Identification of two nuclear factor-binding domains on the chicken cardiac actin promoter: implications for regulation of the gene.

The cis-acting regions that appear to be involved in negative regulation of the chicken alpha-cardiac actin promoter both in vivo and in vitro have been identified. A nuclear factor(s) binding to the proximal region mapped over the TATA element between nucleotides -50 and -25. In the distal region, binding spanned nucleotides -136 to -112, a region that included a second CArG box (CArG2) 5' to the more familiar CCAAT-box (CArG1) consensus sequence. Nuclear factors binding to these different domains were found in both muscle and nonmuscle preparations but were detectable at considerably lower levels in tissues expressing the alpha-cardiac actin gene. In contrast, concentrations of the beta-actin CCAAT-box binding activity were similar in all extracts tested. The role of these factor-binding domains on the activity of the cardiac actin promoter in vivo and in vitro and the prevalence of the binding factors in nonmuscle extracts are consistent with the idea that these binding domains and their associated factors are involved in the tissue-restricted expression of cardiac actin through both positive and negative regulatory mechanisms. In the absence of negative regulatory factors, these same binding domains act synergistically, via other factors, to activate the cardiac actin promoter during myogenesis.

Differential effect of zinc finger deletions on the binding of CTCF to the promoter of the amyloid precursor protein gene.

High levels of transcription from the amyloid precursor protein promoter are dependent on the binding of CTCF to the APBbeta core recognition sequence located between positions -82 and -93 upstream from the transcriptional start site. CTCF comprises 727 amino acids and contains 11 zinc finger motifs arranged in tandem that are flanked by 267 amino acids on the N-terminal side and 150 amino acids on the C-terminal side. Deletion of either the N- or the C-terminal regions outside of the zinc finger domain had no detrimental effect on the binding of CTCF to APBbeta. However, internal deletions of zinc fingers 5-7 completely abolished binding. The binding of full-length CTCF generated a DNase I protected domain extending from position -78 to -116, which was interrupted by a hypersensitive site at position -99. Selective deletions from the N- and C-terminal sides of the zinc finger domain showed that the N-terminal end of the zinc finger domain was aligned toward the transcriptional start site. Furthermore, deletions of zinc fingers peripheral to the essential zinc fingers 5-7 decreased the stability of the binding complex by interrupting sequence-specific interactions.

Upregulation of amyloid precursor protein gene promoter in rat primary hippocampal neurons by phorbol ester, IL-1 and retinoic acid, but not by reactive oxygen species.

The APP gene promoter has multiple regulatory sequences, some of which may contribute to the neuropathology of Alzheimer's disease (AD). In this study, we investigated the effects of phorbol ester (PMA), IL-1, retinoic acid and reactive oxygen species on APP promoter activity in primary hippocampal neurons. We transfected neurons with either of two APP promoter constructs, a -2.8 kb and a shorter -488 bp upstream fragment fused to the chloramphenicol transferase (CAT) reporter gene. We demonstrated that phorbol 12-myristate-13 acetate (PMA), retinoic acid and IL-1 all stimulated both APP promoter constructs in hippocampal neurons after 24 h treatment. PMA and IL-1 treatments led to 2-fold increases of APP promoter activity. Retinoic acid induced a 3-fold increase. In addition, the magnitude of APP promoter responses to PMA and IL-1 treatment was similar between APP -2.8 kb and -488 bp plasmid transfected neurons. This suggests that the AP-1 sequence at -350 to -344 in the APP promoter may mediate the stimulatory effects of PMA and IL-1, as previously observed in endothelial and HeLa cells. In contrast, hydrogen peroxide, which was shown to activate NF-kappaB in primary neurons, failed to stimulate APP promoter activity, suggesting that the regulatory elements in the APP promoter may not respond to reactive oxygen species. Overall, these data indicate that APP expression in primary neurons can be modulated by PMA, IL-1 and retinoic acid. However, the contribution of reactive oxygen to Alzheimer's disease may not be directly related to the activation of the APP gene promoter but instead to neuronal damage associated with oxidative stress. Since elevated levels of IL-1 have been observed in AD brain, IL-1 could contribute to development of Alzheimer's disease by stimulating APP synthesis in primary neurons.

Two nuclear factor binding domains activate expression from the human amyloid beta-protein precursor promoter.

