Abolished Promoter Activity Research Articles

Transcriptional Regulation of the p67phoxGene: ROLE OF AP-1 IN CONCERT WITH MYELOID-SPECIFIC TRANSCRIPTION FACTORS

We have investigated the myeloid-specific transcriptional regulation of p67(phox), an essential component of phagocyte respiratory burst NADPH oxidase. Analysis was carried out on the p67(phox) 5'-flanking region from -3669 to -4 (relative to ATG), including the first exon and intron and part of the second exon. The construct extending from -985 to -4 produced the highest luciferase activity in myeloid HL-60 cells but was not active in HeLa or Jurkat cells, indicating myeloid-specific expression. Four active elements were identified: Sp1/Sp3 at -694, PU.1 at -289, AP-1 at -210, and PU.1/HAF1 at -182, the latter three being in the first intron. These cis elements bound their cognate transacting factors both in vitro and in vivo. Mutation of the Sp1, PU.1, or PU.1/HAF1 site each decreased promoter activity by 35-50%. Mutations in all three sites reduced promoter activity by 90%. However, mutation of the AP-1 site alone nearly abolished promoter activity. The AP-1 site bound Jun and Fos proteins from HL-60 cell nuclear extract. Co-expression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67(phox) promoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function.

Binding of Serum Response Factor to CArG Box Sequences Is Necessary but Not Sufficient to Restrict Gene Expression to Arterial Smooth Muscle Cells

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22alpha promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5'-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

Modulation of the promoter region of prepro-hypocretin by α-interferon

Hypocretins 1 and 2 (also called orexins A and B, respectively) are hypothalamic neuropeptides that have recently been shown to be involved in the sleep disorder narcolepsy and possibly in the normal regulation of sleep and wake functions. These two peptides are derived from a single precursor molecule called prepro-hypocretin, also known as prepro-orexin. We have cloned a 450 bp fragment from the 5′-flanking region of the human prepro-hypocretin gene and demonstrated that this fragment has promoter activity in vitro. Deletions at the 5′ end from −450 to −188 reduced the promoter activity by ∼50%. Further deletion from the 5′-end to −69 almost completely abolished promoter activity. The 450 bp fragment contains a number of potential transcription factor binding sites, including an interferon (IFN) response element. Our studies demonstrate that α-IFN strongly inhibits the promoter activity of both 450 and 188 bp fragments in a dose-dependent manner. The inhibitory effect of α-IFN is consistent with recent studies which suggest that hypocretin 1/orexin A may be involved in modulating arousal states and with the literature indicating involvement of immune-related molecules in sleep regulation.

Proximal Promoter of the Surfactant Protein D Gene: REGULATORY ROLES OF AP-1, FORKHEAD BOX, AND GT BOX BINDING PROTEINS

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following lung injury. Because AP-1 proteins regulate cellular responses to diverse environmental stimuli, we hypothesized that the conserved AP-1 motif (at -109) and flanking sequences in the human SP-D promoter contribute to the regulation of SP-D expression. The AP-1 sequence specifically bound to fra-1, junD, and junB in H441 lung adenocarcinoma nuclear extracts. Mutagenesis of the AP-1 motif in a chloramphenicol acetyltransferase reporter construct containing 285 base pairs of upstream sequence nearly abolished promoter activity, and co-transfection of junD significantly increased wild type but not mutant promoter activity. The sequence immediately downstream of the AP-1 element contained a binding site for HNF-3 (FOXA), and simultaneous mutation of this site (fox-d) and an upstream FoxA binding site (-277, fox-u) caused a 4-fold reduction in chloramphenicol acetyltransferase activity. Immediately upstream of the AP-1-binding site, we identified a GT box-containing positive regulatory element. Despite finding regions of limited homology to the thyroid transcription factor 1-binding site, SP-D promoter activity did not require thyroid transcription factor 1. Thus, transcriptional regulation of SP-D gene expression involves complex interactions with ubiquitous and lineage-dependent factors consistent with more generalized roles in innate immunity.

