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.

Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice.

To identify DNA sequence elements from the human apolipoprotein B (apoB) gene required for high-level, correct tissue-specific expression in transgenic mice, we made several constructs that included one or more of the key regulatory elements that were previously characterized with cultured liver-derived and intestine-derived cell lines. Our data show that the apoB promoter alone (-898 to +121) is not sufficient to direct transcription in transgenic mice. An enhancer located in the second intron is absolutely required to specify transcription by the homologous apoB promoter in the livers of transgenic mice; this enhancer does not direct transcription in the small intestines. Thus, the elements controlling transcriptional activation of the apoB gene in the liver and the intestine in vivo are distinct and separable. Analysis of the DNase I hypersensitivity of the integrated human transgenes in various lines of expressing and nonexpressing mice suggests that the formation of DH4, a strong hypersensitive site in intron 2, may be a prerequisite for hepatic expression of the apoB gene. Nuclear matrix association regions (MARs) of the apoB gene may play a role in transgene expression. Constructs including MAR sequences displayed higher levels of expression than those lacking them. However, these MARs did not completely insulate the associated transgenes from position effects.

Activity of a cloned Xenopus albumin gene promoter in the homologous frog oocyte system.

The activity of the liver-specific promoter from the Xenopus laevis 68 kDa albumin gene in homologous oocytes has been analysed by microinjection. We find that the albumin promoter functions relatively efficiently in oocytes, directing the synthesis of correctly initiated transcripts, and deletion analysis reveals that only a small amount of upstream sequence is required for full activity.

Neonatal lesions of the basal forebrain cholinergic neurons result in abnormal cortical development.

The effect of electrolytic lesions of the neonatal forebrain on the morphogenesis of the mouse neocortex has been examined. Balb/C mice were lesioned unilaterally within 24 h of birth. The development of cortical cytoarchitecture was assessed in Nissl-stained sections, and the levels of presynaptic markers for cholinergic, noradrenergic and serotonergic afferents were measured in the fronto-parietal cortex ipsilateral and contralateral to the lesion at various postnatal ages and in adulthood. The basal forebrain (nBM) lesion resulted in a transient but severe reduction of cortical cholinergic markers and in abnormal cortical cytoarchitecture. Cytoarchitectural abnormalities were expressed as delay in the emergence of differentiated cell populations and affected sequentially more superficial layers with maturation following lesion. Furthermore, the location and extent of these morphologic abnormalities appeared to correlate with the degree of cholinergic denervation. Cortical monoamines were also temporarily reduced as a result of the lesion; however, pharmacologic lesions of the monoaminergic projections alone did not result in the abnormal cortical cytoarchitecture. Thus, the basal forebrain cholinergic projection appears to serve a role in regulating cortical differentiation.

Glycoengineered factor IX variants with improved pharmacokinetics and subcutaneous efficacy.

BACKGROUND: The rapid clearance of factor IX (FIX) necessitates frequent intravenous administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration would be a preferred route of administration but is limited by bioavailability. OBJECTIVES: To improve the pharmacokinetics (PK) and bioavailability of FIX, a screen was performed to identify positions for the introduction of novel glycosylation sites with maximal effect on PK and maintenance of coagulation activity. METHODS: Two hundred fifty-one variants, each containing one additional N-linked glycosylation site, were screened in vitro, and the PK profiles of selected variants mapping to spatially distinct regions of FIX were evaluated in mice. Optimal variants were combined, and their PK and efficacy were determined in mice with hemophilia B. RESULTS: Variants that mapped to spatially distinct regions of the FIX structure exhibited different degrees of improved PK and enabled selection of optimized sites while minimizing the loss of FIX activity. Combining the most effective N-glycan sites in the same FIX molecule resulted in further improvements in PK. An optimized variant containing three novel N-glycan sites (at amino acids 103, 151, and 228), and the activity enhancing 338A variant had double the specific activity of wild-type FIX, exhibited 4.5-fold reduced clearance and 2.4-fold increased subcutaneous bioavailability, and was efficacious at a fivefold lower mass dose than wild-type FIX after subcutaneous injection in a bleeding model in mice with hemophilia B. CONCLUSIONS: Glycoengineering was used to significantly improve the subcutaneous PK and efficacy of FIX and may have advantages for subcutaneous dosing.

Structure and functional expression of a cloned Xenopus thyroid hormone receptor.

