silencing unit suppressor of variegation 3-9 homolog 1 (Suv39H1)/DNA (cytosine 5)-methyltransferase (DNMT1) (7, 8).
Despite lacking E3 ligase activity, EBP1 has been implicated in the ubiquitin proteasome system (UPS) for degradation of its binding partner by linking an E3 ligase to its target protein. For instance, p48 EBP1 physically associates with MDM2 (also known as HDM2) and enhances the interaction between MDM2 and p53, promoting p53 degradation in glioblastoma cells of patients with poor clinical outcome (4). Moreover, p48 EBP1 sustains Akt-dependent MDM2 phosphorylation, confining MDM2 to the nucleus, and thereby preventing self-ubiquitination of MDM2 via upregulation of Akt activity (9). Similarly, p42 EBP1 interacts with regulatory subunit, p85 subunit of phosphatidyl inositol 3-kinase (PI3K) that results in the UPS-dependent degradation of the p85 subunit by recruiting the E3 ligase carboxy terminus of heat shock protein 70 (Hsp70) interacting protein (CHIP) (5), which accounts for the tumor suppressor activity of p42 EBP1. Despite growing evidence of the roles of the EBP1 isoforms in UPS-dependent protein degradation for the regulation of diverse cellular activities over the past decade, it is not known whether EBP1 contributes to transcriptional regulation exclusively by epigenetic control or also via UPS-dependent protein suppression during embryonic development.
Methylation of lysine 9 on histone H3 (H3K9) generates silent domains, a process which is critical for heterochromatin assembly, and is sufficient for the initiation of gene repression (10, 11). Spurred by our finding of dramatic Suv39H1-mediated histone methylation changes in EBP1-deficient cells during embryonic development, we then questioned whether Suv39H1 protein level is also affected by p48 EBP1. We previously reported that gene expression of Suv39H1 and H3K9 trimethylation was upregulated in the absence of EBP1; similarly, in the current study, we not only found transcriptional repression of Suv39H1 by p48 EBP1, but also found that the protein level of Suv39H1 was markedly increased in EBP1-deficient mouse brain and
Our recent study of genetic ablation of
To further assess the role of EBP1 in Suv39H1 regulation, we transfected GFP-tagged Suv39H1 with or without EBP1 and performed cellular fractionation analysis. As anticipated, in the nuclear fraction, we found that the protein level of Suv39H1 was decreased, and taken in accordance with reduced H3K9 methylation in the presence of Flag-EBP1 expression, reflects decreased enzymatic activity of Suv39H1 (Fig. 1E). Moreover, we found decreased binding of Suv39H1 to histone H3 under the condition of
To determine whether EBP1 physically interacts with Suv39H1, we cotransfected GST-Suv39H1 with various fragments of GFP-EBP1 in HEK293T cells. GST pull-down experiments revealed that EBP1 directly interacts with Suv39H1, and its N-terminus 54 amino acid residues are indispensable for the interaction between EBP1 and Suv39H1 (Fig. 2A). To map the specific region of Suv39H1 that binds to EBP1, we generated a series of Suv39H1 fragments (Fig. 2B) and performed mapping analysis. We found that EBP1 weakly binds to full-length Suv39H1 and to the N-terminal domain (amino acids 1-179) that includes an essential chromatin organization modifier domain (chromodomain), which is a major reader of histone methylation tags in proteins, at amino acids 44-88. However, EBP1 robustly bound to the SET domain of Suv39H1, which is a conserved site for lysine methylation (Fig. 2C). To identify the residues involved in this interaction, we first verified the association between EBP1 and the SET domain of Suv39H1 by demonstrating increased binding in response to increased
To explore the molecular mechanisms underlying EBP1-induced reduction of Suv39H1, we first examined the half-life of Suv39H1, and found that it was markedly decreased in
To further evaluate the specificity of Suv39H1 degradation by EBP1, we examined ubiquitination of various truncated forms of Suv39H1. Similar to that found in our protein binding analysis, Suv39H1 ubiquitination was robust in the SET domain, exhibiting a strong binding ability with EBP1, and the full WT protein and chromodomain of Suv39H1 were also ubiquitinated albeit at lower levels. In contrast, areas flanking the SET domain did not interact with EBP1 and were not ubiquitinated (Fig. 3E). Further, the interaction between EBP1 and Suv39H1 was enhanced by MG132 exposure (Fig. 3F). These findings indicate that EBP1 binding is required for Suv39H1 ubiquitination.
