Identification and Functional Characterization of an Intragenic DNA Binding Site for the Spumaretroviral trans-Activator in the Human p57 Gene

Abstract

Expression of the human cyclin-dependent protein kinase inhibitor p57Kip2 gene was previously shown to be specifically and strongly activated by the retroviral trans-activator Bel1 of human foamy virus by means of expression profiling, Northern, and Western blot analysis. Here we report that Bel1-mediated trans-activation was conferred by a Bel1 response element (BRE) located in the second exon of p57Kip2 . The intragenic Kip2-BRE was capable of trans-activating the luciferase reporter gene upon cotransfection with Bel1. In electrophoretic mobility shift assays using 293T nuclear extracts or a purified glutathione S-transferase (GST)⅐Bel1 fusion protein, we identified the 55-nucleotide-long Kip2-BRE site that mainly consists of three direct repeats of 14-mers partially homologous to a functionally active BRE in the viral internal promoter. The specificity of the transactivator-DNA binding was shown by using mutated and shortened Kip2-BRE oligodeoxynucleotides in competition experiments with the authentic viral internal promoter and by Bel1-specific antibody that led to a supershift of the nuclear protein⅐Kip2-BRE and GST⅐Bel1⅐Kip2-BRE complex. The data indicate that Bel1 can directly bind to BRE sites. The cellular Kip2-BRE can be used to predict those human genes that are directly or indirectly activated by the Bel1 trans-activator.

Bel1-mediated Transactivation of the Spumaretroviral Internal Promoter Is Repressed by Nuclear Factor I

“…Plasmids pUC18, pCMV␤-gal, pbel1s (24), pGL3-basic-HFV-IP (Ϫ192 to Ϫ1), pGL3-pro-IP.BRE Ϫ166 to Ϫ116 and Ϫ166 to Ϫ140 derivatives (23) were transfected into 293T cells using the coprecipitation method of Chen and Okayama (25). In general, 6 g of plasmid DNA were transfected into 293T cells grown in 6-cm Petri dishes.…”

“…Identical amounts of proteins were separated by SDS-PAGE, blotted, reacted with monoclonal serum directed against the HA epitope of the four different HA-tagged NFI proteins (Roche) or against hNFI-X1 protein (sc870X, Santa Crux), or polyclonal sera directed against Bel1/Bet, Gag, or RNase H proteins (23), and detected by enhanced chemiluminescence as described previously (23).…”

“…The Kip2-BRE was bound by proteins in nuclear extracts from 293T cells that had been transfected with Bel1 as shown by EMSA and supershift experiments. A purified glutathione S-transferaseBel1 fusion protein was bound specifically to Kip2-BRE, and the formation of the Bel1-Kip2-BRE complexes was effectively blocked by the minimal viral IP.BRE oligonucleotide (10,23). Cotransfection with Sp1 expression plasmids enhanced the Bel1-specific transactivation of Kip2-BRE to a limited extent.…”

“…It is worth noting that the Kip2-BRE primarily consists of direct repeats of three 14 mers that partially overlap with the G base pattern conserved in both the IP.BRE and Kip2-BRE (10,23). The Kip2-BRE was bound by proteins in nuclear extracts from 293T cells that had been transfected with Bel1 as shown by EMSA and supershift experiments.…”

“…Previously, a Kip2-BRE DNA element located within the second exon of the human p57 Kip2 gene was identified that mediates a specific Bel1 transactivation (23). It is worth noting that the Kip2-BRE primarily consists of direct repeats of three 14 mers that partially overlap with the G base pattern conserved in both the IP.BRE and Kip2-BRE (10,23).…”