Amyloid beta-protein is derived from the amyloid beta-protein precursor (APP), and it is a major component of brain amyloid depositions in Alzheimer's disease and Down's syndrome. Overexpression of APP may be one of several factors contributing to amyloid formation. The APP promoter was therefore analyzed to determine the mechanism by which APP gene expression is regulated. Cell type-specific expression from the human APP promoter is accomplished with 94 base pairs upstream from the main transcriptional start site. This promoter region contains two nuclear factor binding domains, designated APB alpha and APB beta. The contribution of these binding domains to promoter activity was analyzed by transient transfection in HeLa and PC-12 cells. Under standard culture conditions, at least 70-90% of the total activity from the APP promoter can be attributed to binding domain APB beta. The recognition domain for this nuclear factor binding site is defined by the sequence GCCGCTAGGGGT (position -93 to -82). The contribution of binding site APB alpha to APP promoter activity is considerably lower and represents 10-30% of the total activity. The recognition site for the nuclear factor that binds to APB alpha is delineated by the sequence GGATCAGCTGAC (position -53 to -42). Elimination of both binding sites causes APP promoter activity in vivo to decline to near background levels.

The amyloid beta-protein precursor promoter. A region essential for transcriptional activity contains a nuclear factor binding domain.

A manifestation of Alzheimer's disease is the presence of amyloid depositions in brains of afflicted individuals. A major component of these depositions is the amyloid beta-protein, which is a truncated form of the larger amyloid beta-protein precursor (APP). To investigate the regulation of APP gene expression, the APP promoter and selected deletions were placed 5' to the reporter gene chloramphenicol acetyltransferase. The promoter deletions were transfected into different cell lines that showed variant levels of endogenous APP transcripts. Transient transfection assays showed that 96 base pairs 5' to the transcriptional start site are sufficient for cell type-specific promoter activity. A nuclear factor that binds to this region in a sequence-specific manner was identified by mobility shift electrophoresis, DNase footprinting, and methylation interference. The DNase-protected region covers about 25 base pairs on both strands (position -31 to -55). Mutations within this domain revealed a sequence of 12 base pairs that is crucial for factor binding. This sequence overlaps with the consensus sequences for transcription factors AP-1 and AP-4. However, competition experiments suggest that the nuclear factor that binds to the APP promoter is distinct from both AP-1 and AP-4. Factor binding to the characterized recognition sequence is observed in nuclear extracts originating from human, mouse, and rat cells, suggesting a high degree of conservation.

Plasma hyaluronan-binding protein is a serine protease.

CTCF is an essential factor for optimal transcription from the amyloid beta-protein precursor promoter. A proteolytic activity detected in bovine, rabbit, horse, and human serum cleaves CTCF at three major sites, resulting in a modified mobility shift pattern of the fragments that retain DNA binding ability. The protease was purified to electrophoretic homogeneity, partially sequenced, and identified as the plasma hyaluronan-binding protein. The proteolytic activity was selectively abolished by various serine protease inhibitors, including the Kunitz-type protease inhibitor domain of amyloid beta-protein precursor. Reduction with beta-mercaptoethanol showed that the 70-kDa protein consists of two polypeptides with apparent molecular masses of 44 and 30 kDa. The serine protease domain was localized to the 30-kDa polypeptide as determined by [(3)H]diisopropylfluorophosphate binding.

The zinc finger protein CTCF binds to the APBbeta domain of the amyloid beta-protein precursor promoter. Evidence for a role in transcriptional activation.

The promoter of the amyloid beta-protein precursor (APP) gene directs high levels of cell type-specific transcription with 94 base pairs 5' to the main transcriptional start site. An essential activator domain in this proximal APP promoter is a nuclear factor binding site designated as APBbeta. The recognition domain for the APBbeta binding factor is located between position -93 and -82 relative to the main transcriptional start site. The nuclear factor that binds to the APBbeta site was partially purified by multiple steps of ion exchange and hydroxyapatite chromatography. Based on UV cross-linking results, a protein with an apparent molecular mass of 140 kDa was selected as the putative APBbeta binding protein. After the final purification step consisting of preparative SDS-polyacrylamide gel electrophoresis, partial peptide sequences were obtained that completely matched the transcriptional factor CTCF. This protein is a known regulator of c-myc and lysozyme gene expression, and it binds to a variety of diverse DNA sequences. The binding of CTCF to the APBbeta domain was further established by competition with CTCF binding oligonucleotides in mobility shift electrophoresis. The identity was also confirmed by the observation that the APBbeta binding factor is recognized by antibodies against C- and N-terminal sequences of CTCF. In addition, oligonucleotide competition during in vitro transcription affirmed that CTCF acts as a transcriptional activator in the APP gene promoter.

USF binds to the APB alpha sequence in the promoter of the amyloid beta-protein precursor gene.