A clone corresponding to a thyroid hormone receptor was isolated from a Xenopus laevis cDNA library prepared from folliculated oocytes. The cDNA encodes a protein of 418 amino acid residues with a domain structure, including a putative DNA binding region with two zinc fingers, similar to other members of the v-erbA-related superfamily of receptors. The encoded protein resembles the TR alpha 1-type receptor of the rat. When expressed in COS cells the protein product binds triiodothyronine with a Kd of 0.12 nM. The receptor mediates thyroid-hormone-inducible expression of a reporter gene which includes a thyroid hormone response element in its upstream region.

Nuclease-hypersensitive sites define a region with enhancer activity in the third intron of the human apolipoprotein B gene.

The positions of several DNase I-hypersensitive (DH) sites have been mapped in the second and third introns of the human apolipoprotein B gene. Two such DH sites, I and V, are present both in human hepatoma (HepG2) and colon carcinoma (CaCo-2) cells that express the gene but absent from HeLa cells that do not express the gene. These DH sites map near sequence elements that have been highly conserved between the human and mouse genes. A PvuII-EcoRI fragment (+1064 to +2977) from the hypersensitive region exhibited enhancer activity, which was further localized by means of deletion experiments to a 155-base pair segment located entirely within the third intron and flanked by two DH sites. Three DNase I footprints were observed within this core enhancer, one of which contains putative binding sites for three liver specific nuclear proteins. Experiments are presented that suggest that this enhancer operates by a similar mechanism as that described previously for the strong second intron enhancer, involving an interaction with the basal transcriptional machinery. Digestions with low levels of micrococcal nuclease were performed to ascertain whether nucleosomes were present in the DNase I sensitive enhancer region. Nine different micrococcal nuclease-hypersensitive (MH) sites were detected in HepG2 cells but not in HeLa cells; one MH site was common to both cell types, and HeLa cells exhibited three unique MH sites. The first six MH sites (I-VI) are spaced approximately 200 base pairs apart, suggesting the presence of positioned nucleosomes in that region. MH sites VI-X are more closely spaced, suggesting either additional cutting sites within the core particle or the absence of one or two nucleosomes in this segment of the third intron enhancer.

Identification of a negative regulatory region 5' of the human apolipoprotein B promoter.

We have identified a negative regulatory region between positions -1802 and -3211 of the human apolipoprotein B gene that reduces expression of the gene. This "reducer" effect was detected in transient transfection experiments performed with hepatic (HepG2 and Hep3B) cells as well as intestinal (CaCo-2) cells. It appears to be specific for the apolipoprotein B promoter because it did not affect expression from the heterologous thymidine kinase promoter. This reducer segment operated in the presence or absence of the transcriptional enhancer from the second intron of the apolipoprotein B gene, suggesting that the protein factors involved in the enhancer and reducer effect can interact with the transcriptional machinery independently of each other. Deletion experiments further localized the positions of the negative regulatory elements in HepG2 cells: sequences between positions -2738 and -2470 together with those from -2118 to -1802 are required for the reducer activity. DNase I footprinting of these two reducer DNA segments identified several DNA sequences that are bound by nuclear proteins from HepG2 cells. Analysis of the nucleotide sequences within these footprints demonstrated a high degree of similarity with sequences within negative regulatory regions of other genes.

Characterization of tissue-specific enhancer elements in the second intron of the human apolipoprotein B gene.

We report the identification and characterization of tissue-specific transcriptional enhancer elements that influence the expression of the human apolipoprotein B gene. A 704-base pair PstI fragment comprising sequences from the first and second introns of the human apolipoprotein B gene (positions +360 to +1064) possesses tissue-specific transcriptional enhancer elements when assayed in transient transfection experiments using either the apolipoprotein B or thymidine kinase promoter. The majority of the enhancer activity, which was observed in transcriptionally active HepG2 and CaCo-2 cells, but not in transcriptionally inactive Chinese hamster ovary or HeLa cells, was subsequently localized to a 443-base pair SmaI-PvuII fragment (positions +621 to +1064) within the second intron of the apolipoprotein B gene. Gel retention experiments demonstrated that sequence motifs within this region interact with a number of nuclear proteins from HepG2, CaCo-2, and HeLa cells. The actual sequence elements that bound to nuclear proteins from HepG2 cells were identified by DNase I footprinting. Deletion experiments were performed to distinguish those protein-binding regions involved in the enhancer effect. Our data demonstrate that sequences between positions +806 and +940 are essential for this enhancer activity. This segment contains one large 97-base pair footprint, whose sequence has been conserved between the human and mouse genes. Binding sites for the liver-specific transcription factors HNF-1 and HNF-3 are present within this footprint.

