Adenovirus L-E1A Activates Transcription through Mediator Complex-Dependent Recruitment of the Super Elongation Complex

Abstract

The adenovirus large E1A (L-E1A) protein is a prototypical transcriptional activator, and it functions through the action of a conserved transcriptional activation domain, CR3. CR3 interacts with a mediator subunit, MED23, that has been linked to the transcriptional activity of CR3. Our unbiased proteomic analysis revealed that human adenovirus 5 (HAdv5) L-E1A was associated with many mediator subunits. In MED23-depleted cells and in Med23 knockout (KO) cells, L-E1A was deficient in association with other mediator subunits, suggesting that MED23 links CR3 with the mediator complex. Short interfering RNA (siRNA)-mediated depletion of several mediator subunits suggested differential effects of various subunits on transcriptional activation of HAdv5 early genes. In addition to MED23, mediator subunits such as MED14 and MED26 were also essential for the transcription of HAdv5 early genes. The L-E1A proteome contained MED26-associated super elongation complex. The catalytic component of the elongation complex, CDK9, was important for the transcriptional activity of L-E1A and HAdv5 replication. Our results suggest that L-E1A-mediated transcriptional activation involves a transcriptional elongation step, like HIV Tat, and constitutes a therapeutic target for inhibition of HAdv replication. Adenovirus E1A is the immediate-early viral gene that plays an essential role in viral replication by driving quiescent host cells into the cell cycle and by transcriptional activation of other viral early genes (reviewed in references 1 and 2). The E1A gene codes for two major protein isoforms, designated small E1A (S-E1A) and large E1A (L-E1A). The two protein isoforms differ only by the presence of a 46-amino-acid unique region (designated conserved region 3, or CR3) that is conserved among different adenovirus serotypes. While both E1A proteins can promote proliferation of quiescent target cells by activation of S phase genes, L-E1A is required for transcriptional activation of other viral early genes (3, 4). The role of CR3 in the transcriptional activation of viral early genes was established on the basis of results that showed a substantial decrease in expression of viral early genes in cells infected with different human adenovirus 5 (HAdv5) mutants with lesions in CR3 (3, 4). Further, protein microinjection and transcriptional tethering studies also demonstrated that a synthetic CR3 peptide (5, 6) or a CR3-Gal4 DNA binding domain (DBD) chimeric gene (7,8) can activate viral early promoters. Although E1A CR3 constitutes a powerful trans-activation domain of a viral transcriptional activator and has been intensely investigated as a model trans-activation domain, the mechanism by which it mediates transcriptional activation of other viral early genes remains incompletely understood. The N-terminal 40-aminoacid region of CR3 which forms the C-4 zinc finger (9) has been postulated to function as the trans-activation subdomain, as it is sufficient for transcriptional activation as a Gal4 DNA binding domain fusion protein. The N-te...

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC

“…A number of previous immunoprecipitation studies using the well-defined hAd5 E1A mutant dl1102 (18) have identified the interaction of TRRAP (8, 10) and p400 (11) with the E1A domain encompassing residues 26 to 35. During our unbiased proteomic analyses of cellular proteins associated with L-E1A and S-E1A (20,22), we detected associations of several proteins (in addition to TRRAP, p400, and GCN5) that we reasoned might be constituents of the TRRAP/p400 and TRRAP-GCN5 complexes. These proteins included Tip49, Tip50, BAF53, c-MYC, MYCBP, and ENY2.…”

“…The cell lysates were passed through a 26-gauge needle attached to a 1-ml syringe 10 times and treated with DNase for immunoprecipitation of TRRAP and p400. The lysates were precleared using protein A-agarose fast flow (P3476 Sigma) and immunoprecipitated with the antibodies indicated in the figures, as described previously (22). The bound proteins were resolved by NuPAGE Novex (Life Technologies) 3 to 8% Tris-acetate gels with Tris-acetate running buffer (for TRRAP and p400), 10% Bis-Tris gels with MOPS (morpholinepropanesulfonic acid) running buffer (for c-MYC), or 4 to 12% Bis-Tris gel with MES (morpholineethanesulfonic acid) running buffer (for all other proteins) and analyzed by Western blotting.…”

The human papillomavirus E7 oncoprotein as a regulator of transcription

“…HPV16 E7, like E1A, can also bind to another component of TFIID, TAF110 (Mazzarelli, Atkins et al 1995), although the consequences of this interaction are not known. E1A can also interact directly with the Mediator complex and with the transcriptional elongation machinery, recruiting them to promoters to drive transcription (Vijayalingam and Chinnadurai 2013). Whether E7 has a similar activity is not known.…”

Deregulations in the Cyclin-Dependent Kinase-9-Related Pathway in Cancer: Implications for Drug Discovery and Development

“…The CDK9-related pathway regulates a wide range of functions in mammalian cell biology [1–12, 24] and the replication program of numerous viral agents, such as the human immunodeficiency virus type 1 (HIV-1) and HIV-2 [25, 56], Epstein-Barr virus (EBV) [57], human T-lymphotropic virus type 1 (HTLV-1) [58, 59], human cytomegalovirus (hCMV) [60, 61], herpes simplex virus 1 (HSV-1) [62, 63], human adenovirus [64], and influenza A virus [65] (Table 2). Moreover, dysfunctions in the CDK9-related pathway are related with several forms of human tumors [9, 13, 26–28, 66–68] and cardiac hypertrophy [69–75].…”