Transcriptional Regulation of Rat Scavenger Receptor Class B Type I Gene

The scavenger receptor class B type I (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells and plays an important role in the reverse uptake of cholesterol to the liver and in the delivery of substrates for steroidogenesis in steroidogenic organs. We report here on the isolation and characterization of the upstream promoter region of the rat SR-BI gene. The transcription start site for rat SR-BI was mapped, and DNA sequence analysis revealed the presence of binding sites for the Sp1 family in the proximal 5'-flanking region. Analysis of deletion mutants with different 5' lengths revealed that the region between -121 and -90 base pairs from the transcription start site is essential for the efficient transcription of SR-BI. Both Sp1 and Sp3 bind to three GC boxes in the region (-141 to -1 base pairs) in a sequence-specific manner. Mutations in any of the GC boxes decreased efficient transcription from this promoter in MA-10 mouse Leydig tumor cells. The overexpression of Sp1 or Sp3 protein enhanced the rat SR-BI promoter activity. These results indicate that Sp1 family members of transcription factors are essential for transcription of the rat SR-BI gene.

Methylation-dependent Silencing of the Testis-specific Pdha-2 Basal Promoter Occurs through Selective Targeting of an Activating Transcription Factor/cAMP-responsive Element-binding Site

In this study, we demonstrate that methylation-dependent repression of the Pdha-2 core promoter is mediated regionally through a consensus activating transcription factor/cAMP-responsive element-binding site located between nucleotides -54 and -62 upstream of the major transcriptional start site. Targeting of the CpG dinucleotide within this cis-element significantly disrupts the ability of this basal promoter to activate gene expression in vitro and completely abolishes promoter activity in vivo. DNase I footprinting experiments indicated that availability of the nuclear factor(s) binding this element is limiting in sexually immature mouse testis, and as such, these factors may play an important role in the coordinate activation of early spermatogenic gene expression. Interestingly, CpG dinucleotides associated with the hypersensitive region flanking the activating transcription factor/cAMP-responsive element-binding site appear to confer some conformational structure on the promoter since mutations at these specific CpG dinucleotides result in elevated basal levels of transcription. This raises the possibility of a potential bifunctional role for CpG dinucleotides in either methylation-dependent or -independent processes. Our data support the notion that hypomethylation and transcription factor recruitment are necessary events that precede gene activation at the early stages of spermatogenesis.

Structure and Regulation of the Envoplakin Gene

Envoplakin, a member of the plakin family of proteins, is a component of desmosomes and the epidermal cornified envelope. To understand how envoplakin expression is regulated, we have analyzed the structure of the mouse envoplakin gene and characterized the promoters of both the human and mouse genes. The mouse gene consists of 22 exons and maps to chromosome 11E1, syntenic to the location of the human gene on 17q25. The exon-intron structure of the mouse envoplakin gene is common to all members of the plakin family: the N-terminal protein domain is encoded by 21 small exons, and the central rod domain and the C-terminal globular domain are coded by a single large exon. The C terminus shows the highest sequence conservation between mouse and human envoplakins and between envoplakin and the other family members. The N terminus is also conserved, with sequence homology extending to Drosophila Kakapo. A region between nucleotides -101 and 288 was necessary for promoter activity in transiently transfected primary keratinocytes. This region is highly conserved between the human and mouse genes and contains at least two different positively acting elements identified by site-directed mutagenesis and electrophoretic mobility shift assays. Mutation of a GC box binding Sp1 and Sp3 proteins or a combined E box and Krüppel-like element interacting with unidentified nuclear proteins virtually abolished promoter activity. 600 base pairs of the mouse upstream sequence was sufficient to drive expression of a beta-galactosidase reporter gene in the suprabasal layers of epidermis, esophagus, and forestomach of transgenic mice. Thus, we have identified a regulatory region in the envoplakin gene that can account for the expression pattern of the endogenous protein in stratified squamous epithelia.

Sp1/Sp3 and PU.1 Differentially Regulate β5Integrin Gene Expression in Macrophages and Osteoblasts

Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.

Functional analysis of the promoter region of the human phosphotyrosine phosphatase activator gene: Yin Yang 1 is essential for core promoter activity

The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5' untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.

Differential activation of the SMalphaA promoter in smooth vs. skeletal muscle cells by bHLH factors.

