REGULATION OF PARATHYROID HORMONE-RELATED PROTEIN (PTHRP) GENE-EXPRESSION - SP1 BINDS THROUGH AN INVERTED CACCC MOTIF AND REGULATES PROMOTER ACTIVITY IN COOPERATION WITH ETS1

Citation
J. Dittmer et al., REGULATION OF PARATHYROID HORMONE-RELATED PROTEIN (PTHRP) GENE-EXPRESSION - SP1 BINDS THROUGH AN INVERTED CACCC MOTIF AND REGULATES PROMOTER ACTIVITY IN COOPERATION WITH ETS1, The Journal of biological chemistry, 269(34), 1994, pp. 21428-21434
Citations number
55
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
269
Issue
34
Year of publication
1994
Pages
21428 - 21434
Database
ISI
SICI code
0021-9258(1994)269:34<21428:ROPHP(>2.0.ZU;2-I
Abstract
We have previously shown that mutations in the GGAA core motif of the Ets1 binding site, EBSI, or deletion of EBSI, reduced basal and Tax, t ransactivation of the PTHrP P2 promoter. Here we demonstrate that, in addition to EBSI, a CACCC-like motif located between -53 and -58 is re quired for full basal activity of this promoter in Jurkat T-cells. Sit e-specific mutations in the CACCC motif decreased promoter activity ap proximately 5-fold. In an effort to identify transcription factors tha t bind to the CACCC element, we found that purified human Sp1, as well as Sp1 in HeLa nuclear extract, can specifically bind to a DNA probe that corresponds to the PTRrP-specific sequence between -94 and -34. G el shift competition studies and DNase I footprinting analyses reveale d that Sp1 specifically interacts with the CACCC motif. In the presenc e of Ets1, the mobility of the Sp1-specific gel shift complex with the PTHrP DNA decreased. DNase I footprint analysis of this gel shift com plex showed an extended footprint over both the Sp1 and the Ets1 bindi ng site, demonstrating that Sp1 and Ets1 form a ternary complex with t he PTRrP DNA. Cotransfection of an Ets1 and Sp1 expression vector into Drosophila Schneider cells demonstrated that Sp1 can functionally coo perate with Ets1 to transactivate the PTHrP promoter. We conclude from these data that Ets1 and Sp1 can cooperatively regulate PTHrP P2 prom oter activity.