Analysis of molecular mechanisms controlling neuroendocrine cell specific transcription of the chromogranin A gene.

Chromogranin A (CgA), a member of the granin/secretogranin family of acidic glycoproteins that play multiple roles in the process of regulated secretion of peptide hormones and neurotransmitters, is specifically expressed in endocrine and neuroendocrine cells. We previously cloned and characterized the human (h) CgA gene and showed that nucleotides -55 to +32 relative to the transcriptional start site that contain a consensus cAMP element (CRE) and TATA-box motif were sufficient for neuroendocrine cell-specific expression. Here, we examined the role of the well conserved CRE in basal and cAMP-stimulated transcription in neuroendocrine cells. Transient transfection studies with hCgA gene promoter/chloroamphenical acetyl transferase (CAT) reporter constructs were conducted in a panel of neuroendocrine cell lines as well as in nonendocrine cell lines. Deletion or mutation of the CRE resulted in loss of neuroendocrine cell specific transcriptional activity. Mutation of a well conserved region (the TG-box) located between the CRE and the TATA box had no effect or resulted in only a modest decrease in activity. Mutation of the CRE in 5'-extended (-2300 to +32 and -700 to +32) constructs resulted in a 50-75% decrease in basal activity in neuroendocrine cells. This emphasized the importance of the CRE in basal transcription and also suggested that other elements between -700 and -55 may act independently of the CRE to contribute to full basal activity in some neuroendocrine cells. Dibutyryl cAMP stimulated transcriptional activity in neuroendocrine cells, and this was abolished by mutation of the CRE. In the presence of a PKA inhibitor, dibutyryl cAMP-induced activity was completely abolished and basal activity was decreased by up to 85%. Similar protein-DNA complexes were formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A predominant complex that was supershifted by addition of a CREB antibody was identical in all cell types. By immunoblot analysis, levels of total CREB protein and phosphorylated (Ser 133) CREB did not differ between neuroendocrine and nonendocrine cells. Phosphorylated CREB was increased by forskolin treatment, an effect that was blocked by a PKA-inhibitor. Expression of the transcriptional cointegrator, CREB-binding protein (CBP), assessed by both RT-PCR and Western blot analysis, did not differ between neuroendocrine and nonendocrine cells. In summary, the CRE in the hCgA gene proximal promoter is critical for both basal and cAMP-induced expression in neuroendocrine cells via a PKA-mediated pathway. However, the neuroendocrine specificity of hCgA gene transcription mediated by the CRE is not a function of levels of total CREB or phosphorylated CREB or its cointegrator CBP. Specificity may be achieved by a PKA-responsive CRE-binding protein other than CREB expressed specifically in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or may lie downstream of a CRE-binding protein, e.g. in the activity or amount of cointegrators other than CBP, which are required to couple transactivators to the basal transcriptional machinery.

Identification and characterization of a novel transcriptional activation domain in the CREB-binding protein.

The CREB-binding protein (CBP) plays a central role in the regulation of gene expression by several different second messenger pathways including serum growth factors, cAMP and phorbol esters. CBP specifically binds to the phosphorylated forms of CREB and c-Jun and is thought to activate transcription through a C-terminal activation domain. In this report, we demonstrate that the C terminus of CBP is dispensable for its ability to stimulate phospho-CREB activity, and, further, that the deletion of this domain produces highly active, mutant forms of CBP. The novel N-terminal activation identified by this deletional analysis consists of the first 714 amino acids of CBP and is sufficient for high levels of transcriptional activity. This domain is also capable of stimulating the activity of a second cAMP-regulated factor, ATF-1. Surprisingly, ATF-1 activity is not significantly stimulated by full-length CBP suggesting that the C-terminal domain of CBP may also serve to regulate ATF-1/CBP activity. Additionally, the demonstration that one of our hyperactive CBP mutants is able to activate a nonphosphorylatable mutant of CREB (M1 CREB) provides the first evidence that CBP may play a role in regulating the basal transcriptional activity of CREB.

Engineered leucine zippers show that hemiphosphorylated CREB complexes are transcriptionally active.

The ability of basic/leucine zipper transcription factors to form homo- and heterodimers potentially increases the diversity of signaling pathways that can impinge upon a single genetic element. The capacity of these proteins to dimerize in various combinations complicates the analysis of their functional properties, however. To simplify the functional analysis of CREB dimers, we mutated selected residues within the leucine zipper region to generate proteins that could only heterodimerize. These mutants allowed us to determine whether phosphorylation of both CREB subunits was necessary for transcriptional activation. Our results reveal that hemiphosphorylated CREB dimers are half as active as fully phosphorylated dimers. It is possible, therefore, that the degree of phosphorylation of CREB complexes could modulate the transcriptional responses of specific genes to cAMP.

3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line.

The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)

A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo.

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.

Site-specific binding constants for actinomycin D on DNA determined from footprinting studies.

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A.

Many promoters respond transcriptionally to elevated levels of cAMP through the cAMP-responsive enhancer (CRE). Several proteins have been characterized which bind to the CRE and presumably modulate CRE-dependent transcription. Of these CRE-binding proteins, only CREB has been shown to be activated by cAMP-dependent protein kinase A (PKA), and as such, CREB represents the only basis for our understanding of cAMP-regulated transcriptional activity. In this report, we describe the complete cDNA sequence of another CRE-binding protein, ATF-1. This protein contains a consensus phosphorylation site for PKA and shares extensive homology with CREB in the region surrounding and carboxyl-terminal to the PKA site. ATF-1 does not contain sequences homologous to the glutamine-rich amino-terminal domain found in CREB, however. ATF-1, like CREB, is expressed in a wide variety of cell types, and ATF-1 is capable of dimerizing with CREB. Both ATF-1 homodimers and ATF-1/CREB heterodimers bind to the CRE but not to the related phorbol ester response element. ATF-1 is as active as CREB in its ability to mediate the transcriptional effects of PKA, and, because ATF-1 has a smaller effect on basal expression, it is actually more responsive than CREB to cAMP. These findings indicate that CREB is not unique in its ability to mediate cAMP-dependent transcriptional regulation.