Functional analysis of the human cyclin D2 and cyclin D3 promoters.

The D-type cyclins promote progression through the G1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5'-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions -306 and -114 relative to the ATG codon at +1. Additional positive elements were localized between -444 and -345, whereas sequences that reduced transcription were identified between nucleotides -1624 and -892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides -366 and -167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides -306 and -1624 of the cyclin D2 gene were necessary for serum inducibility.

Characterization of the human cyclin-dependent kinase 2 gene. Promoter analysis and gene structure.

Cyclin-dependent kinase 2 is a serine/threonine protein kinase essential for progression of the mammalian cell cycle from G1 to S phase. CDK2 mRNA has been shown to be induced by serum in several cultured cell types. Therefore, we set out to identify elements that regulate the transcription of the human CDK2 gene and to characterize its structure. This paper describes the cloning of approximately 2.4-kilobase pair genomic DNA fragment from the upstream region of the human CDK2 gene. This fragment contains five transcription initiation sites within a 72-nucleotide stretch. A 200-base pair sub-fragment that confers 70% of maximal basal promoter activity was shown to contain two synergistically acting Sp1 sites. However, a much larger DNA fragment containing approximately 1.7 kilobase pairs of upstream sequence is required for induction of promoter activity following serum stimulation. The intron exon boundaries of seven exons in this gene were also identified, and this information will be useful for analyzing genomic abnormalities associated with CDK2.

Members of the caudal family of homeodomain proteins repress transcription from the human apolipoprotein B promoter in intestinal cells.

Apolipoprotein B (apoB) is the major protein component of low density lipoproteins, and plays a central role in cholesterol transport and metabolism. The apoB gene is transcribed in the liver and in the intestine in humans. Although much is known about the DNA sequence elements and protein factors that are important for transcription of the human apolipoprotein B gene in the liver, less is known about the mechanisms that control transcription of this gene in the intestine. The sucrose isomaltase gene (SI), is expressed exclusively in the intestine. Two sequences from the promoter region of the SI gene, namely SIF-1 and SIF-3, are essential for promoter activity of the SI gene in intestinal cells. Sequences displaying a high degree of similarity to those of SIF-1 and SIF-3 are present in the third intron of the apoB gene. Rather than stimulating apoB promoter activity, the BSIF-1 and BSIF-3 sequences repressed transcription in CaCo-2 cells. Gel retardation studies demonstrated that BSIF-1, like SIF-1, binds to proteins related to the caudal family of proteins such as mCdx-4 and mCdx-2. These proteins appear to repress transcription from the apoB promoter by a mechanism that involves an interaction with members of the C/EBP family of proteins, that bind to a target sequence for the repressor in the segment from -139 to -111 of the apoB promoter. On the other hand, BSIF-3, like SIF-3, binds to HNF-1 and also represses transcription from the apoB promoter.

A polymorphism in a region with enhancer activity in the second intron of the human apolipoprotein B gene.

A 443-base pair fragment (+622 to +1064) from the second intron of the human apolipoprotein B gene was shown to contain a tissue-specific enhancer when placed in front of an apolipoprotein B promoter-chloramphenicol acetyltransferase construct in transfection experiments. To identify potential regulatory mutations in this region of the gene, DNA from various subjects was examined for the presence of point mutations by means of chemical cleavage of mismatched heteroduplexes. An A----G substitution within the second intron of the gene at position +722 was identified in three unrelated subjects and confirmed by DNA sequencing. Although the base substitution was contained within a nuclear protein-binding site, as determined by DNase I footprinting, it did not appear to affect the protein/DNA interaction in its vicinity, as shown by gel retardation experiments. The single base substitution at position +722 abolishes a StyI restriction site, thus creating a StyI polymorphism. Using allele-specific oligonucleotides, we screened the DNA of 172 subjects for the presence of this polymorphism: two other subjects carrying the polymorphism were found. In each of the five unrelated subjects, the polymorphism was associated with the same haplotype.