Paclitaxel activation of the GADD153 promoter through a cellular injury response element containing an essential Sp1 binding site.
The GADD153 promoter is transcriptionally activated by paclitaxel-induced injury. Promoter deletion from -786 to -85 base pairs relative to the start of transcription had no significant effect on activation, but deletion to the TATA box abolished it. Placement of the 39 bases from -74 to the TATA box (cellular injury response element, CIRE) upstream of the adenovirus E4 TATA box conferred paclitaxel inducibility. The only consensus sequence present in the CIRE is an Sp1 site; mutation of this site inhibited paclitaxel activation. Paclitaxel failed to activate a SV40-driven luciferase construct containing five Sp1 sequences, and Sp1 sites further upstream in the GADD153 promoter were not essential for activation. Pure Sp1 and nuclear extracts from uninjured and paclitaxel-injured cells protected the same region from -62 to -48 bases on the noncoding strand and -74 to -53 on the coding strand. Nuclear extracts shifted the CIRE to the same extent as purified Sp1 but had no effect on a CIRE with a mutated Sp1 site in gel shift assays. Immunodepletion of Sp1 abolished the shift; antibody to Sp1 produced a supershift. These data indicate that paclitaxel activates the GADD153 promoter through a constitutively occupied Sp1 site at -61 bases.
Gately
D P
DP
Department of Biomedical Sciences, University of California, San Diego, La Jolla, California 92093-0812, USA.
Howell
S B
SB
D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004249:DNA Damage; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000502:physiology; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; C497249:DDIT3 protein, human; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051742:Transcription Factor CHOP; D017239:Paclitaxel