Somatostatin gene regulation: an overview.

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimer's disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Quantitative footprinting analysis. Binding to a single site.

The theory for measuring ligand binding constants from footprinting autoradiographic data associated with a single binding site is derived. If the ligand and DNA cleavage agent compete for a common site, the spot intensities are not proportional to the amount of DNA not blocked by ligand. The analysis of a single site is experimentally illustrated by using results for the anticancer drug actinomycin D interacting with the duplex d(TAGCGCTA)2 as probed with the hydrolytic enzyme DNase I.

Molecular mechanisms of cAMP-regulated gene expression.

The ability of many genes to be induced by cAMP is dependent on the presence of enhancers located in the regions of DNA upstream of the start sites to the genes. The two best characterized enhancers are the CRE (5'-TGACGTCA-3') and the AP-2 site (5'-CCCCAGGC-3'). The activity of the CRE is modulated by sequences adjacent to the consensus sequence as well as by promoter context and cell type. The complex control of the CRE is reflected in the large number of cloned CRE binding proteins that arise both from unique genes and from splice variants. These factors are leucine zipper proteins that must dimerize before binding to DNA. Although all of the factors isolated can form active homodimers, many are also able to form heterodimers. The amino termini of these proteins contain consensus phosphorylation sites through which these factors trans-activate their cognate promoters. The diversity of the trans-acting factors and their cis-acting sequences reflects the precise control that cells require in the modulation of gene expression by cAMP.

Esperamicins, a class of potent antitumor antibiotics: mechanism of action.

The esperamicins represent a class of antitumor antibiotics characterized by an unusual chemical core structure and extremely potent cytotoxicity. The mechanism by which these drugs produce cytotoxicity was investigated and found to be related to the formation of single- and double-strand DNA breaks. Using five structurally related analogs, we defined a structure-activity relationship for cytotoxicity in various eukaryotic and DNA-repair-deficient prokaryotic cell lines, for DNA breakage in a human colon carcinoma cell line, and for DNA breakage in vitro in pBR322 DNA. Mild reducing agents such as dithiothreitol greatly increased the DNA breakage potency of these analogs in vitro. Results suggest that the pendant aromatic chromophore of esperamicin A1 may contribute to the uptake of the drug into cells but may also hinder double-strand DNA break formation. Little DNA breakage specificity was observed for the drug in a 139-base-pair fragment of pBR322 DNA. Evidence supports a previously proposed mechanism whereby esperamicins may produce the observed DNA breaks through reduction of the methyl trisulfide group to a thiolate anion followed by a Michael addition of the anion across the alpha,beta-unsaturated ketone. This addition may result in the saturation of the bridgehead double bond, thus allowing the two triple bonds to approach each other, causing cyclization of the diyn-ene to form a phenylene diradical. It is likely that this diradical is the active form of the drug responsible for single- and double-strand DNA breakage produced by this class of antitumor agents.

Actinomycin D induced DNase I cleavage enhancement caused by sequence specific propagation of an altered DNA structure.

Two DNA hexadecamers containing one central 5'-GC-3' base step have been examined by footprinting methodology in the presence and absence of actinomycin D. The results of these studies, coupled with imino proton NMR measurements indicate that the antitumor drug causes a change in DNA conformation at a distance from the actinomycin intercalation site in a molecule of sequence d[ATATATAGCTATATAT] that does not occur in d[AAAAAAAGCTTTTTTT]. The experiments demonstrate that DNase I rate enhancements associated with actinomycin D binding are caused by ligand alteration of equilibrium DNA structure.

Determination of netropsin-DNA binding constants from footprinting data.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

Rate enhancements in the DNase I footprinting experiment.

Footprinting experiments for DNase I digests of a 139-base-pair segment of pBR-322 DNA in the presence of either netropsin or actinomycin D were carried out. Plots of oligonucleotide concentration as a function of drug concentration were analyzed to study the enhancement in cleavage rates at approximately 30 sites, accompanying drug binding at other sites. The pattern of enhancements is not consistent with drug-induced DNA structural changes, but agrees with a redistribution mechanism involving DNase I. Since the total number of enzyme molecules per fragment remains unchanged, drug binding at some sites increases the enzyme concentration at other sites, giving rise to increased cleavage. The consequences of the redistribution mechanism for analysis of footprinting experiments are indicated.

Quantitative footprinting analysis of the netropsin-DNA interaction.

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.

Actinomycin D facilitates transition of AT domains in molecules of sequence (AT)nAGCT(AT)n to a DNAse I detectable alternating structure.

The interaction of actinomycin D with (AT)nAGCT(AT)n (where n = 2, 3, or 4) was investigated using a combination of imino proton NMR and DNAse I digestion. The stoichiometry of the interaction appears to be one:one with the actinomycin chromophore intercalated between the two GC base pairs. This binding event facilitates the conversion of the flanking repetitive AT regions to an alternating conformation characterized by induced sensitivity of the ApT sequences to attack by DNAse I. The neighboring TpA sequences do not exhibit rate changes as a function of binding of the drug. The potential relevance of such ligand induced DNA structural alterations is discussed.