title: Roles of USF, Ikaros and Sp proteins in the transcriptional regulation of the human reduced folate carrier B promoter.
abstract: The hRFC (human reduced folate carrier) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to seven non-coding regions (A1, A2, A, B, C, D and E) and at least four promoters. For the hRFC-B basal promoter, regulation involves binding of Sp (specificity protein) transcription factors to a critical GC-box. By transiently transfecting HT1080 cells with 5'- and 3'-deletion constructs spanning 1057 bp of upstream sequence, a transcriptionally important region was localized to 158 bp flanking the transcriptional start sites. By gel shift and chromatin immunoprecipitation assays, USF (upstream stimulatory factor), Sp1 and Ikaros-related proteins were bound to consensus elements (one E-box, two GC-box and three Ikaros) within this region. The functional importance of these elements was confirmed by transient tranfections of HT1080 cells with hRFC-B reporter constructs in which they were mutated, and by co-transfections of Drosophila Mel-2 cells with wild-type hRFC-B promoter and expression constructs for USF1, USF2a, Sp1 and Ikaros 2 and 8. Both USF1 and Sp1 proteins transactivated the hRFC-B promoter. Sp1 combined with USF1 resulted in a synergistic transactivation. Identical results were obtained with USF2a. Ikaros 2 was a repressor of hRFC-B promoter activity whose effects were partly reversed by the dominant-negative Ikaros 8. In HT1080 cells, transfection with Ikaros 2 decreased endogenous hRFC-B transcripts, whereas USF1 and Sp1 increased transcript levels. Ikaros 2 also decreased reporter gene activity and levels of acetylated chromatin associated with the endogenous promoter. Collectively, these results identify transcriptionally important regions in the hRFC-B promoter that include multiple GC-box, Ikaros and E-box elements. Our results also suggest that co-operative interactions between transcription factors Sp1 and USF are essential for high-level hRFC-B transactivation and imply that these effects are modulated by the family of Ikaros proteins and by histone acetylation.
authors.affiliation: Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201, USA.
authors.forename: Mingjun
authors.initials: M
authors.lastname: Liu
authors.forename: Johnathan R
authors.initials: JR
authors.lastname: Whetstine
authors.forename: Scott G
authors.initials: SG
authors.lastname: Payton
authors.forename: Yubin
authors.initials: Y
authors.lastname: Ge
authors.forename: Robin M
authors.initials: RM
authors.lastname: Flatley
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D051740:Ikaros Transcription Factor; D026901:Membrane Transport Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D020218:Response Elements / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D051720:Upstream Stimulatory Factors
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; C497239:IKZF1 protein, human; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D051740:Ikaros Transcription Factor