An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.

FIG. 1.

Butyrate induces ORF50 expression. (A) ORF50 protein expression in three PEL cell lines treated with PMA, butyrate, or TSA. Western blot analysis shows the expression of ORF50 protein in BC1, BC3, and HH-B2 cells 24 h after chemical treatment. The same immunoblot was probed with antibody to acetylated histone H4. (B) Kinetics of induction of ORF50 mRNA expression by butyrate. HH-B2 cells were treated with butyrate in the presence (+) or absence (−) of cycloheximide; cells were harvested at intervals. ORF50 mRNA was detected by an RT-PCR assay. Lanes: M, DNA length marker; 1, untreated samples; 2 to 8, butyrate-treated samples; 9 to 15, butyrate- and cycloheximide (CHX)-treated samples; 2 and 9, 20 min; 3 and 10, 45 min; 4 and 11, 2 h; 5 and 12, 4 h; 6 and 13, 8 h; 7 and 14, 13 h; 8 and 15, 24 h.

FIG. 2.

Identification of the butyrate-responsive region of the ORF50 promoter. (A) Schematic diagram of ORF50 promoter-luciferase reporter constructs. Numbers indicate the length of the ORF50 promoter in base pairs upstream of the ATG translation initiation codon. (B and C) Luciferase reporter assays. HH-B2 cells (1.5 × 10⁷) were transfected by electroporation with equal molar amounts of the indicated luciferase constructs. Transfected cells were incubated at 37°C for 24 h under 5% CO₂ before the addition of the indicated drugs. Cells were harvested 24 h after drug treatment and analyzed for luciferase activity. C, control (untreated); B, butyrate; T, TSA.

FIG. 3.

An Sp1 site confers butyrate responsiveness to the ORF50 promoter. (A) Schematic diagram of sequence replacement mutants in the ORF50 promoter. Numbers indicate the corresponding positions of nucleotides in the ORF50 promoter map upstream of the translation start site. The putative Sp1 site is located at 103 to 112; the putative TATA box is from 61 to 68. The sequence in boldface type represents linker DNA used to replace various regions in the ORF50 promoter to generate mutant constructs (m1 to m5). The putative Sp1 site is in italics; the putative TATA element is underlined. Wt, wild type; m, mutant. (B) Luciferase assay of ORF50 promoter mutants. HH-B2 cells were transfected and treated with drugs as described in the legend to Fig. 2. C, control (unreated); B, butyrate; T, TSA. The numbers below the bars indicate the induction (n-fold) of reporter activity in butyrate-treated cells compared to untreated HH-B2 cells.

FIG. 4.

Sp1 activates the ORF50 promoter in Drosophila Schneider 2 cells and HKB5/B5 cells. (A) Schneider cells were transfected with equal molar amounts of ORF50 promoter luciferase reporter (p134Luc) or mutant reporters (p134m1 to p134m5) in the presence or absence of an Sp1 or Sp3 expression construct. p134m1 to p134m5 contain the same mutations shown in Fig. 3A. Cells were harvested 2 days after transfection and assayed for luciferase activity. Con, control (transfected with salmon sperm carrier DNA). (B) HKB/B5 cells were transfected with p2500Luc and different amounts of pCMVHA-Sp1. Cells were harvested 1 day after transfection and assayed for luciferase activity.

FIG. 5.

Sp1 and Sp3 bind to the ORF50 promoter in vitro. (A) Schematic diagram showing the region of ORF50 promoter sequences used as wild-type (WT) and linker-scanning mutant competitors in the EMSA illustrated in panel B. The numbers indicate the positions of the nucleotides upstream of the ATG site. For example, mutant 1 (GSM1) contains sequences ranging from 87 to 102 and 113 to 131. (B) EMSA with nuclear extracts (N.E.) from HH-B2 cells that were untreated or treated with sodium butyrate for 24 h. Lanes 4 to 17 represent competitions with a 50-fold excess of cold oligonucleotides. Lanes 18 to 21 represent supershifts (ss) with antibodies (Ab) to Sp3 and Sp1. (C) DNA sequences of point mutant competitors used in the EMSA illustrated in panel D. (D) EMSA with nuclear extracts of untreated or butyrate-treated HH-B2 cells. Lanes 4 to 7, supershifts with antibodies to Sp1 and Sp3; lanes 8 to 27, competition with a 50-fold molar excess of wild-type or mutant oligonucleotide from the ORF50 promoter (−69 to −118). +, present; −, absent.

FIG. 6.

Butyrate does not alter the abundance of Sp1 or Sp3. Immunoblots of extracts of untreated or butyrate-treated HH-B2 cells reacted with antibody to Sp1 and Sp3 are shown. M.W., molecular mass.

FIG. 7.

Butyrate induces transient association of Sp1 and CBP/p300 with the ORF50 promoter. (A) ChIP experiments exploring the temporal association of Sp1, CBP, and p300 with the ORF50 promoter in HH-B2 cells after butyrate induction. Immunoprecipitation was performed with normal goat immunoglobulin G (control [Ctrl]) (lanes 1 to 4), goat antibody to Ku-70 (lanes 5 to 8), goat antibody to Sp1 (lanes 9 to 12), or input DNA representing 5% of the sample (lanes 13 to 16). Cells were harvested at 0 h (lanes 1, 5, 9, 13), 4 h (lanes 2, 6, 10, 14), 11 h (lanes 3, 7, 11, 15), or 24 h (lanes 4, 8, 12, 16) after butyrate treatment. PCR primers detected the ORF50 promoter (I) or the K8 coding sequence (II). (B) The samples immunoprecipitated with antibody to Sp1 were analyzed with primers from the ORF50 promoter by using increasing numbers of PCR cycles. Samples were obtained 0 h (lane 1), 4 h (lane 2) 11 h (lane 3), or 24 h (lane 4) after butyrate treatment. (C) ChIP experiment exploring the association of Sp3 and CBP with the ORF50 promoter. M, marker; lanes 1 to 4, rabbit anti-Sp3; lanes 5 to 8, rabbit anti-CBP; lanes 9 to 12, 5% input. Samples were harvested at 0 h (lanes 1, 5, 9), 4 h (lanes 2, 6, 10), 11 h (lanes 3, 7, 11) and 24 h (lanes 4, 8, 12) after butyrate treatment. Primers specific for ORF50 promoters (I) or the K8 coding sequence (II) were used. (D) Transient association of p300 with the ORF50 promoter. ChIP experiment with antibody to p300. Samples were taken 0, 4, 11, and 24 h (lanes 1 to 4, respectively) after butyrate treatment.