No abstract

Efficient transcription of the adenovirus Ela-inducible E4 gene is mediated by an Ela-dependent enhancer in vivo. In vitro, the enhancer functions constitutively in the absence of Ela, conferring high levels of transcription on the E4 and heterologous promoters. Binding of a cellular transcription factor, E4F1, to two sites within the E4 enhancer and to one other functionally important site located directly upstream of the E4 TATA box is required for transcriptional activity. The relationship between the enhancer and the TATA box proximal site is further demonstrated by the fact that the E4 enhancer can be functionally substituted by two copies of the TATA box proximal site. These and other results suggest that the E4 promoter may be comprised solely of multiple E4F1 binding sites and a TATA box. In addition to the E4 promoter, E4F1 interacts with other adenovirus early promoters and thus may be involved in the co-ordinate expression of Ela-inducible early viral genes.

We have examined the relationship between sequence-specific DNA-binding proteins that activate transcription of ElA-inducible adenovirus early promoters. Factors previously referred to as E4F1 and E2A-EF bind to the E4 and E2A promoters, respectively. We demonstrate here that E4F1 and E2A-EF have identical DNA-binding specificity. Moreover, E4F1 and E2A-EF both activate transcription of the E4 and E2A promoters in vitro. These findings demonstrate that E4F1 and E2A-EF are the same factor, which we have designated activating transcription factor, or ATF. In addition to the E4 and E2A promoters, ATF binds to an important functional element of the ElA-inducible E3 promoter. Interaction of a common activator protein, ATF, with multiple ElA-inducible early viral promoters, suggests a significant role for ATF in ElA-mediated transcriptional activation.The EIA gene of adenovirus produces closely related 13S and 12S mRNAs that encode nuclear-localized phosphoproteins with diverse transcriptional regulatory properties (1-3). The EMA 13S gene product coordinately activates a set of viral early genes (EIA, EIB, E2A, E3, and E4) during a productive infection of permissive human cells (4-7). The ElA 12S gene product encodes a transcriptional repression function that appears to act through transcriptional enhancer elements (8-10). In addition to regulating viral transcription, ElA activates or represses transcription of a limited number of cellular genes (11,12), and activates polymerase III-dependent promoters (for review, see ref. E4 and E3 transcription (18, 19). For the EIB promoter (22) and the cellular /-globin promoter (23), the "TATA" box has been implicated as an ElA-responsive promoter element. Thus activation of a variety of ElA-inducible promoters appears to involve different cellular factors and may occur through divergent pathways, ultimately linked by ElA. Two independent studies have identified additional factors that interact with early viral promoters. A factor referred to as E4F1 binds to the E4 promoter and also interacts with the EIA, E2A, and E3 promoters (18). Similarly, a factor referred to as E2A-EF binds to the E2A, EIA, E3, and E4 promoters (17). We show here that E4F1 and E2A-EF have the same DNA-binding specificity and that both factors activate transcription of the E4 and E2A promoters in vitro. These results demonstrate that E4F1 and E2A-EF are the same factor, which we refer to as ATF, for activating transcription factor. In addition to the E4 and E2A promoters, ATF interacts with an important functional element of the adenovirus E3 promoter. The interaction of ATF with multiple ElA-inducible promoters suggests a significant role for ATF in E1A-mediated transcriptional activation. MATERIALS AND METHODSPlasmids. pE4WT contains the adenovirus type 5 genome between map units 100 and 89, including the entire E4 gene cloned into pBR322 between the EcoRI and Pvu II sites.

Chromosomal translocations involving the N-terminal Ϸ250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.Ewings sarcoma oncogene ͉ EWS/ATF1 ͉ intrinsically disordered proteins ͉ EWS activation domain ͉ molecular recognition feature T he Ewings sarcoma (EWS) protooncogene together with TLS/FUS and hTAF II 68 form a subgroup (the TET family; ref. 1) within the RNP family of RNA-binding proteins. Aberrant chromosomal translocations involving EWS produce EWS fusion proteins (EFPs) that cause human cancers (2, 3) and EFPs share the following common features (Fig. 1A). First, all EFPs contain at least the N-terminal 7 exons of EWS (residues 1-264) encoding the EWS activation domain (EAD). Second, the EWS fusion partner is one of several cellular transcription factors containing a sequence-specific DNA-binding domain that specifies the tumor type. Third, EFPs are potent transcriptional activators that depend on both the EAD and the fusion partner. Gene-specific transcriptional deregulation is likely to be a primary route for tumorigenesis (2, 3). However, some biological effects of EFPs might occur by alternative mechanisms including perturbation of pre-mRNA splicing (3).Progress in determining structure/function relations for the EAD has been limited for several reasons. The EAD has a highly repetitive primary sequence (Fig. 1B) containing multiple copies of a degenerate hexap...