Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

Abstract

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription factor activator protein 1 (AP1). This mutation simultaneously reduces polyomavirus transcription and DNA replication. Reversion of this mutation simultaneously restores binding of PEA1 and both DNA replication and transcription. Binding of a second protein, PEA2, adjacent to the PEA1 site at nt 5147-5155 is enhanced by PEA1 binding, suggesting that these proteins interact. A third protein, PEA3, binds to the sequence AGGAAG (nt 5133-5138) adjacent to the PEA1 binding site; integrity of this late-proximal PEA3 binding site or an additional earlyproximal site (nt 5228-5233) is important for enhancer function. We correlate binding of PEA1 and PEA2 with the induction of a DNase 1-hypersensitive site in polyomavirus minichromosomes isolated from mouse fibroblasts.The polyomavirus enhancer activates the viral early promoter in vivo and is required for viral DNA replication (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). It is composed of multiple functionally redundant DNA elements whose activities vary with cell type and growth state (2, 5-7, 9, 11, 12). These elements serve as binding sites for cellular proteins (13-20) that most likely help form initiation complexes at cis-linked origins and promoters (5,14,21).Two cellular proteins (15) bind to the polyomavirus enhancer at nucleotides (nt) 5139-5155 of the A3 strain (22) or nt 5114-5130 of the A2 strain (23). Polyomavirus enhancer A binding protein 1 (PEA1) binds to nt 5139-5145 (A2 strain nt 5114-5120), which make up the consensus sequence for the HeLa cell transcription factor activator protein 1 (AP1) (TGACTAA). AP1 activates the human metallothionein and simian virus 40 enhancers (24); it is most likely encoded by the human protooncogene c-jun (25), and it shares substantial sequence homology with the DNA-binding domains of yeast . PEA1 is probably the murine homolog of AP1 (21, 30).PEA2 binds to nt 5147-5154 (A2 strain nt 5122-5129), adjacent to the PEA1 binding site. An additional factor, polyomavirus enhancer B binding protein 1 (PEB1), binds to other enhancer sequences between nt 5180 and 5220 (A2 strain nt 5155-5195) (13,14,21), and several proteins- Because of the functional redundancy of the polyomavirus enhancer, we chose to inactivate multiple important elements by introducing numerous random point mutations. Using this approach, we identified several polyomaviruses whose DNA replication and transcription were greatly reduced because of point mutations in the enhancer (5). In this report we provide evidence for the involvement of PEA1 and PEA2, with a third factor, PEA3, ...

Control of HPV 18 DNA replication by cellular and viral transcription factors

“…The auxiliary elements of replication are also commonly involved in the control of viral transcription. In polyomavirus, the transcriptional enhancer is an important auxiliary element, and binding of the API factor to this enhancer is required for activation of both early transcription and DNA replication in contrast to HPV 18 (46,47). Moreover, both processes in studies involving polyomavirus show identical cell specificity.…”

Transcription factors junB and c-jun are selectively up-regulated and functionally implicated in fibrosarcoma development.

“…The AP-1 site is a heptameric sequence contained in the regulatory regions of various viral and cellular genes (Garcia-Carranca et al 1988;Martin et al 1988;Sch6nthal et al 1988) and is specifically recognized by either fun-fun homodimers or Jun-Fos heterodimers (Bohmann et al 1987;Angel et al 1988;Halazonetis, et al 1988;Nakabeppu et al 1988;Rauscher et al 1988a, b;Hirai, et al 1989;Ryseck and Bravo 19911. A radiolabeled oligonucleotide probe (33-mer) carrying the AP-1 consensus sequence (TGACTCA) was incubated with nuclear extract prepared from a normal skin fibroblast culture, two mild fibromatosis cultures, three aggressive fibromatosis cell lines, and four fibrosarcoma cell lines.…”

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

“…The essential origin sequences are shown (striped bars). DNAinding sites for putative enhancer-specific proteins (open boxes) include PEAl, 2, and 3 (Martin et al 1988); EFC (Fujimura 1986;Ostapuck et al 1986); PEBl (Bohnlein and Gruss 1986;Piette and Yaniv 1986); and F441 (Kovesdi et al 1987). The enhancer region of three PyV host-range mutants selected for growth in undifferentiated mouse embryonal carcinoma F9 cells (Fujimura et al 1981) contains a point mutation at nucleotide 5233 (solid bar, F441), a 31-bp tandem duplication of the mutated region (crosshatched box.…”

“…The abihty of an enhancer to stimulate ori-core dependent DNA replication in a particular mouse cell type correlates closely with its ability to stimulate transcription (Veldman et al 1985;Campbell and Villarreal 1986;Hassell et al 1986;Mueller et al 1988). Mutations that alter one function also alter the other (Linney and Donerly 1983;Tang et al 1987;Martin et al 1988;Satake et al 1988), and cis-acting alterations of the PyV enhancer region can allow viral DNA replication in mouse cells that are normally permissive (Amati 1985). However, some characteristics of enhancers in the PyV origin of replication differ from those of enhancers coupled to promoters.…”