Next, we hypothesized that EBP1 recruits an E3 ligase for Suv39H1 in the nucleus based on the following: 1) our cellular fractionation analysis after EBP1 depletion revealed that stabilization of Suv39H1 was prominently in the nucleus, 2) we also found enhanced methyltransferase activity as shown by H3K9 trimethylation (Fig. 4A), and 3) EBP1 does not possess E3 ligase activity. Considering our previous findings that EBP1 physically associates with MDM2 and enhances the interaction between MDM2 and p53 (4), and that EBP1 confines MDM2 in the nucleus by sustaining MDM2 phosphorylation and inhibiting self-ubiquitination (9), in addition with the finding that Suv39H1 is a substrate of MDM2 (12), we investigated whether EBP1 contributes to MDM2-dependent Suv39H1 degradation. Overexpression of MDM2 elicits a >50% reduction of Suv39H1, and that this effect was facilitated by co-expression with EBP1 up to a 70% reduction, although
In agreement with a previous finding that MDM2 binds to the chromodomain of Suv39H1 and induces subsequent ubiquitination of Suv39H1 (12), our protein binding analysis showed that MDM2 binds to the chromodomain of Suv39H1, and in fact, exhibits a strong interaction with the chromodomain containing the N-terminal domain (amino acids 1-179) of Suv39H1, but not the SET domain or any other domain of the protein (Fig. 4E). In addition, our ubiquitination experiments showed that the Δchromo mutant exhibited approximately 60% of the ubiquitination found for WT-Suv39H1 (Fig. 4F). However, this decrease in ubiquitination was reversed by overexpression of
In the current study, we uncovered an additional role of EBP1 for the regulation of SUV39H1-mediated H3K9 methylation in a post-translational modification process. In
We have shown that EBP1 controls Suv39H1 protein levels by enhancing polyubiquitination with the E3 ligase MDM2, which is an interesting finding considering the role of MDM2 as the main regulator of p53 and its involvement in p53 pathway modulation in various cellular contexts (13). In fact, Suv39H1 forms a complex with MDM2 and p53 that negatively regulates p53 function (14). In p53-activating conditions, binding of Suv39H1 to MDM2 is inhibited. Previously, we have shown that EBP1 forms a complex with MDM2 and p53, facilitating p53 degradation as well as inhibition of p53 function in certain types of cancer cells (4). In the absence of EBP1, the level of phospho-MDM2 is decreased (Fig. 4D), a finding which is consistent with reduced ubiquitination of Suv39H1 (Fig. 3D), and reflects the increased binding affinity of Suv39H1 and MDM2 under conditions of p48 EBP1 overexpression (Fig. 4B). Although EBP1 promotes access of MDM2 to target proteins, such as p53 and Suv39H1, and enhances the ubiquitination and degradation of target proteins, it has not yet been determined whether EBP1-mediated degradation of Suv39H1 is a spatio-temporal event during development as H3K9 modification appears to have a critical role in embryogenesis. Furthermore, this importance is highlighted by the finding that mutations in genes of H3K9-modifying enzymes cause severe embryonic growth defects (15) as well as our finding that aberrant upregulation of Suv39H1 contributes to embryonic lethality (8). In addition, whether EBP1 links MDM2 to p53 or Suv39H1 with any preference to the presence of p53 expression, and whether regulation of Suv39H1 levels by EBP1 ultimately modulates the p53 pathway, is unknown and should be addressed in the future.
Chromosome instability is involved in tumor initiation and progression. Suv39H1 deficiency impairs H3K9 methylation at pericentromeric heterochromatin and leads to chromosome instability. For instance, Suv39H1-deficient mice exhibit spontaneous B cell lymphoma and meiosis defects (16). We found that overexpression of
Overall, our results demonstrate that p48 EBP1 is a key regulator of Suv239H1 through UPS-dependent degradation by modulating accessibility of the E3 ligase MDM2 during embryonic development. These findings provide a molecular explanation for the role of EBP1 in the regulation of epigenetic control and furthers a possible role of EBP1 in the regulation of chromosome instability.
Materials and methods are available in the supplemental materials.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government [Ministry of Science, Information and Communication Technology and Future Planning (MSIP)] (2016R1A5A2945889 and 2020R 1A2C2003268).
The authors have no conflicting interests.