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

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.     

Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.