E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha-actin (SMalphaA) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalphaA promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

Integrin αvPromoter Activity in Keratinocytes

Background.During reepithelialization keratinocytes show increased expression of the integrin subunit αv. We have investigated the promoter region of the αvintegrin subunit to learn more about its regulation.Methods.The promoter region of the human integrin αvgene was cloned into a luciferase reporter vector. Deletional mutants were created using PCR. Computerized sequence analysis was performed using the Wisconsin Package. Gel-shift analysis was performed using keratinocyte nuclear extracts and oligonucleotides spanning the regions of interest.Results.Deletion from −522 bp to −235 resulted in no discernible effect on promoter activity. In contrast deletion of the next 22 bp, which included a putative ets binding site, reduced activity by approximately half. Further deletion to −139 bp essentially abolished promoter activity. Computer searching of this region of the integrin αvpromoter revealed two tandemly repeated motifs, TCCTCCTCC, that had previously been implicated in the function of the epidermal growth factor receptor (EGFR) promoter. Comparison of the αvintegrin promoter to the EGFR promoter revealed an area of high homology in this region. Gel-shift analysis revealed binding of a single-strand specific DNA binding protein to single-stranded oligos comprising these motifs, but no binding of factors to the double-stranded oligo containing the ets binding site.Conclusions.In keratinocytes αvintegrin expression is controlled by a region of the promoter with high homology to the epidermal growth factor receptor promoter. This region binds single-strand specific DNA binding proteins that are likely to be important in controlling transcription.

Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes.

MHC class I molecules are normally expressed at very low levels in the brain and their up-regulation in response to cytokines and viral infections has been associated with a number of neurological disorders. Here we demonstrate that the down-regulation of surface class I molecules in differentiated primary rat oligodendrocytes was accompanied by reduced steady-state levels of class I heavy-chain mRNA. Transient expression assays were performed in oligodendrocytes and fibroblasts, using a mouse H-2Kb class I promoter chloramphenicol acetyltransferase plasmid termed pH2KCAT (which contained 5'-flanking sequences from -2033 to +5 bp of the H-2Kb gene relative to the transcriptional start site at +1 bp). These assays showed that H-2Kb promoter activity was reduced in oligodendrocytes but not in class I-expressing fibroblasts. H-2Kb promoter activity was up-regulated in oligodendrocytes co-transfected with a plasmid expression vector encoding the transcriptional activator tax of human T-cell leukaemia virus type I, showing that down-regulation of promoter activity was reversible. Deletion mutant analysis of the H-2Kb promoter revealed the presence of negative regulatory elements that were functional in oligodendrocytes at -1.61 to -1.07 kb and -242 to -190 bp. Deletion of sequences in pH2KCAT encompassing the downstream element totally abolished promoter activity in both oligodendrocytes and fibroblasts, whereas a deletion within the upstream negative regulatory element increased promoter activity specifically in oligodendrocytes. The upstream negative regulatory element also down-regulated a linked heterologous herpes simplex virus thymidine kinase promoter in oligodendrocytes, but not in fibroblasts. Gel retardation assays using overlapping DNA probes that spanned the entire -1.61 to -1.07 kb region revealed the presence of a number of DNA-binding activities that were present in oligodendrocyte, but not in fibroblast nuclear extracts.

DNA methylation accounts for the inhibition of collagen VI expression in transformed fibroblasts.

The expression of collagen VI, an adhesive glycoprotein of the extracellular matrix, is completely inhibited in virally transformed fibroblasts and in many cell lines derived from spontaneous mesenchymal tumors. Here we present evidence that DNA methylation plays an important role in this inhibition: (a) The mRNA level for DNA methyltransferase is highly increased in simian virus 40 (SV40)-transformed fibroblasts compared with normal cells and this increase correlates with the decrease of the mRNA level for collagen VI. (b) Methylation of the alpha2(VI) collagen promoter in vitro abolishes promoter activity in a transient transfection assay. (c) Genomic sequencing reveals extensive methylation of the promoter region in SV40-transformed cells, but virtually no methylation of the corresponding region in normal cells. Increased methylation is also observed in a rhabdomyosarcoma cell line. (d) Two of the cis-acting elements of the alpha2(VI) collagen promoter lose their affinity for transcription factor AP2 when methylated in vitro as demonstrated by gel retardation experiments. DNA methylation is therefore involved in the silencing of the alpha2(VI) collagen gene. It seems likely that the same mechanism is also responsible for the repression of other transformation-sensitive proteins.

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.

