title: The SANT domain of human MI-ER1 interacts with Sp1 to interfere with GC box recognition and repress transcription from its own promoter.
abstract: To gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within-68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters.
authors.affiliation: Terry Fox Cancer Research Laboratories, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Phillip Drive, St. John's, Newfoundland A1B 3V6, Canada.
authors.forename: Zhihu
authors.initials: Z
authors.lastname: Ding
authors.forename: Laura L
authors.initials: LL
authors.lastname: Gillespie
authors.forename: F Corinne
authors.initials: FC
authors.lastname: Mercer
authors.forename: Gary D
authors.initials: GD
authors.lastname: Paterno
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D018899:CpG Islands; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; C470480:MIER1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase; D006655:Histone Deacetylases