Ad E1A 243R oncoprotein promotes association of proto-oncogene product MYC with the NuA4/Tip60 complex via the E1A N-terminal repression domain

Abstract

The adenovirus E1A 243R oncoprotein targets TRRAP, a scaffold protein that assembles histone acetyltransferase (HAT) complexes, such as the NuA4/Tip60 complex which mediates transcriptional activity of the proto-oncogene MYC and helps determine the cancer cell phenotype. How E1A transforms cells through TRRAP remains obscure. We performed proteomic analysis with the N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) and showed that E1A 1-80 interacts with TRRAP, p400, and three other members of the NuA4 complex - DMAP1, RUVBL1 and RUVBL2 - not previously shown to associate with E1A 243R. E1A 1-80 interacts with these NuA4 components and MYC through the E1A TRRAP-targeting domain. E1A 243R association with the NuA4 complex was demonstrated by co-immunoprecipitation and analysis with DMAP1, Tip60, and MYC. Significantly, E1A 243R promotes association of MYC/MAX with the NuA4/Tip60 complex, implicating the importance of the MYC/NuA4 pathway in cellular transformation by both MYC and E1A.

Keywords: 243R; DMAP1; E1A 1-80; NuA4; RUVBL1; RUVBL2; TRRAP; Tip60.

Figure 1. E1A 1-80FH is active in transcriptional repression and interaction with p300

A. Lentiviral expression constructs for E1A 1-80FH and FH-GFP. B. Luciferase reporter assays with pHER2-Luc and the lentiviral constructs. HeLa cells were co-transfected with pHER2-Luc, phRL-0 (renilla luciferase), and the lentiviral constructs as indicated, and dual luciferase assays performed as described previously (Loewenstein and Green, 2011). Data plotted represent average of three experiments. C. Interaction of E1A 1-80FH with p300. Lentiviral particles from the constructs in A were used to infect HeLa cells to establish blasticidin-resistant cells. Cell lysates were co-immunoprecipitated with Flag antibody beads, and the bound proteins eluted and examined by Western blots for p300 (upper panel) or for FH-GFP and E1A 1-80FH using HA antibody (lower panel). Lanes 2 and 3 represent two samples of eluted E1A 1-80FH complexes with high (lane 2) and low (lane 3, below detection limit in this Western blot) quantities of E1A 1-80FH.

Figure 2. Interaction of E1A 1-80 and E1A 243R with NuA4 components through the TRRAP targeting domain

A. Illustration of expression constructs. B. Adenoviral-expressed E1A 1-80FH interacts with components of the NuA4 complex identified by proteomic analysis. HeLa cells were infected with Ad expression vectors as indicated, and cell lysates co-immunoprecipitated with Flag antibody beads. Bound proteins were identified by Western blots with indicated antibodies. E1A Western blot analysis was with a rabbit polyclonal antibody. The apparent low quantity of E1A Δ2-11FH in the E1A Western blot analysis was possibly due to weak binding of the protein to the PVDF membrane during the assay. E1A Western blot analysis with Flag and HA antibodies revealed the same result (not shown). C. Adenoviral-expressed E1A 243R interacts with the identified components of the NuA4 complex. HeLa cells were infected with Ad-lacZ (lane 1) or Ad-E1A 243R (lane 2), and co-immunoprecipitation was with E1A M73 antibody beads (SCBT, Inc.). Western blot analysis was as in B, except E1A 243R Western blot analysis was with M58 antibody (SCBT, Inc.)

Figure 3. E1A 243R and E1A 1-80 promote association of MYC/MAX with the NuA4/Tip60 complex

A. Experimental model. The relative position of each protein in the NuA4/Tip60 complex remains to be elucidated. B. Flag-DMAP1 interacts with E1A 1-80 and E1A 243R as well as components of the NuA4 complex identified by proteomic analysis. Flag-DMAP1 was expressed in HeLa cells using a lentivirus vector (lanes 1–4). The cells were then infected with Ad-lacZ (lane 2), Ad-E1A 1-80 C+ (lane 3), or Ad-E1A 243R (lane 4) as indicated. Cell lysates were immunoprecipitated with Flag antibody beads and immunoprecipitates examined by Western blots with indicated antibodies. Lanes 5 and 6 are controls with HeLa cells expressing FH-GFP. Expressed DMAP1-Flag immunoprecipitated by the Flag antibody is indicated by a dashed oval. Expression of E1A 1-80 C+ or E1A 243R recruited MYC/MAX into the NuA4 complex (lanes 3 and 4). C. E1A 243R and E1A 1-80 promote the association of MYC/MAX with the NuA4/Tip60 complex. Experimental conditions are the same as in B, except that Flag-Tip60 (lanes 1–3) and FH-MYC (lanes 4–6) are expressed by lentivirus vectors. When the cells were not infected with an Ad vector (not shown), Flag-Tip60 and FH-MYC had the same patterns of binding as when Ad-lacZ was used (lanes 1 and 4, respectively). Expressed Flag-Tip60 or FH-MYC immunoprecipitated are indicated by a dashed oval to differentiate from cellular endogenous proteins. Expression of both E1A 1-80 C+ and E1A 243R recruited MYC/MAX into the NuA4/Tip60 complex when Flag-Tip60 was used for analysis (lanes 2 and 3). When FH-MYC was immunoprecipitated for analysis, E1A 1-80 and E1A 243R both enhanced the association of FH-MYC with the examined components of the NuA4/Tip60 complex (lanes 5 and 6).