The APB alpha domain in the amyloid beta-protein precursor (APP) promoter contains a nuclear factor binding domain with the core recognition sequence TCAGCT-GAC. Proteins in nuclear extracts from brain and numerous cell lines bind to this domain and it contributes approximately 10-30% to the basal APP promoter activity. Included in this domain is the CANNTG motif, which is recognized by basic helix-loop-helix transcription factors. The same motif is also present in the CDEI element of the yeast centromere and in the adenovirus major late promoter (AdMLP). Here we present evidence based on thermostability, relative binding affinity, eletrophoretic mobility and antibody recognition that the cellular proteins that bind to the APB alpha and CDEI motifs are USF. However, the relative binding affinity for the motifs is different. The affinity of USF for AdMLP is approximately 20-fold higher than for the APB alpha sequence and 5-fold higher than for the CDEI sequence. Mutational analysis suggested that the primary determinant for USF binding affinity resides within the octamer CAGCTGAC, which is composed of the E-box consensus sequence CANNTG followed by the dinucleotide AC. The human homolog of the mouse CDEI binding protein did not bind to either the CDEI sequence or APB alpha.

The initiator element and proximal upstream sequences affect transcriptional activity and start site selection in the amyloid beta-protein precursor promoter.

The TATA-less human amyloid beta-protein precursor promoter contains an initiator element with the sequence CGTCA+1GTT. Primary transcriptional start sites were identified at positions +1 and -4. Deletion of the upstream activator elements APBbeta and APBalpha did not affect the selection of transcriptional start sites, although total transcriptional activity was reduced both in vitro and in vivo. Mutations within the initiator element shifted the transcriptional start sites and reduced transcriptional activity. Mutations between positions -6 and -35 changed the relative utilization of start sites +1 and -4 without affecting the total level of transcriptional activity. A 10-base pair deletion between position -40 and -31 increased in vitro transcriptional activity with a preeminent utilization of the start site at position -4. In contrast, a 20-base pair deletion between position -40 and -21 resulted in a reduction in transcriptional activity and in the primary utilization of the start site at position +1. Furthermore, transactivation by APBbeta and APBalpha was eliminated. DNase I footprinting provided evidence for the existence of two binding domains designated UE (position -12 to -30) and Inr (position +7 to -7). The positions of these binding domains are altered in mutations and deletions that affect transcriptional activity.

CTCF is essential for up-regulating expression from the amyloid precursor protein promoter during differentiation of primary hippocampal neurons.

The transcriptional mechanism underlying amyloid precursor protein (APP) regulation in primary neurons during development was investigated. We observed an approximately threefold elevation of APP mRNA levels in differentiating rat hippocampal neurons between day 1 and day 7 in culture and in rat brain hippocampi between embryonic day 18 and postnatal day 3. When an APP promoter construct extending to position -2,832 upstream from the main transcriptional start site was transfected into primary rat hippocampal neurons, promoter activity increased from day 1 until reaching a maximum on day 7 in culture. This increase in APP promoter activity was correlated more closely with the time course of expression of the synaptic vesicle protein synaptophysin, an indicator of synaptogenesis, than with neurofilament accumulation, an indicator of neuritogenesis. Transfection of 5' APP promoter deletions and internal block mutations indicated that the CTCF binding domain designated APBbeta was the primary contributor to the increase in APP promoter activity. Furthermore, the binding of transcription factor CTCF to the APBbeta element increased approximately fivefold between day 1 and day 7, whereas the binding of USF to the APBalpha sequence increased only twofold. These results suggest that CTCF is pivotal for the up-regulation of APP expression during synaptogenesis in primary neurons.

The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor.

Although beta actin mRNA is down-regulated during myogenesis, the beta actin promoter confers constitutive expression when joined to heterologous genes transfected into a variety of different cell backgrounds, including differentiated muscle. Normal promoter activity is dependent upon the binding of a ubiquitous factor to the CCAAT-box element. Loss or reduction in factor binding correlates with a major reduction in promoter activity both in vivo and in vitro. The binding domain covers approximately 23 base pairs as determined by DNase footprinting. Methylation of A and G residues in and adjacent to the CCAAT box results in the loss of factor binding. Mutations across the binding domain indicate that the sequence GCCAATCAG within the domain is sufficient as a recognition sequence for factor binding. This binding is not competed by the alpha cardiac actin CCAAT sequence. Bandshift experiments demonstrate a predominant single band of similar mobility in nuclear extracts from various cells and tissues, with the exception of HeLa cells. The prevalence of the factor and its recognition sequence in a variety of promoters suggests that this factor has a common role in the transcriptional activation of several eukaryotic promoters.

Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.