Human apolipoprotein B gene intestinal control region.

Recently, we reported that a 315 bp enhancer, located over 55 kilobases (kb) upstream of the transcriptional start site of the human apolipoprotein B (apoB) gene, was sufficient to direct high-level expression of human apoB transgenes in mice. In this report, we expand our analysis of the distant apoB intestinal control region (ICR), by examining the function of segments in the vicinity of the 315 bp intestinal enhancer (315 IE). DNaseI hypersensitivity (DH) studies of a 4.8 kb segment from the ICR revealed three new DH sites, in addition to the previously described DH1 region present within the 315 IE. DH2 mapped to a 485 bp segment (485 IE) immediately upstream of the 315 IE that exhibited strong intestinal enhancer activity in transient transfection experiments with intestine-derived CaCo-2 cells. Within the DH2 region, an HNF-4/ARP-1 binding site was demonstrated by gel retardation experiments. A 1.8 kb segment incorporating the 485 IE was capable of driving expression of human apoB transgenes in the intestines of mice. Additionally, a third component of the apoB ICR was found about 1.2 kb downstream of the 315 IE, within a 1031 bp segment (1031 IE) that also harbored two DH sites, DH3 and DH4. This segment did not display enhancer activity but was capable of driving transgene expression in the intestine. The three components of the ICR displayed a similar pattern of apoB mRNA expression along the horizontal axis of the intestine. The previously characterized in vivo liver-specific elements of the apoB gene, namely, the second intron enhancer and the 5' upstream liver enhancer, did not play a role in intestinal expression of apoB transgenes in mice.

The 5' boundary of the human apolipoprotein B chromatin domain in intestinal cells.

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.

HNF-3 beta, C/EBP beta, and HNF-4 act in synergy to enhance transcription of the human apolipoprotein B gene in intestinal cells.

Recently, we identified a 315-bp intestinal enhancer (IE), localized over 55 kb upstream from the transcriptional start of the human apolipoprotein B (apoB) gene, that confers expression of human apoB transgenes in the intestines of mice. Four functional binding sites for the intestine-enriched transcription factors hepatocyte nuclear factor (HNF)-3beta, CAAT enhancer binding protein (C/EBP)beta, and HNF-4 were demonstrated within the 315-bp IE. In this report, we extend these earlier studies and examine the relative contributions of these three transcription factors to the activity of the enhancer as well as their mechanism of interaction with one another. Cotransfection experiments with the expression vectors for HNF-3beta, C/EBPbeta, and HNF-4 revealed that HNF-3beta bound to Site 1, C/EBPbeta bound to Site 2, and HNF-4 bound to Site 3 within the 315-bp IE and that the sites act synergistically to enhance intestinal expression of apoB. Each one of these four binding sites was mutated, and mutant constructs were transfected into intestine-derived CaCo-2 cells to evaluate the role of each of these binding sites in enhancer activity. The results of the mutagenesis experiments confirmed that the HNF-3beta and HNF-4 sites are most important for the enhancer activity, followed by C/EBPbeta Site 2. All three factors bound to Sites 1, 2, and 3 must act synergistically for optimal activity of the apoB IE.

Identification and characterization of a 315-base pair enhancer, located more than 55 kilobases 5' of the apolipoprotein B gene, that confers expression in the intestine.

We recently reported that an 8-kilobase (kb) region, spanning from -54 to -62 kb 5' of the human apolipoprotein B (apoB) gene, contains intestine-specific regulatory elements that control apoB expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal control region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity studies uncovered a prominent DNaseI hypersensitivity site, located within a 315-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcriptionally active CaCo-2 cells but not in transcriptionally inactive HeLa cells. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and analysis of the DNA sequence revealed putative binding sites for the tissue-specific transcription factors hepatocyte nuclear factor 3beta, hepatocyte nuclear factor 4, and CAAT enhancer-binding protein beta. Binding of these factors to the 315-bp enhancer was demonstrated in gel retardation experiments. Transfection of deletion mutants of the 315-bp enhancer revealed the relative contributions of these transcription factors in the activity of the apoB intestinal enhancer. The corresponding segment of the mouse apoB gene (located -40 to -83 kb 5' of the structural gene) exhibited a high degree of sequence conservation in the binding sites for the key transcriptional activators and also exhibited enhancer activity in transient transfection assays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp enhancer conferred intestinal expression to human apoB transgenes.

The nuclear matrix protein CDP represses hepatic transcription of the human cholesterol-7alpha hydroxylase gene.

To date, the molecular mechanisms that govern hepatic-specific transcription of the human cholesterol 7alpha-hydroxylase (CYP7A1) gene are poorly understood. We recently reported that the region extending from -1888 to +46, which includes the promoter, is not capable of conferring expression to human CYP7A1 promoter lacZ transgenes in the livers of mice, but that expression is observed with transgenes containing the entire structural gene. To locate liver-specific elements in other segments of the human gene, DNase I hypersensitivity studies were performed with transcriptionally active, liver-derived HepG2 cells and with transcriptionally inactive HeLa cells. Three DNase I hypersensitivity sites were detected within the first intron of the human CYP7A1 gene, but only in HepG2 cells. Transient transfection experiments with HepG2 cells revealed a transcriptional repressor within intron 1. Five binding sites for the CAAT displacement protein (CDP) were detected within intron 1. Since CDP is a nuclear matrix protein, two methods were employed to localize nuclear matrix attachment sites within intron 1 of the human CYP7A1 gene. A matrix attachment site was found throughout the entirety of intron 1. Gel retardation experiments and cell transfection studies provided evidence for the repression mechanism. Repression is achieved by displacement by CDP of two hepatic activators, namely HNF-1alpha and C/EBPalpha, that bind to three different sites within intron 1. Additionally, CDP represses transactivation mediated by these two activators.

An open chromatin structure in a liver-specific enhancer that confers high level expression to human apolipoprotein b transgenes in mice.

A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (-5262 to -899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints.