Sp and GATA factors are critical for Apolipoprotein AI downstream enhancer activity in human HepG2 cells.
The factors that bind to the hepatic-specific human apolipoprotein AI (apoAI) 48-bp downstream enhancer (DSE) were identified and characterized by electrophoretic mobility shift assays. A significant homology was shown between the histone 4 (H4) promoters and the hepatic-specific human apoAI DSE at Sp1 and H4TF2 binding sites. Human HepG2 nuclear extracts were used to form four specific complexes with the DSE (referred to as apoAI DSE-1, -2, -3, and -4). The apoAI DSE-1 and -2 complexes showed similar binding specificity to the Sp/H4TF1 consensus site within the apoAI DSE. The apoAI DSE-1 complex was predominantly recognized by anti-Sp1 and Sp3 sera in gel shift assays, indicating that the DSE was recognized by multiple Sp family members. Nuclear extracts that were prepared from retinoic acid treated HepG2 cells showed increased levels of Sp factors in gel shift and Western blot assays. The apoAI DSE-2 complex was identified as H4TF1 and formed in the absence of magnesium chloride. The apoAI DSE-3 complex bound to a consensus GATA element within the DSE that was recognized by recombinant human GATA-6 as well. The apoAI DSE-3 complex was completely disrupted by a GATA-4 antibody in EMSA. GATA-4 and -6 were detected in nuclear extracts prepared from retinoic acid treated HepG2 cells using Western blot assays. The highest apoAI DSE-3 levels were observed with retinoic acid treated HepG2 cell nuclear extracts in EMSA. ApoAI DSE-4 is a multi-factor complex that includes an Sp/H4TF1 factor and either H4TF2 or apoAI DSE-3. Because apoAI DSE mutations revealed transcription defects in transient transfection assays, we conclude that the entire DSE sequence is required for full apoAI transcriptional activity in HepG2 cells.
Ivanov
Gleb S
GS
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, 715 Albany Street, Building R-618, Boston, MA 02118, USA.
Kater
Jessie M
JM
Jha
Shivkumar H
SH
Stutius
Erica A
EA
Sabharwal
Ravleen
R
Tricarico
Marisa D
MD
Ginsburg
Geoffrey S
GS
Ozer
Josef S
JS
D016632:Apolipoprotein A-I / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D016632:Apolipoprotein A-I; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; C494759:GATA6 protein, human; D009841:Oligonucleotides; D016329:Sp1 Transcription Factor; D014157:Transcription Factors