Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein

Abstract

The papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element but with the centromeric element replaced by E2-binding sites. E2 expression was not sufficient to maintain such plasmids, but plasmid stability could be rescued by expression of the mammalian protein Brd4. In the presence of both Brd4 and E2 proteins, plasmids with multiple E2-binding sites were stable without selection. S. cerevisiae encodes a homolog of Brd4 named Bdf1 that does not contain the C-terminal domain that interacts with the E2 protein. A fusion protein of Bdf1 and the Brd4 C-terminal "tail" could support E2-mediated plasmid maintenance in yeast. Using a panel of mutated E2 proteins, we determined that plasmid stability required the ability of E2 to bind DNA and to interact with Brd4 and mammalian mitotic chromosomes but did not require its replication initiation and transactivation functions. The S. cerevisiae-based plasmid maintenance assays described here are invaluable tools for dissecting mechanisms of episomal viral genome replication and screening for additional host protein factors involved in plasmid maintenance.

Fig. 1.

Reporter liquid culture (A) and red/white sectoring (B) assay plasmids.

Fig. 2.

E2 does not maintain E2-binding site plasmids in S. cerevisiae. (A) Plasmid-loss assay of W303a transformants with indicated reporter plasmids and the E2 expression plasmid pRB16-E2. Transformants were grown in selective medium and transferred for growth for 11 generations in medium nonselective for the reporter plasmids. Aliquots of 10-fold serial dilutions were plated onto media selective and nonselective for the reporter plasmids. (B)A red/white colony sectoring assay. YMB1670 transformants containing the reporter plasmids shown in Fig. 1B and E2 expression plasmid p416CYC1-E2 (or empty vector p416CYC1) were streaked on media selective and nonselective for the reporter plasmids.

Fig. 3.

Mammalian Brd4 protein rescues E2-mediated plasmid maintenance. Red/white sectoring assay of YMB1670 transformants containing p416CYC1-E2, indicated reporter plasmids (see Fig. 1B), and empty vectors (p425/PGK and pADNS) or expression vectors for mammalian candidate proteins p425PGK-hEBP2 (p40), pADNS-Brd2, or pADNS-Brd4. Transformants were streaked onto media nonselective for the reporter plasmids.

Fig. 4.

Brd4 maintains plasmids containing E2RE1 or LCR elements. (A) Red/white sectoring assay of YMB1670 transformants containing p416CYC1E2, pADNS-Brd4, and individual reporter plasmids as indicated (see Fig. 1B). Transformants were streaked onto media selective and nonselective for the reporter plasmids. (B) Individual colonies of transformants shown in A on nonselective medium. (C) Plasmid-loss assay of YMB1670 transformants shown in A. After eight generations of growth in nonselective media, 10-fold serial dilutions were plated on media selective and nonselective for the reporter plasmids.

Fig. 5.

Requirements for E2-mediated plasmid maintenance. Red/white sectoring assay of YMB1670 transformants with pADNS-Brd4, individual reporter plasmids (see Fig. 1B), and p416CYC1 plasmids expressing WT or mutated E2 proteins. Transformants were streaked on media nonselective for the reporter plasmids. The phenotypes of the E2 proteins are indicated as transactivation (ta), replication (rep), DNA binding (db), and chromosome binding (cb) and are listed in more detail in Table 2.

Fig. 6.

Bdf1–BRD4 fusion protein supports E2-mediated plasmid maintenance. (A) Diagram of BET family members S. cerevisiae Bdf1 and mouse Brd4. Regions indicated are bromodomains (BD1 and BD2), nuclear localization signal (NLS), ATP-binding motif (ATP), extraterminal domain (ET), and SEED motif. The Brd4 C-terminal region was fused to Bdf1 to generate Bdf1Mtail. (B) Red/white sectoring assay of YMB1670 transformants with p416CYC-E2, individual reporter plasmids (see Fig. 1B), and pADNS-Brd4, pADNSpl-Bdf1, and pADNSpl-Bdf1rcMtail expression plasmids. Transformants were streaked on medium nonselective for the reporter plasmids.