Promoter activity of the Campoletis sonorensis polydnavirus (CsPDV) WHv1.6 gene was analysed by transient transfection assays in insect cell culture using constructs expressing the CAT gene. Deletions of the WHv1.6 gene promoter were used to define promoter regions important for expression. Progressive deletion of the regions upstream of the TATA box reduced the promoter activity, whereas deletions eliminating the TATA box abolished promoter activity. Cis-activating elements were detected up to 1 kb upstream of the WHv1.6 transcription initiation site (TIS). Promoter elements increasing transcription were detected between -444 and -550 bp and between -831 and -1035 bp relative to the TIS. Analysis of the 3' flanking sequences of the WHv1.6 gene indicated that the polyadenylation signals were the only important elements affecting expression in the constructs. Comparison of promoter regions of four cysteine-rich CsPDV genes revealed homologous sequences that may be important for transcriptional regulation of polydnavirus gene expression in parasitized Heliothis virescens larvae.

Sequencing and Functional Analysis of the SNRPN Promoter: In Vitro Methylation Abolishes Promoter Activity

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.

Mutational analysis of the RNA-fork model of the influenza A virus vRNA promoter in vivo.

The genome of influenza A virus consists of eight negative-stranded RNA segments which have partially complementary non-coding terminal sequences. Previous transcription studies of the virion RNA promoter in vitro have shown that the 5' terminus forms an integral part of the promoter and an 'RNA-fork' model has been proposed for the initiation of transcription. According to this model part of the promoter is formed by an RNA-duplex which involves complementary residues 10 to 1 2 of the 3' end and residues 11' to 13' of the 5' end. With a reverse genetics system, based on the chloramphenicol acetyltransferase (CAT) gene, we have now tested this part of the promoter in vivo. Single mutations of the conserved residues at positions 11 and 12 of the 3' terminus and at positions 12' and 13' of the 5' terminus abolished promoter activity. The introduction of complementary mutations into both termini partially restored activity. On the other hand, mutations at positions 10 of the 3' terminus and 11' of the 5' terminus inhibited activity independently of whether a base-pair was formed or not. Thus, at these positions, the nature of the residues is apparently more important than their ability to form base-pairs. These results extend our previous virion 'RNA-fork' model and are consistent with in vitro findings that the 5' terminus is involved in the initiation of transcription.

Interaction of Sp1 with the Growth- and Cell Cycle-Regulated Transcription Factor E2F

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type specificity of the promoter. The functional activity of the CRE site was confirmed through studies of the endogenous chromogranin A gene. Northern mRNA analysis showed that expression of the endogenous chromogranin A gene was stimulated seven- to eightfold by cAMP in PC12 cells, whereas no induction occurred in the NIH3T3 cells. Similar cAMP induction was obtained with the transfected chromogranin A promoter in PC12 cells, and abolition of the CRE site (by deletion or point mutation) eliminated the induction. Thus, the CRE site in the chromogranin A proximal promoter is functional and plays a crucial, indeed indispensable, role in neuroendocrine-specific expression of the gene. These results also provide insight into transcriptional mechanisms governing acquisition of the neuroendocrine secretory phenotype.

Tissue-specific expression and temporal regulation of the rice glutelin Gt3 gene are conferred by at least two spatially separated cis-regulatory elements.

The rice glutelin Gt3 promoter was fused to a beta-glucuronidase (GUS) reporter gene and its expression evaluated in transgenic tobacco plants. Histochemical analysis revealed that the expression of the introduced Gt3 promoter/GUS (beta-glucuronidase) chimeric gene was confined to endosperm tissue of developing seeds. 5'-promoter deletion analysis revealed that two domains of the Gt3 promoter, -346 to -263 bp (domain I) and -945 to -726 bp (domain II) from the transcriptional start site, were essential for optimum expression of the GUS reporter gene. Removal of 5' sequences upstream of -726 resulted in a reduction in overall promoter activity and a shift in temporal expression from a maximum of 16-20 days after flowering to 24 days. Removal of DNA sequences from the 5' end to -346 yielded a promoter fragment that was still able to confer endosperm-specific expression, although a further deletion to -263 abolished promoter activity. These data suggest that at least two cis-regulatory elements are required for endosperm specificity and temporal regulation of glutelin Gt3 gene expression.