Hepatocyte nuclear factor 1 and C/EBP are essential for the activity of the human apolipoprotein B gene second-intron enhancer.
The tissue-specific transcriptional enhancer of the human apolipoprotein B gene contains multiple protein-binding sites spanning 718 bp. Most of the enhancer activity is found in a 443-bp fragment (+621 to +1064) that is located entirely within the second intron of the gene. Within this fragment, a 147-bp region (+806 to +952) containing a single 97-bp DNase I footprint exhibits significant enhancer activity. We now report that this footprint contains four distinct protein-binding sites that have the potential to bind nine distinct liver nuclear proteins. One of these proteins was identified as hepatocyte nuclear factor 1 (HNF-1), which binds with relatively low affinity to the 5' half of a 20-bp palindrome located at the 5' end of the large footprint. A binding site for C/EBP (or one of the related proteins that recognize similar sequences) was identified in the center of the 97-bp footprint. This binding site is coincident or overlaps with the binding sites for five other proteins, two of which appear to be distinct from the C/EBP-related family of proteins. The binding site for a nuclear factor designated protein I is located between the HNF-1 and C/EBP binding sites. Finally, the 3'-most 15 bp of the footprinted sequence contain a binding site for another nuclear protein, which we have called protein II. Mutations that abolish the binding of either HNF-1, protein II, or the C/EBP-related proteins severely reduce enhancer activity. However, deletion experiments demonstrated that neither the HNF-1-binding site alone, nor the combination of binding sites for HNF-1, protein I, and C/EBP, nor the C/EBP-binding site plus the protein II-binding site is sufficient to enhance transcription from a strong apolipoprotein B promoter. Rather, HNF-1 and C/EBP act synergistically with protein II to enhance transcription of the apolipoprotein B gene.
Brooks
A R
AR
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, California.
Levy-Wilson
B
B
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*
D001055:Apolipoproteins B; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA